Anti-CCR8 antibodies, conjugates, and their use

Abstract

Anti-CCR8 antibodies, conjugates, and their uses. Disclosed herein, in certain embodiments, are anti-hCCR8 antibodies, anti-hCCR8 antibody conjugates, and pharmaceutical compositions comprising anti-hCCR8 antibodies or conjugates. In some embodiments, methods for delivering a payload using the anti-hCCR8 antibodies described herein and methods for therapeutic use of the anti-hCCR8 antibodies described herein are also disclosed. In some embodiments, isolated antibodies comprising a chemokine receptor 8 (CCR8) binding domain containing one or more complementarity-determining regions (CDRs) are disclosed herein.

Description

[Technical Field]

[0001] cross reference This application is incorporated herein by reference to and claims the benefits of U.S. Provisional Application No. 63 / 498,977, filed April 28, 2023. [Background technology]

[0002] Regulatory T (Treg) cells are a type of T cell that normally controls the immune response in mammals and prevents the development of autoimmunity. These cells accumulate in certain tumors, establishing a suppressive tumor microenvironment (TME), which allows the tumor to evade the immune response mediated by effector T cells (Teff). This immune evasion mechanism promotes tumor progression and metastasis. Depleting Treg cells in the TME is an attractive approach to reverse immunosuppressive activity and enhance anti-tumor immunity. Therefore, there is still a need for therapies that can specifically target and inhibit the activity of Treg cells in the tumor microenvironment.

[0003] The G protein-coupled receptor (GPCR) CCR8 is differentially upregulated in tumor Treg cells. However, developing antibodies against GPCR antigens such as CCR8 is generally difficult due to their low immunogenicity. [Overview of the project] [Means for solving the problem]

[0004] In some embodiments, the Specified herein discloses isolated antibodies comprising a chemokine receptor 8 (CCR8) binding domain containing one or more complementarity-determining regions (CDRs), wherein one or more CDRs are selected from the group consisting of CDR1, CDR2, and CDR3 regions, the CDR1 region comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residues to any one of SEQ ID NOs: 2, 3, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214, and the CDR2 region comprising SEQ ID NOs: 4, 5, 6, 13, 14, 15, 48, 50 The CDR3 region includes an amino acid sequence in which the percentage of identical residues for any one of 51, 55, 59, 60, 120-125, 146-151, or 216-224 is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and the CDR3 region is in sequence numbers 8, 9, 17, 49, 52 The CCR8 binding domain includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the following: 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246. In some cases, the CCR8 binding domain includes the CDR1, CDR2, and CDR3 regions. In some cases, the CDR1 region contains an amino acid sequence in which the percentage of identical residues to a sequence selected from SEQ ID NOs. 2, 3, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214 is at least 85%, 90%, 95%, 99%, or 100%. In some cases, the CDR2 region contains an amino acid sequence in which the percentage of identical residues to a sequence selected from SEQ ID NOs. 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 216-224 is at least 85%, 90%, 95%, 99%, or 100%.In some cases, the CDR3 region contains an amino acid sequence with at least 85%, 90%, 95%, 99%, or 100% identical residue rate to a sequence selected from SEQ ID NOs. 8, 9, 17, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residue rate to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residue rate to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 13, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 56. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 6, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 8. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 13, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 157. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 52. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 53. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 50, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 52. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 51, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 243. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 50, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 53. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 51, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 244.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 50, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 245. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 50, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 246. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 56. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 61. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 59, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 62. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 60, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 63.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 60, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 64. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 65. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 57, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 66. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 58, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 67. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 68. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 60, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 69.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 51, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and CDR. The three regions contain amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 227. In some cases, the CDR1 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 47, the CDR2 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 48, and the CDR3 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 210, the CDR2 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 48, and the CDR3 region contains amino acid sequences that are at least 80% identical to any one of SEQ ID NOs. 226. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 216, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 227. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 217, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 227. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 218, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 227.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 219, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 211, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 220, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 228. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 212, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 221, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 222, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 227. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 47, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 223, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 213, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 51, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 226.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 214, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 226. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 212, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 221, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 212, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 48, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 212, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 221, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 226. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 212, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 221, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 49. In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 56.In some cases, the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 55, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 229; or the CDR1 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 54, the CDR2 region contains an amino acid sequence with at least 80% identical residues to any one of SEQ ID NOs. 224, and the CDR3 region contains an amino acid sequence with at least 80% identity to any one of SEQ ID NOs. 56.

[0005] In some cases, the CCR8-binding domain further comprises one or more framework regions (FRs), which are selected from the group consisting of FR1, FR2, FR3, and FR4 regions. In some cases, the isolated antibody contains FR1, FR2, FR3, and FR4 regions. In some cases, the FR1, FR2, FR3, and FR4 regions are human. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 19, 23, 163, 164, 175, 176, or 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR2 region contains an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, or 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR3 region contains an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR4 region contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 22, 26, 172, 173, or 185 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NOs. 163 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NOs. 165 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NOs. 169 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NOs. 172 is at least 80%, 85%, 90%, 95%, 99%, or 100%.In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 166 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 170 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 167 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%.In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 175 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 178 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 182 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 185 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 176 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 179 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 183 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 180; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 184; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173.In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 181; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 184; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 164; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 231; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 171; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173.In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 164; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 232; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 171; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%.In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%, and the FR4 region. The FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%.In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 168; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 234; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the FR1 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region contains an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%.In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 168; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 235; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173. In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 181; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 236; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173. In some cases, the FR1 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177; the FR2 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 181; the FR3 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 236; and the FR4 region contains an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 173.In some cases, the FR1 region contains an amino acid sequence with an identical residue rate of at least 80%, 85%, 90%, 95%, 99%, or 100% with respect to SEQ ID NO: 177, the FR2 region contains an amino acid sequence with an identical residue rate of at least 80%, 85%, 90%, 95%, 99%, or 100% with respect to SEQ ID NO: 181, the FR3 region contains an amino acid sequence with an identical residue rate of at least 80%, 85%, 90%, 95%, 99%, or 100% with respect to SEQ ID NO: 184, and the FR4 region contains an amino acid sequence with an identical residue rate of at least 80%, 85%, 90%, 95%, 99%, or 100% with respect to SEQ ID NO: 173.

[0006] In some cases, FR1 contains an IgG1, IgG2, IgG3, or IgG4 framework region. In some cases, FR2 contains an IgG1, IgG2, IgG3, or IgG4 framework region. In some cases, FR3 contains an IgG1, IgG2, IgG3, or IgG4 framework region. In some cases, the CCR8 binding domain contains an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 27-30, 70-86, 106-111, or 186-209 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the CCR8 binding domain contains an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 27-30 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the CCR8-binding domain contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 70–86 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the CCR8-binding domain contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 106–111 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the CCR8-binding domain contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 186–209 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the percentage of identical residues is determined by BLASTp. In some cases, the isolated antibody further contains an Fc region. In some cases, the Fc region is a human Fc region. In some cases, the Fc region is an Fc region of IgG1, IgG2, IgG3, IgG4, IgGA1, or IgGA2. In some cases, the isolated antibody contains one or more further mutations within the Fc region. In some cases, one or more mutations modulate the effector function of the isolated antibody compared to a corresponding antibody lacking at least one mutation. In some cases, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, and / or complement-dependent cell-mediated cytotoxicity (CDC). In some cases, one or more mutations reduce the Fc-γ receptor binding of the isolated antibody compared to a corresponding antibody lacking at least one mutation.In some cases, one or more mutations increase the cytolytic activity of the isolated antibody compared to the corresponding antibody lacking at least one mutation (e.g., increasing ADCC activity and / or CDC activity). In some cases, one or more mutations are located in amino acid residues selected from the group consisting of L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, 1332, E333, K334, and P396. In some cases, one or more mutations are fucosylation. In some cases, the Fc region contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 34, 44, 238, or 239 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the Fc region contains one or more mutations relative to the wild-type Fc region containing the amino acid sequence of SEQ ID NO: 34 or 237. In some cases, one or more mutations contain amino acid substitutions, deletions, insertions, or combinations thereof. In some cases, one or more mutations contain an amino acid substitution at S252, an amino acid substitution at I351, or a combination thereof, compared to the wild-type Fc region containing the amino acid sequence of SEQ ID NO: 34, numbered according to the Kabat scheme. In some cases, the amino acid substitution at S252 is an amino acid substitution at S252D. In some cases, the amino acid substitution at I351 is an amino acid substitution at I351E. In some cases, the Fc region contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 34, 44, 238, or 239 is at least 80%, 85%, 90%, 95%, 99%, or 100%. In some cases, the isolated antibody contains an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 31-33, 45-46, or 87-103 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

[0007] In some embodiments, the Specified herein discloses isolated antibodies containing a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain is a wild-type CCR8 binding domain containing the amino acid sequence of SEQ ID NO: 108, numbered according to the IMGT scheme, with amino acid substitutions at G64, L110, I39, G40, R55, R38, R108, R55, A25, N82, G63, S58, G64, S28, T65, A25, R38, and S57. This includes amino acid substitutions at S58, S35, R37, R108, R55, N83, A25, S57, S58, S35, G64, R108, R37, R55, R108, N83, A25, R38, R57, R55, R108, N83, A25, R38, S57, N83, R55, R57, R108, G81, S28, or combinations thereof.In some cases, the CCR8 binding domain is subject to amino acid substitutions at G64, L110, I39, G40, R55, R38, R108, R55, A25, R55, N82, G63, L110, T65, L110, G64, S28, G64, L110, A25, R38, S57, G64, L110, S58, and S35. Amino acid substitutions include amino acid substitutions at R37, amino acid substitutions at G64, amino acid substitutions at R108, amino acid substitutions at R37, amino acid substitutions at R55, amino acid substitutions at R108, amino acid substitutions at N83, amino acid substitutions at A25, amino acid substitutions at R38, amino acid substitutions at S57, amino acid substitutions at N83, amino acid substitutions at A25, amino acid substitutions at R38, amino acid substitutions at R55, amino acid substitutions at N83, amino acid substitutions at A25, amino acid substitutions at R38, amino acid substitutions at S57, amino acid substitutions at R108, amino acid substitutions at A25, amino acid substitutions at R28, amino acid substitutions at S57, amino acid substitutions at R108, or amino acid substitutions at A25, amino acid substitutions at R38, amino acid substitutions at S571, and amino acid substitutions at G81.In some cases, the amino acid substitution at G64 is G64S or G64T, the amino acid substitution at L110 is L110V or L110Y, the amino acid substitution at I39 is I39Y, the amino acid substitution at G40 is G40S, the amino acid substitution at R55 is R55W, R55H, or R55G, the amino acid substitution at R38 is R38T, the amino acid substitution at R108 is R108I, the amino acid substitution at A25 is A25V, the amino acid substitution at N82 is N82K, the amino acid substitution at G63 is G63I, the amino acid substitution at S58 is S58G, the amino acid substitution at G64 is G64S or G64T, the amino acid substitution at T65 is T65I, the amino acid substitution at A25 is A25V, the amino acid substitution at R38 is R38G, the amino acid substitution at S57 is S57I, the amino acid substitution at S58 is S58G, the amino acid substitution at S35 is S35T, the amino acid substitution at R37 is R37V, the amino acid substitution at R108 is R108I, the amino acid substitution at R55 is R55H, the amino acid substitution at N83 is N83K, the amino acid substitution at A25 is A25V, the amino acid substitution at S57 is S57I, the amino acid substitution at R37 is R37V, the amino acid substitution at R108 is R108I, the amino acid substitution at G81 is G81A, and the amino acid substitution at S28 is S28F.

[0008] In some embodiments, what is disclosed herein is an isolated antibody comprising a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain comprises an amino acid substitution at D81, an amino acid substitution at M108, an amino acid substitution at P65, or a combination thereof, relative to the wild-type CCR8 binding domain comprising the amino acid sequence of SEQ ID NO: 111 numbered according to the IMGT scheme. In some cases, the CCR8 binding domain comprises an amino acid substitution at D81, an amino acid substitution at D81 and M108, or an amino acid substitution at P65. In some cases, the amino acid substitution at D81 is D81G, the amino acid substitution at M108 is M108K, and the amino acid substitution at P65 is P65L.

[0009] In any of the embodiments described above or related embodiments, the isolated antibody comprises a full-length antibody, a chimeric antibody, a single-domain antibody, a single-chain antibody (scFv), a heavy-chain-only antibody (HcAb), (e.g., a heavy-chain-only antibody derived from a camelid or a heavy-chain-only antibody derived from a shark), or a functional fragment thereof. In some cases, the functional fragment comprises Fab, Fab', Fab'-SH, Fv, or F(ab')2. In some cases, the isolated antibody comprises Fab, Fab', Fab'-SH, Fv, or F(ab')2. In some cases, the isolated antibody is a single-domain antibody. In some cases, the single-domain antibody is a VHH antibody or a VNAR antibody. In some cases, the single-domain antibody is a VHH derived from a camelid. In some cases, the single-domain antibody is a single-domain antibody derived from a llama.

[0010] In any of the embodiments described above or related embodiments, the isolated antibody comprises a first single-domain antibody and a second single-domain antibody, wherein the first single-domain antibody comprises a CCR8-binding domain and the second single-domain antibody comprises an antigen-binding domain. In some cases, the antigen-binding domain is a CCR8 antigen-binding domain. In some cases, the antigen-binding domain is a cancer antigen-binding domain.

[0011] In any of the embodiments described above or related embodiments, the isolated antibody is a bispecific antibody comprising a first single-domain antibody and a second single-domain antibody, wherein the first single-domain antibody is ligated to the second single-domain antibody, the first single-domain antibody comprises a CCR8-binding domain, and the second single-domain antibody comprises an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen).

[0012] In any of the embodiments described above or related embodiments, the isolated antibody is a bispecific antibody, which comprises a CCR8-binding domain and further comprises an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen).

[0013] In any of the embodiments described above or related embodiments, the isolated antibody is a single-chain antibody.

[0014] In any of the embodiments described above or related embodiments, the isolated antibody is humanized, monoclonal, deimmunized, bispecific, multispecific, polyvalent, or a combination thereof. In some cases, the isolated antibody is humanized.

[0015] In any of the embodiments described above or related embodiments, the isolated antibody includes IgG-scFv, nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2.scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody.

[0016] In any of the embodiments described above or related embodiments, the isolated antibody specifically binds to the CCR8 polypeptide or a portion thereof. In some cases, the isolated antibody specifically binds to the human CCR8 polypeptide or a portion thereof. In some cases, the isolated antibody specifically binds to the extracellular domain of the CCR8 polypeptide (e.g., human CCR8 polypeptide) or a portion thereof. In some cases, the isolated antibody specifically binds to the transmembrane domain of the CCR8 polypeptide (e.g., human CCR8) or a portion thereof. In some cases, the isolated antibody specifically binds to the cytoplasmic domain of the CCR8 polypeptide (e.g., human CCR8) or a portion thereof.

[0017] In any of the embodiments described above or related embodiments, the isolated antibody further comprises a payload which is conjugated to or coupled to the isolated antibody. In some cases, the payload is selected from the group consisting of therapeutic agents, small molecules, polypeptides, polynucleotides, enzymes, substrates, cofactors, fluorescent markers, chemiluminescent markers, peptide tags, magnetic particles, drugs, therapeutic agents, siRNA, antisense oligonucleotides, toxins, radionuclides, binding sites for secondary antibodies, metal-binding domains, or combinations thereof.

[0018] In any of the embodiments described above or related embodiments, the isolated antibody is cytolytic. In some cases, the isolated antibody exhibits ADCC activity. In some cases, the isolated antibody is cytolytic against regulatory T cells (Tregs). In some cases, the isolated antibody is cytolytic against tumor-infiltrating regulatory T cells. In some cases, the isolated antibody induces ADCC in regulatory T cells (Tregs), such as tumor-infiltrating regulatory T cells.

[0019] In any of the embodiments described above or related embodiments, the isolated antibody has the ability to (a) enhance the immune response against a tumor; (b) reduce, deplete, or kill Treg cells, such as tumor-infiltrating regulatory T ("Treg") cells; (c) induce internal migration of CCR8 polypeptide or a portion thereof into Treg cells, such as tumor-infiltrating regulatory T ("Treg") cells; (d) bind to CCR8 polypeptide or a portion thereof (e.g., human CCR8 polypeptide or a portion thereof); (e) bind to CCR8 polypeptide or a portion thereof (e.g., human CCR8 polypeptide or a portion thereof) by a KD of 10 nM or less as measured by BIACORE®; (f) induce antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing CCR8 polypeptide or a portion thereof; or (g) any combination thereof.

[0020] In some embodiments, antibody conjugates comprising an isolated antibody and a payload as described herein are disclosed. In some cases, the payload is a therapeutic agent. In some cases, the payload (e.g., therapeutic agent) comprises a small molecule, peptide, protein, or polynucleotide (e.g., DNA, RNA, mRNA). In some cases, the payload (e.g., therapeutic agent) comprises an immunomodulator, an anticancer agent, a cytotoxic drug, an NSAID, siRNA, an oligonucleotide (e.g., antisense oligonucleotide), a corticosteroid, a nutritional supplement such as an antioxidant, or a chemotherapeutic agent. In some cases, the immunomodulator comprises a cytokine, a chemokine, or an interferon. In some cases, the payload is covalently or noncovalently conjugated to the isolated antibody. In some cases, the payload is conjugated to the isolated antibody by a linker. In some cases, the antibody conjugate comprises A-(X) of formula (I). 1 -B) n The formula includes, where A comprises an isolated antibody as described herein, B comprises a payload, and X 1 `n` includes a join or linker, and `n` is an average value selected from 1 to 12.

[0021] In some embodiments, this specification discloses isolated nucleic acid molecules or antibody conjugates that encode the isolated antibodies described herein.

[0022] In some embodiments, vectors comprising nucleic acid molecules described herein are disclosed herein.

[0023] In some embodiments, in vitro cells are disclosed herein, which include (a) an isolated antibody as described herein, or an in vitro cell that expresses and / or secretes an antibody as described herein, (b) an isolated nucleic acid molecule as described herein, and / or (c) a vector as described herein.

[0024] In some embodiments, a pharmaceutical composition is disclosed herein, comprising (a) an isolated antibody or antibody conjugate as described herein, and (b) a pharmaceutically acceptable carrier, adjuvant, or diluent. In some cases, the pharmaceutical composition further comprises at least one additional therapeutic agent. In some cases, the pharmaceutical composition is formulated for administration via a subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intraventricular, intracranial, intracavitary, or intracerebellar route of administration. In some cases, the pharmaceutical composition is in an aqueous or lyophilized form. In some cases, the pharmaceutical composition is contained in a delivery device selected from the group consisting of syringes, blunt-tip syringes, catheters, and implantable pumps.

[0025] In some embodiments, the use of isolated antibodies or antibody conjugates described herein for the treatment of cancer or infectious diseases (e.g., pathogenic infections such as microbial or viral infections).

[0026] In some embodiments, the use of isolated antibodies or antibody conjugates described herein in the manufacture of a drug is disclosed. In some cases, the drug is for a disease, disorder, or condition involving CCR8 polypeptide-expressing cells (e.g., Treg cells, e.g., tumor-infiltrating Treg cells). In some cases, the drug is for the treatment of cancer or infection (e.g., pathogenic infections such as microbial or viral infections).

[0027] In some embodiments, the use of the antibody conjugates described herein for delivering a payload to a target site is disclosed. In some cases, the target site is a tumor site.

[0028] In some embodiments, this specification discloses fusion proteins comprising the isolated antibodies described herein.

[0029] In some embodiments, the Specified herein discloses chimeric antigen receptors (CARs) comprising the isolated antibodies described herein.

[0030] In some embodiments, the Specified herein discloses T cell receptors (TCRs) comprising the isolated antibodies described herein.

[0031] In some embodiments, the Specified Information discloses a method for delivering a payload to a target site within a subject, the method comprising delivering the payload to the target site by administering an effective amount of the antibody conjugate described herein to the subject. In some cases, the target site is an immune cell or cell expressing the CCR8 polypeptide or a portion thereof. In some cases, the target site is a tumor site.

[0032] In some embodiments, methods for treating a subject of interest are disclosed herein, the methods comprising treating the subject by administering an effective amount of (a) an isolated antibody as described herein, (b) a pharmaceutical composition as described herein, or (c) an antibody conjugate as described herein.

[0033] In some embodiments, methods for reducing or inhibiting Treg cells are disclosed herein, the methods comprising administering an effective amount of (a) an isolated antibody as described herein, (b) a pharmaceutical composition as described herein, or (c) an antibody conjugate as described herein to a subject requiring such reduction, or contacting a population of Treg cells (e.g., a population of invasive Treg cells) with an effective amount of (a) an isolated antibody as described herein, (b) a pharmaceutical composition as described herein, or (c) an antibody conjugate as described herein, thereby reducing and / or inhibiting Treg cells.

[0034] In some embodiments, methods for enhancing an immune response in a target subject are disclosed herein, the methods comprising treating the target by administering an effective amount of (a) an isolated antibody as described herein, (b) a pharmaceutical composition as described herein, or (c) an antibody conjugate as described herein.

[0035] In any of the embodiments described above or related embodiments, the subject has or is at risk of having cancer or an infection. In some cases, the cancer is a solid tumor. In some cases, the cancer is a hematological malignancy. In some cases, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer. In some cases, the subject is a human. In some cases, the method further includes administering an effective dose of a second therapeutic agent. In some cases, administration or contact may result in (a) enhancement of an immune response (e.g., an immune response against a tumor), (b) reduction, depletion, or killing of Treg cells (e.g., tumor-infiltrating regulatory T ("Treg") cells), (c) induction of internal migration of CCR8 polypeptide within Treg cells (e.g., tumor-infiltrating regulatory T ("Treg") cells), (d) binding to CCR8 polypeptide or a portion thereof (e.g., human CCR8), (e) binding to CCR8 polypeptide or a portion thereof (e.g., human CCR8) by a KD of ≤ 10 nM as measured by BIACORE®, (f) induction of antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing CCR8 polypeptide or a portion thereof (e.g., Treg cells such as tumor-infiltrating Treg cells), or (g) any combination thereof. In some cases, administration may be via subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intraventricular, intracranial, intracavitary, or intracerebellar routes of administration. In some cases, the contact is in vivo or in vitro.

[0036] In some embodiments, this specification discloses a method for producing an isolated antibody as described herein, the method comprising (a) culturing an in vitro cell as described herein in a culture medium under conditions that allow for the expression of a polypeptide encoded by an isolated nucleic acid molecule and the assembly of an isolated antibody, and (b) purifying an antibody isolated from the cultured cell or in vitro cell medium.

[0037] In some embodiments, the Specified Publicly Discloses a kit comprising an isolated antibody, an antibody conjugate, an isolated nucleic acid molecule, or a vector as described herein. In some cases, the kit further comprises an effective amount of one or more additional therapeutic agents. In some cases, one or more additional therapeutic agents include anticancer agents, cytotoxic drugs, NSAIDs, corticosteroids, dietary supplements such as antioxidants, siRNAs, small molecules, oligonucleotides, chemotherapeutic agents, or combinations thereof.

[0038] In some embodiments, the Specified Specified Antibodies Containing a Chemokine Receptor 8 (CCR8) Binding Domain are disclosed herein, wherein the CCR8 binding domain includes amino acid substitutions at Q1, Q5, A15, V24, Q49, R50, L52, F70, A83, K95, P96, Q123, or combinations thereof, relative to the wild-type CCR8 binding domain comprising the amino acid sequence of Sequence ID No. 27, numbered according to the IMGT scheme. In some cases, the CCR8 binding domain includes amino acid substitutions at Q1, Q5, A15, V24, Q49, R50, L52, F70, A83, K95, P96, and Q123. In some cases, the CCR8 binding domain includes amino acid substitutions at Q1, Q5, A15, V24, Q49, A83, K95, and Q123. In some cases, the amino acid substitution at Q1 is Q1V, the amino acid substitution at Q5 is Q5V, the amino acid substitution at A15 is A15P, the amino acid substitution at V24 is V24A, the amino acid substitution at Q49 is Q49G, the amino acid substitution at R50 is R50L, the amino acid substitution at L52 is L52W, the amino acid substitution at F70 is F70S, the amino acid substitution at A83 is A83S, the amino acid substitution at K95 is K95R, the amino acid substitution at P96 is P96A, and the amino acid substitution at Q123 is Q123T.

[0039] In some embodiments, the Specified herein discloses isolated antibodies comprising a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain comprises amino acid substitutions at Q1, Q5, A15, Q49, R50, L52, R77, A83, R84, S85, A86, L120, Q123, or combinations thereof, relative to the wild-type CCR8 binding domain comprising the amino acid sequence of Sequence ID No. 28, numbered according to the IMGT scheme. In some cases, the CCR8 binding domain includes amino acid substitutions at Q1, Q5, A15, Q49, R50, L52, R77, A83, R84, S85, A86, L120, and Q123. In some cases, the amino acid substitution at Q1 is Q1V, at Q5 is Q5V, at A15 is A15P, at Q49 is Q49G, at R50 is R50L, at L52 is L52W, at R77 is R77T, at A83 is A83S, at R84 is R84K, at S85 is S85N, at A86 is A86Y, at L120 is L120Q, and at Q123 is Q123T.

[0040] Reference All publications, patents, and patent applications described herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is explicitly and individually incorporated by reference.

[0041] Various aspects of this disclosure are described in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of exemplary embodiments utilizing the principles of this disclosure, and by referring to the appended drawings. [Brief explanation of the drawing]

[0042] [Figure 1] Flow cytometry analysis shows that VHH-1 (SEQ ID NO: 27) and VHH-3 (SEQ ID NO: 29), derived from llamas immunized with hCCR8, specifically bind to hCCR8 overexpressed in HEK293 cells, but do not bind to human CCR7 (SEQ ID NO: 39). [Figure 2A] This study evaluates representative cell binding of bivalent VHH-Fc antibodies in hCCR8-expressing cells, with each antibody containing a unique fusion of a short hinge (SEQ ID NO: 34) of the IgG1 Fc domain to the VHH domain. The antibodies are VHH-1-Fc (SEQ ID NO: 31), VHH-10-Fc (SEQ ID NO: 92), VHH-11-Fc (SEQ ID NO: 93), and VHH-12-Fc (SEQ ID NO: 94). BMS-4A19 was used as a comparative positive control. [Figure 2B] Similar representative binding evaluations for VHH-2-Fc (SEQ ID NO: 33), VHH-17-Fc (SEQ ID NO: 99), and VHH-18-Fc (SEQ ID NO: 100) are shown. [Figure 3]A-C show the determination of binding epitope sites using flow cytometry data for two VHH-Fc fusion antibodies, namely VHH-1-Fc (SEQ ID NO: 31) and VHH-2-Fc (SEQ ID NO: 33), in HEK293 T cells transiently transfected with either hCCR8 (SEQ ID NO: 37) or a chimeric construct between hCCR8 (SEQ ID NO: 37) and hCCR1 (SEQ ID NO: 39). A shows conceptual diagrams of the two receptor chimeric constructs employed in the binding study on the left, which are composed of different polypeptide regions obtained from hCCR8 and hCCR1. B shows the binding of the selected VHH-Fc antibody to HEK293 cells expressing chimeric A (SEQ ID NO: 42), demonstrating interaction with the transmembrane domain of hCCR8. C shows the binding of a selected VHH-Fc antibody to HEK293 cells expressing chimeric B (SEQ ID NO: 42) to evaluate interaction with only the N-terminus of hCCR8. [Figure 4A] This report presents evaluation of VHH antibodies formatted as VHH-Fc fusions by flow cytometry regarding their binding to hCCR8 in the presence or absence of known non-antibody hCCR8 binders. It also shows VHH-1-Fc binding in the presence of the CCR8 antagonist ML604086, increasing its concentration until it competes with antibody binding. [Figure 4B] This report presents evaluation of VHH antibodies formatted as VHH-Fc fusions by flow cytometry regarding their binding to hCCR8, both in the presence and absence of known non-antibody hCCR8 binders. It shows that both ML604086 and CCL1 compete for antibody binding to the receptor. [Figure 4C] This report presents evaluation of VHH antibodies formatted as VHH-Fc fusions by flow cytometry regarding their binding to hCCR8 in the presence or absence of known non-antibody hCCR8 binders. Cell binding of VHH-1-Fc (SEQ ID NO: 31), VHH-9-Fc (SEQ ID NO: 91), VHH-10-Fc (SEQ ID NO: 92), and VHH-12-Fc (SEQ ID NO: 94) to hCCR8-expressing HEK293 cells shows complete competition with the binding of 10 μM ML604086 (indicated as +ML), but does not affect the binding of BMS-4A19. [Figure 5] VHH-1-Fc (SEQ ID NO: 31) and VHH-2-Fc (SEQ ID NO: 33) potently inhibit CCL1-induced hCCR8 signaling via the β-arrestin pathway on hCCR8-expressing cells, similar to the potent anti-CCR8 binding antibody BMS-4A19. The inhibition curve shows the inhibition of CCL1 activation by CCR8 as antibody concentration increases. [Figure 6] This shows the identification of KABAT and IMGT in the CDR and FR regions of the VHH antibodies VHH-1 (SEQ ID NO: 27), VHH-2 (SEQ ID NO: 28), VHH-3 (SEQ ID NO: 29), and VHH-4 (SEQ ID NO: 30). [Figure 7] The ADCC reporter bioassay demonstrated the activity of VHH-1-Fc-DE (SEQ ID NO: 45) and VHH-2-Fc-DE (SEQ ID NO: 46). During the assay, activation of human FcγRIIIa on reporter effector cells generated a luminescence signal (RLU) upon association with anti-CCR8 VHH-Fc bound to CCR8 expressed on target HuT-78 cells. [Figure 8A] This shows the alignment of the amino acid sequences of VHH-1 (SEQ ID NO: 27) or VHH-2 (SEQ ID NO: 28) with the human germline heavy chain IGHV3-23*04 (SEQ ID NO: 104) and the human J gene joining region IGHJ6*01 (SEQ ID NO: 105), based on IMGT sequence numbering. The CDR region is shown in bold and underlined font, and its boundaries are defined by the IMGT definition. Asterisks indicate amino acid differences between the respective parental llama-derived VHH amino acid sequences within the framework region and the aligned human V and J gene sequences. The alignment of VHH-1 (SEQ ID NO: 27) with human IGHV3-23*04 is shown, with bolded residues indicating the positions where parental llama-derived VHH residues were substituted to produce humanized versions of VHH-1 (SEQ ID NO: 27), represented as VHH-1.1 (SEQ ID NO: 106), VHH-1.2 (SEQ ID NO: 107), and VHH-1.3 (SEQ ID NO: 108). [Figure 8B]This shows the alignment of the amino acid sequences of VHH-1 (SEQ ID NO: 27) or VHH-2 (SEQ ID NO: 28) with the human germline heavy chain IGHV3-23*04 (SEQ ID NO: 104) and the human J gene joining region IGHJ6*01 (SEQ ID NO: 105), based on IMGT sequence numbering. The CDR region is in bold and underlined font, and the boundaries are defined by the IMGT definition. Asterisks indicate amino acid differences between the respective parental llama-derived VHH amino acid sequences within the framework region and the aligned human V and J gene sequences. The alignment of VHH-2 (SEQ ID NO: 28) with IGHV3-23*04 is shown, with bold residues indicating residues substituted to generate the humanized VHH-1 domain, shown as VHH-2.1 (SEQ ID NO: 109), VHH-2.2 (SEQ ID NO: 110), and (VHH-2.3) (SEQ ID NO: 111). [Figure 9] Figures A and B show the evaluation of humanized VHH domains formatted as bivalent Fc fusions to the short hinge of the Fc domain (SEQ ID NO: 34) of human IgG1 during binding assays to HEK293 cells expressing hCCR8. Figure A shows cell binding curves for VHH-Fc containing VHH domains from the following groups: VHH-1 (SEQ ID NO: 27), VHH-1.2 (SEQ ID NO: 107), and VHH-1.3 (SEQ ID NO: 108). Figure B shows the binding of VHH-Fc containing VHH domains from the following groups: VHH-2 (SEQ ID NO: 28), VHH-2.2 (SEQ ID NO: 110), and VHH-2.3 (SEQ ID NO: 111). [Figure 10] To identify mutation-tolerant CDR paratope sites, binding curves measured by flow cytometry in HEK293 cells expressing hCCR8 are shown for alanine scanning mutations in the CDR3 region of VHH-1 (SEQ ID NO: 27) using the Fc fusion antibody format. The mutation site (IMGT) within the CDR3 region is included in the antibody name. [Figure 11]This shows cell binding analysis of a representative set of humanized VHH-Fc fusions, including human IgG Fc (SEQ ID NO: 34) fused to humanized VHH optimized by yeast display. The binding and curves to HuT-78 cells, measured by flow cytometry, show the case without 10 μM ML604086 for VHH-Fc fusions of VHH-1.3-13 (SEQ ID NO: 198), VHH-1.3-19 (SEQ ID NO: 204), VHH-1.3-20 (SEQ ID NO: 205), VHH-1.3-21 (SEQ ID NO: 206), and VHH-1 (SEQ ID NO: 27). [Figure 12] This shows the evaluation of VHH-Fc fusion antibodies in an assay for functional inhibition of CCL1-induced hCCR8 signaling via the β-arrestin pathway. Inhibition curves for VHH-Fc antibodies containing VHH fusion to the Fc domain (SEQ ID NO: 34) are shown for VHH-1 (SEQ ID NO: 27) and humanized VHH domains, VHH-1.3 (SEQ ID NO: 108), VHH-1.3-19 (SEQ ID NO: 204), and VHH-2.3-2 (SEQ ID NO: 208). [Figure 13A] This report presents an evaluation of the ADCC effector function of representative VHH-Fc antibodies, measured through activation of signaling mediated by either mouse-derived FcγRIV or human FcγRIIIa on effector cells in response to antibodies bound to HuT-78 target cells. Each VHH-Fc antibody contains a fusion of a distinct VHH domain to the hinge of either the indicated mouse-derived IgG2a Fc (mFc) (SEQ ID NO: 238) or human IgG1 Fc (SEQ ID NO: 34). The mFc fusion activity of VHH-1 (SEQ ID NO: 27), VHH-1.3-7 (SEQ ID NO: 192), VHH-1.3-19 (SEQ ID NO: 204), VHH-1.3-20 (SEQ ID NO: 205), or VHH-1.3-21 (SEQ ID NO: 206) is shown in the absence or presence (+ML) of the hCCR8 antagonist ML604086 (10 μM). [Figure 13B]This report presents an evaluation of the ADCC effector function of representative VHH-Fc antibodies, measured through activation of Fc-mediated signaling on effector cells via either mouse-derived FcγRIV or human FcγRIIIa in response to antibodies bound to HuT-78 target cells. Each VHH-Fc antibody contains a fusion of a distinct VHH domain to the hinge of either mouse-derived IgG2a Fc (mFc) (SEQ ID NO: 238) or human IgG1 Fc (SEQ ID NO: 34). The antibodies exhibit activity of human Fc fusions of VHH-1.3-17 (SEQ ID NO: 202), VHH-1.3-19 (SEQ ID NO: 204), VHH-1.3-20 (SEQ ID NO: 205), or VHH-1.3-21 (SEQ ID NO: 206). [Figure 14] This graph shows the growth curves of MC38 tumors in hCCR8 knock-in mice under therapeutic treatment with VHH-1-mFc antibody (SEQ ID NO: 239) or an IgG2a Fc isotype-negative control. Points on the plot represent the mean measured tumor volume (10 mice per point) at the indicated number of days after the start of treatment. Measurements are shown as the average of all surviving mice in each treatment group at each time point. [Modes for carrying out the invention]

[0043] This disclosure addresses the challenge of producing antibodies against the structured extracellular domain of CCR8 by employing a strategy involving immunization of llamas with optimized human CCR8 antigens and subsequent isolation of novel hCCR8 antibody binders derived from their specific antibody repertoire. Camelids such as llamas, alpacas, and camels possess both conventional quadruple-chain IgG antibodies consisting of paired heavy and light chain components, and unique heavy-chain-only antibodies lacking a light chain. The two independent, identical antigen-binding domains (e.g., VHH) of these antibodies are each formed from a single heavy chain. The specificity and affinity of VHH are comparable to those of human IgG, even though VHH is only about 15 kDa in size. On average, single-domain antibodies, such as VHH or heavy-chain-only antibodies, have longer complementarity-determining region 3 (CDR3), which facilitates binding to deeper cavities found on the extracellular surface of GPCRs like hCCR8. Furthermore, such antibodies can be readily formatted into a multitude of configurations to produce biopharmaceutical molecules with properties optimal for use as therapeutic agents. This specification discloses, in certain embodiments, antibodies and antibody conjugates that specifically bind to human CCR8, and pharmaceutical compositions comprising antibodies or antibody conjugates that specifically bind to human CCR8. In some embodiments, this specification also discloses methods for delivering a payload using the antibodies described herein, and methods of therapeutic treatment using the antibodies described herein.

[0044] It should be understood that this application is not limited to specific formulation or process parameters, as these can naturally vary. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them. Furthermore, it should be understood that several methods and materials similar to or equivalent to those described herein can be used in carrying out the present invention.

[0045] According to this application, conventional molecular biology, microbiology, and recombinant DNA techniques, as well as well described in the art, may be used.

[0046] The following definitions supplement those of the art and apply to this application, and do not apply to any related or unrelated cases, such as any patent or application owned by the same applicant. Accordingly, the terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0047] In this application, the use of the singular form includes the plural form unless otherwise explicitly stated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include the plural referent unless otherwise explicitly indicated by the context. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limited.

[0048] As used herein, the terms “and / or” and “any combination thereof,” as well as their grammatical equivalents, may be used interchangeably. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually, B individually, C individually, A and B, B and C, A and C, as well as A, B, and C.”

[0049] The term "or" can be used conjunctively or disjunctively unless the context specifically indicates disjunctive use.

[0050] The terms “approximately” or “about” mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and depend in part on the method of measuring or determining the value, i.e., the limits of the measuring system. For example, “approximately” can mean within one or more standard deviations, according to convention in the art. Alternatively, “approximately” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term can mean within five orders of magnitude of the value, preferably more than two orders of magnitude. Unless otherwise stated, where a particular value is described in this application and claims, the term “approximately” is considered to mean that the particular value is within an acceptable margin of error.

[0051] Where used herein and in the claims, “comprising” (and any form of “comprise” such as “comprise” and “comprises”), “having” (and any form of “have” and “has”), “including” (and any form of “includes” and “include”), or “containing” (and any form of “contains” and “contain”) are comprehensive or open-ended and do not exclude additional unlisted elements or method steps. Any embodiment discussed herein can be implemented with respect to any method or composition of the present invention, and vice versa. Furthermore, compositions of the present invention can be used to achieve the methods of the present invention.

[0052] As used herein, the term “essentially consisting of” refers to an element required for a given embodiment. This term allows for the presence of elements that do not substantially affect the basic and novel or functional features of such embodiment of the invention.

[0053] As used herein, the term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not listed in the description of the embodiments.

[0054] In this specification, references to “several embodiments,” “embodiment,” “one embodiment,” or “other embodiments” mean that certain features, structures, or characteristics described in relation to the embodiments are included in at least some embodiments, but not necessarily in all embodiments of the present invention.

[0055] In this specification, the terms “disease,” “disorder,” or “condition” are used interchangeably and refer to any change in the state of the body or any of the organs, interruption or impairment of the performance of function, and / or symptoms, such as discomfort, dysfunction, pain, or even death, caused to the patient or to those in contact with the person. Disease or disorder may also be associated with distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, or affection.

[0056] When used in the context of therapeutic or preventive measures, the term "needs it" means having a disease, being diagnosed with a disease, or needing to prevent a disease, for example, being at risk of developing the disease. Therefore, those who need it may be those who need treatment or prevention of a disease.

[0057] The term "cancer" refers to a disease characterized by the rapid and uncontrolled proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0058] The term “antitumor effect” refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in regulatory T cell number, a reduction in immunosuppressive cell number, a reduction in metastasis, an extension of life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or remission of various physiological symptoms associated with the cancerous state. “Antitumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumor development in the initial stages. In some embodiments, the antibody induces the antitumor effect by inducing cytotoxicity. In some embodiments, the antibody of this disclosure is cytotoxic, e.g., cytolytic or cell division inhibitory. As used herein, “cytolytic” refers to the depletion, elimination, and / or killing of target cells, e.g., cells expressing an antigen or fragment thereof to which the antibody disclosed herein can bind (e.g., CCR8 polypeptide). In some embodiments, the cells are immunosuppressive cells. In some embodiments, the cells are Treg cells.

[0059] As used herein, “subject,” “patient,” “individual,” and similar terms are interchangeable and refer to vertebrates, mammals, primates, or humans. Mammals include, but are not limited to, humans, primates, rodents, wild animals, or domesticated animals, such as feral domesticated animals, livestock, sporting animals, and pets. Primates include, for example, chimpanzees, crab-eating macaques, spider monkeys, and macaques (e.g., rhesus macaques). Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domesticated and game animals include, for example, cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, and canine species, such as dogs, foxes, wolves, birds, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. The terms “individual,” “patient,” and “subject” are used interchangeably herein. A subject may be male or female.

[0060] In some embodiments, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects representing animal models of conditions or disorders associated with uncontrolled cell proliferation (e.g., cancer). Non-limiting examples include mouse tumor models. Furthermore, the compositions and methods described herein may be used to treat domestic animals and / or pets. The subject may be one that has been previously diagnosed with cancer or identified as having cancer. The subject may be one that has been diagnosed and is currently being treated, or is seeking treatment, monitoring, adjustment, or modification of existing therapeutic treatment, or is at risk of developing a given disorder (e.g., cancer).

[0061] As used herein, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably to refer to a series of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein,” “peptide,” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to smaller polypeptides; however, the use of these terms in the art is overlapping. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. These terms include, for example, natural and artificial proteins of protein sequences, protein fragments, and polypeptide analogs (mutations, variants, and fusion proteins), as well as post-translational or otherwise covalently or non-covalently modified proteins. Peptides, polypeptides, or proteins can be monomers or polymers. Polypeptides may have amino acid sequences of naturally occurring polypeptides derived from any mammal. Such naturally occurring polypeptides can be isolated from nature or produced by recombinant or synthetic means. In some embodiments, the polypeptide is a “variant.” A “variant” means a bioactive polypeptide that, after the sequence has been aligned as necessary and gaps introduced to achieve the maximum percentage of identical residues, has at least about 80% amino acid identical residues compared to the naturally occurring polypeptide, but does not consider any conservative substitutions as part of the identical residue rate. Examples of such variants include polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid identical residues.In some embodiments, the variant has at least about 90% amino acid identity. In some embodiments, the variant has at least about 95% amino acid identity with respect to the native sequence polypeptide. A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has undergone chemical modification, phosphorylation, and glycosylation, for example, by conjugation to another chemical moiety (e.g., polyethylene glycol or albumin, e.g., human serum albumin).

[0062] The terms “increased,” “increased,” or “enhanced” are all used herein to mean, in general, an increase of a statically significant amount, and to avoid any doubt, the terms “increased,” “increased,” or “enhanced” mean an increase of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to a reference level, or a decrease of less than 100% or more than 100%, or an increase of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or more.

[0063] The terms “reduce,” “decrease,” “reduce,” “lower,” or “inhibit” are all used herein to generally mean a statistically significant reduction. For example, “reduce,” “decrease,” “reduce,” or “inhibit” means a reduction of at least 10% compared to a reference level, e.g., at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or up to 100% (e.g., post-treatment tumor size compared to a reference level), or any reduction between 10% and 100% compared to a reference level. In the context of markers or symptoms, these terms mean a statistically significant reduction at such levels. This reduction can be, for example, at least 10%, at least 20%, at least 30%, or at least 40% or more, and preferably a reduction to a level acceptable as within the normal range for a given disease-free individual. Reduction or inhibition may refer to the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, or the presence or size of dormant tumors. In some embodiments, the reference level is the level in the absence of the antibody disclosed herein. In some embodiments, the reference level is the level before administration of the antibody disclosed herein, contact with the antibody, and / or treatment with the antibody. In some embodiments, the reference level is the level in the absence of administration of the antibody, contact with the antibody, and / or treatment with the antibody disclosed herein.

[0064] As used herein, the term “fusion protein” refers to a polypeptide comprising the amino acid sequence of an antibody or a fragment thereof and the amino acid sequence of a heterologous polypeptide (i.e., an unrelated polypeptide).

[0065] Where used herein, the terms “regulatory T cells,” “regulatory T cells,” “Treg,” or “Treg” refer to a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs are known to induce effector T cell lysis, support the formation of immune-tolerant dendritic cells, support the formation of M2 macrophages, produce immunosuppressive metabolites and cytokines, function as IL-2 sinks, and promote neoangiogenesis. There are many types of Tregs, but many Tregs express CD4 and FOXP3, and in many cases FOXP3 serves as a marker for Tregs.

[0066] As used herein, the terms “synthetic polynucleotide,” “synthetic gene,” or “synthetic polypeptide” mean that the corresponding polynucleotide sequence or part thereof, or the amino acid sequence or part thereof, is derived from a designed, newly synthesized, or modified sequence compared to an equivalent naturally occurring sequence. Synthetic polynucleotides (antibodies or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including but not limited to the chemical synthesis of nucleic acids or amino acid sequences. Unlike naturally occurring genes, synthetic genes are usually located within the context of a synthetic expression control sequence, typically at the level of either amino acids or polynucleotides (or both). The polynucleotide sequences of synthetic genes may not necessarily encode different proteins of different amino acids compared to naturally occurring genes; for example, they may also include synthetic polynucleotide sequences that incorporate different codons but encode the same amino acids (i.e., nucleotide changes represent silent mutations at the amino acid level).

[0067] As used herein, the term “Fc receptor” refers to a polypeptide found on the surface of immunoeffector cells that is bound by the Fc region of an antibody. In some embodiments, the Fc receptor is the Fcγ receptor. There are three subclasses of the Fcγ receptor: FcγRI(CD64), FcγRII(CD32), and FγcRIII(CD16). All four IgG isotypes (IgG1, IgG2, IgG3, and IgG4) bind to and activate the Fc receptors FcγRI, FcγRIIA, and FcγRIIIA. FcγRIIB is an inhibitory receptor, and therefore antibodies that bind to this receptor do not activate complement and cellular responses. FcγRI is a high-affinity receptor that binds to IgG in monomeric form, while FcγRIIA and FcγRIIA are low-affinity receptors that bind to IgG only in multimeric form and have slightly lower affinity. The binding of antibodies to the Fc receptor and / or C1q is governed by specific residues or domains within the Fc region. Furthermore, binding also depends on residues located within the hinge region and within the CH2 moiety of the antibody. In some embodiments, the agonist and / or therapeutic activity of the antibodies described herein depends on the binding of the Fc region to an Fc receptor (e.g., FcγR). In some embodiments, the agonist and / or therapeutic activity of the antibodies described herein is enhanced by the binding of the Fc region to an Fc receptor (e.g., FcγR).

[0068] The term “specifically binding” to an antigen or epitope is a well-understood term in the art, and methods for determining such specific binding are also well-known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts with or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with higher affinity than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to the CCR8 epitope is an antibody that binds to this epitope with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other CCR8 epitopes or non-CCR8 epitopes. Furthermore, reading this definition, it can be understood that, for example, an antibody (or partial or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (but may include) exclusive binding. Generally, though not always, references to binding imply preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0069] As used herein, the term “complementarity-determining regions” (CDRs, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The CDRs of the variable heavy chain can be CDR-H1, CDR-H2, and CDR-H3. The CDRs of the variable light chain can be CDR-L1, CDR-L2, and CDR-L3. An example of a hypervariable loop occurs 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)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) arise at amino acid residues 24-34 for L1, 50-56 for L2, 89-97 for L3, 31-35B for H1, 50-65 for H2, and 95-102 for H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991)). Therefore, HV can be contained within the corresponding CDR, and references to the "hypervariable loops" of the VH and VL domains herein should be interpreted as also encompassing the corresponding CDR, and vice versa, unless otherwise indicated. Regarding the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31–35 (CDR1), 50–65 (CDR2), and 95–102 (CDR3), which can optionally include one or two additional amino acids following position 35 (referred to as positions 35A and 35B in the Kabat numbering scheme). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3).As is well known to those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or elongation of the FR and / or CDR, and therefore the Kabat number of an amino acid is not necessarily the same as its linear amino acid number.

[0070] With respect to the Chothia numbering system, CDRs within an antibody heavy chain molecule are typically located at amino acid positions 26–31 (CDR1), 52–56 (CDR2), and 95–102 (CDR3), and may optionally contain one or two additional amino acids following position 31 (referred to as positions 31A and 31B in the Chothia numbering scheme). Using the Chothia numbering system, CDRs within an antibody light chain molecule are typically located at amino acid positions 24–34 (CDR1), 50–56 (CDR2), and 89–97 (CDR3). As is well known to those skilled in the art, using the Chothia numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or additional amino acids due to shortening or elongation of FRs and / or CDRs; therefore, the Chothia number of an amino acid is not necessarily the same as its linear amino acid number.

[0071] The more conserved regions of the variable domain are called framework regions (FRs), as defined below. The variable domains of the natural heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) that primarily take on a [beta]-sheet configuration, linked by three hypervariable loops. The hypervariable loops within each chain are held together in close proximity by the FRs and, together with the hypervariable loops of other chains, contribute to the formation of the antibody's antigen-binding site. Structural analysis of antibodies has revealed the relationship between the sequence and the shape of the binding site formed by the complementarity-determining regions (Chothia et al., J.Mol.Biol.227:799-817 (1992)); Tramontano et al., J.Mol.Biol,215:175-182 (1990)). Despite their high sequence variability, five of the six loops employ only a limited repertoire of back-chain conformations, known as the "canonical structure." These conformations are all determined, firstly, by the length of the loops, and secondly, by the presence of key residues that determine the conformation through their packing, hydrogen bonding, or ability to exhibit abnormal back-chain conformations at specific locations within the loops and framework regions.

[0072] The "variable domain" of an antibody is represented either by the variable domain of the antibody light chain or the variable domain of the antibody heavy chain, either alone or in combination. The variable domains of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held together in close proximity by the FRs and, together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Allazikani et al (1997) J. Molec. Biol. 273:927-948). CDR may refer to a CDR defined by either approach or a combination of both approaches. In some embodiments, the antibodies of this disclosure include a single variable domain (e.g., a heavy chain variable domain, VH).

[0073] As used herein, “antigen-binding domain” refers to a binding domain from an antibody or non-antibody that can bind to an antigen. An antigen-binding domain may be a CCR8 antigen-binding domain, a cancer antigen-binding domain, or a binding domain that can bind to an antigen (such as a molecule) on an antigen-presenting cell. If there are more than one antigen-binding domains in a given complex or antibody construct, the antigen-binding domains may be numbered (e.g., a first antigen-binding domain, a second antigen-binding domain, a third antigen-binding domain, etc.). Different antigen-binding domains in the same complex or construct may target the same or different antigens (e.g., a first antigen-binding domain that can bind to a tumor antigen, a second antigen-binding domain that can bind to a molecule on an antigen-presenting cell (APC antigen), and a third antigen-binding domain that can bind to an APC antigen). The term “antigen-binding domain” refers to a fragment of an antibody that includes an area that specifically binds to an epitope and is complementary to some or all of the antigen. An antigen-binding domain may be provided, for example, by one or more antibody-variable domains (also called antibody-variable regions). In some embodiments, the antigen-binding domain includes a single variable domain, such as a heavy chain variable domain (VH). In some embodiments, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0074] The "constant region" of an antibody is defined as either the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. The constant region does not change with respect to antigen specificity.

[0075] As used herein, the term “heavy chain region” includes the amino acid sequence derived from the constant domain of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain region includes at least one of the following: the CH1 domain, the hinge (e.g., upper, middle, and / or lower hinge regions), the CH2 domain, the CH3 domain, or variants or fragments thereof. In some embodiments, the antibody or its antigen-binding fragment may include the Fc region of the immunoglobulin heavy chain (e.g., the hinge region, the CH2 domain, and the CH3 domain). In other embodiments, the antibody or its antigen-binding fragment lacks at least a region of the constant domain (e.g., all or part of the CH1 domain). In certain embodiments, at least one, preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain region includes a complete human hinge domain. In other preferred embodiments, the heavy chain region includes a complete human Fc region (e.g., the hinge, CH2, and CH3 domain sequences derived from human immunoglobulin). In certain embodiments, the constant domains constituting the heavy chain region are derived from different immunoglobulin molecules. For example, the heavy chain region of a polypeptide may include a domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain containing regions from different immunoglobulin molecules. For example, the hinge may include a first region derived from an IgG1 molecule and a second region derived from an IgG3 or IgG4 molecule. As described above, those skilled in the art will understand that the constant domains of the heavy chain region may be modified to deviate in amino acid sequence from naturally occurring (wild-type) immunoglobulin molecules. That is, the polypeptides of the present invention disclosed herein may include changes or modifications to one or more of the heavy chain constant domains (CH1, hinge, CH2, or CH3) and / or to the light chain constant domain (CL). Exemplary modifications include the addition, deletion, or substitution of one or more amino acids within one or more domains.

[0076] The antibodies or antigen-binding fragments of the present disclosure may include a CDR3 region having a length of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The antibodies or antigen-binding fragments of the present disclosure may include a CDR3 region having a length of at least about 18 amino acids.

[0077] As used herein, the term “hinge region” includes the region of the heavy chain molecule that links the CH1 domain to the CH2 domain. The hinge region can contain approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: an upper, middle, and lower hinge domain (Roux et al. J.Immunol. 1998 161:4083).

[0078] The terms “antibody fragment,” “antigen-binding fragment,” or “antibody-binding domain” refer to at least a portion of an antibody, or a recombinant variant thereof, that includes an antigen-binding domain, i.e., the antigen-determining variable region of an intact antibody, which is sufficient to give the antibody fragment recognition and specific binding to a target such as an antigen and its defined epitope. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single-domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments. In another embodiment, the antibody is a full-length antibody, e.g., an intact IgG1 antibody or other antibody class or isotype as described herein. (See, for example, Hudson et al. Nat. Med. 9:129-134 (2003); Pluckthiin, The Pharmacology of Monoclonal Antibodies, vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)). A full-length antibody, intact antibody, or whole antibody is an antibody having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region, as defined herein. Antibody fragments can be prepared by a variety of techniques, including, but not limited to, the proteolysis of intact antibodies and the production of recombinant host cells (e.g., E. coli or phages), as described herein.

[0079] As used herein, the term "Fv" refers to a minimal antibody fragment containing a complete antigen recognition and antigen-binding site. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly bound together by a non-covalent bond.

[0080] The folding of these two domains creates six hypervariable loops (three from the H chain and three from the L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0081] The “heavy chain variable region” or “VH” in relation to an antibody refers to a fragment of the heavy chain that comprises three CDRs and, in some embodiments, is inserted between adjacent segments (flanking stretches) known as framework regions. These framework regions are generally more conserved than the CDRs and form a scaffold supporting the CDRs.

[0082] The six hypervariable loops (three from the heavy chain and three from the light chain) contribute amino acid residues for antigen binding, giving the antibody antigen-binding specificity. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0083] "Framework" or FR residues are variable domain residues other than hypervariable region residues.

[0084] The "Fc region" (crystallizable region of a fragment), "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including those located within the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the CH2 and CH3 domains of the two antibody heavy chains, while the Fc regions of IgM and IgE are located within each polypeptide chain and contain three heavy chain constant domains (CH domains 2-4). Although the boundaries of the Fc region of immunoglobulin heavy chains can vary, the Fc region of human IgG heavy chains is generally defined as the stretch from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, and this numbering follows the EU index, as in Kabat. As used herein, the Fc region may be the native sequence Fc or the variant Fc.

[0085] "Fv" is the smallest antibody fragment containing the complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in a tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0086] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of several residues to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is the herein designation for Fab' in which the cysteine ​​residue(s) of the constant domain support a free thiol group. The Fab' fragment is produced by reducing the heavy chain disulfide crosslink of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0087] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0088] As used herein, the terms “monoclonal antibody” and “mAb” refer to antibodies obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations that may be present in small amounts.

[0089] The term "humanized antibody" refers to an antibody of non-human origin whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans.

[0090] The term "chimeric" antibody refers to an antibody in which a portion 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.

[0091] The antibodies disclosed herein In this specification, the terms “antibody” or “immunoglobulin” are used in their broadest sense to refer to any polypeptide containing an antigen-binding domain (e.g., a CCR8-binding domain) with a complementarity-determining region (CDR). This term encompasses a wide range of antibody structures, including, but not limited to, monospecific antibodies, multispecific antibodies (e.g., bispecific (e.g., bispecific T-cell engagers) and trispecific antibodies), humanized antibodies, chimeric antibodies, human antibodies, single-chain antibodies, heavy-chain-only antibodies (e.g., VHH), llama-derived antibodies, single-domain antibodies, and nanobodies (e.g., VHH).

[0092] The term antibody also includes, but is not limited to, fragments (antibody fragments, or antigen-binding fragments) that can bind to antigens, such as Fv, single-chain Fv(scFv), Fab, Fab', di-scFv, sdAb (single-domain antibodies), and (Fab')2 (including chemically linked F(ab')2). Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and can still crosslink antigens. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, and cynomolgus monkey. Furthermore, for all antibody constructs provided herein, variants having sequences from other organisms are also intended. Therefore, if a human version of an antibody is disclosed, a person skilled in the art will understand how to transform a human sequence-based antibody into sequences from mouse, rat, cat, dog, horse, etc. Antibody fragments also include any orientation, such as single-stranded scFv, tandem di-scFv, diabody, tandem tri-sdcFv, and minibody. Antibody fragments also include single-domain antibodies (sdAb), which are antibodies having a single monomeric domain, such as a variable domain pair of the heavy chain that does not contain a light chain. In some embodiments, an antibody fragment may be referred to as a specific species (e.g., human scFv or mouse-derived scFv). This refers to the sequence of at least a portion of the non-CDR region, rather than the source of the construct.

[0093] In some embodiments, the antibodies disclosed herein are full-length antibodies. Full-length antibodies are heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain containing 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic quadruple-chain units that can be combined with J chain polymerization to form a polyvalent assembly. In the case of IgG, a quadruple-chain unit is typically about 150,000 daltons. Each light chain is linked to a heavy chain by one disulfide covalent bond, while two heavy chains are linked to each other by one or more disulfide bonds depending on the heavy chain isotype. Each heavy and light chain also has intrachain disulfide crosslinks at regular intervals. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each alpha and gamma chain) and four (CH1, CH2, CH3, and CH4 for the mu and epsilon isoforms) constant domains (CH), and a hinge region (hinge) between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the opposite end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. The VH and VL pair together to form an antigen-binding site. For the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, Eighth Edition, eds. by Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, pp. 71 and 6 chapters. Light chains derived from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain (CH) of their heavy chain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with their heavy chains called alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes can be further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. The antibodies in this disclosure are also sometimes called anti-hCCR8 antibodies.

[0094] This disclosure provides antibodies that specifically bind to human CCR8. In some embodiments, the antibody or its antigen-binding fragment induces the lysis of cells expressing CCR8 (e.g., Treg cells) and / or cancer cells. Lysis can be induced by any mechanism, for example, by mediating effector functions such as C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or direct induction of cell apoptosis.

[0095] In some embodiments, the antibodies disclosed herein are modified to have an increase in at least one effector function compared to an unmodified parent antibody. Effector functions are biological activities that may be attributable to the Fc region of the antibody, varying depending on the antibody isotype. These effector functions may also interchangeably be referred to as “Fc receptor-mediated effector functions.” In some embodiments, the antibodies disclosed herein include an Fc region. In some embodiments, the antibodies disclosed herein include at least one mutation within the Fc region. In some embodiments, at least one mutation within the Fc region modulates (e.g., increases) the effector function of the antibody compared to a corresponding antibody lacking at least one mutation. Examples of effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. For example, the antibodies disclosed herein can be glycoengineered to have an increase in at least one effector function compared to a non-glycoengineered parent. Antibody-dependent cell-mediated cytotoxicity (ADCC) is the result of the formation of a complex between the Fab portion of an antibody and an antigen on the cell surface, and the binding of the Fc portion to the Fc receptor (FcγR) on effector cells. Increased effector function can be increased binding affinity to the Fc receptor, increased ADCC, increased cell-mediated immunity, increased binding to cytotoxic CD8 T cells, increased binding to NK cells, increased binding to macrophages, increased binding to polymorphonuclear cells, increased binding to monocytes, increased binding to macrophages, increased binding to macrogranulocytes, increased binding to granulocytes, direct signaling that induces apoptosis, increased maturation of dendritic cells, or increased T cell priming.

[0096] In some embodiments, the antibodies disclosed herein include an antigen-binding domain (CCR8-binding domain) containing one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of CDR1, CDR2, and CDR3 regions, wherein the CDR1 region contains an amino acid sequence in which the identical residue rate to any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214 is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the CDR2 region contains an antigen-binding domain (CCR8-binding domain) containing one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of CDR1, CDR2, and CDR3 regions, wherein the CDR1 region contains an amino acid sequence in which the identical residue rate to any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214 is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 5 The CDR3 region contains amino acid sequences in sequence numbers 7, 8, 9, 16, 17, 18, 49, 52, 91, 8, 9, 16, 17, 18, 49, 52, 91, 16, 17, 18, 49, 52, 91, 16, 17, 18, 16, 17, 18, 16, 17, 18, 16, 17, 18, 19 It contains an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the following: 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246.

[0097] In some embodiments, the antibodies disclosed herein include an amino acid sequence in which the percentage of identical residues to any one of the amino acid sequences SEQ ID NOs: 27-30, 70-86, 106-111, 186-209, 31-33, 45-46, or 87-103 is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the antibody includes an antigen-binding domain (e.g., including a CCR8-binding domain) comprising an amino acid sequence in which the percentage of identical residues to any one of the amino acid sequences of SEQ ID NOs. 27-30, 70-86, 106-111, or 186-209 is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the antibodies disclosed herein further comprise an Fc region. In some embodiments, the antibodies disclosed herein are single-domain antibodies (e.g., VHH), which have a percentage of identical residues to any one of the amino acid sequences of SEQ ID NOs. 27-30, 70-86, 106-111, 186-209, 31-33, 45-46, or 87-103 of at least about 50%, 60%, 70%, 75%, 80%, and 85%. The antibody comprises an amino acid sequence having an amino acid content of %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the antibody of the present disclosure comprises a single-domain antibody and a fusion of Fc regions, the antibody having an identical residue rate of at least about 50%, 60%, 70%, 75%, 80%, 85%, or 90% for any one of the amino acid sequences of SEQ ID NOs. 31-33, 45-46, or 87-103. The amino acid sequence comprises an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the Fc region comprises an amino acid sequence having at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to SEQ ID NOs. 34, 44, 238, or 239.

[0098] In one embodiment, an antibody disclosed herein having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages with respect to any one of sequence numbers 27-30, 70-86, 106-111, 186-209, 31-33, 45-46, or 87-103, wherein the antibody containing such sequence has the ability to bind to an antigen (e.g., CCR8). In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in one of the amino acid sequences of SEQ ID NOs: 27-30, 70-86, 106-111, 186-209, 31-33, 45-46, or 87-103. In some embodiments, the substitution, insertion, or deletion occurs in the region outside the CDR (e.g., within the FR). Optionally, the antibody contains an amino acid sequence in which the percentage of identical residues to one of the following amino acid sequences is at least approximately 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and includes post-translational modifications of that sequence.

[0099] In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of sequence numbers 27-30, 70-86, 106-111, 186-209, 31-33, 45-46, or 87-103.

[0100] In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, and selected CDR1, CDR2, and CDR3 are paired according to Table 2. In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, and selected CDR1, CDR2, and CDR3 are paired according to Table 2, further comprising FR1 region, FR2 region, FR3 region, and FR4 region, and the FR1 region, FR2 region, FR3 region, and FR4 region are paired according to Table 14. In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, and selected CDR1, CDR2, and CDR3 are paired according to Table 4. In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, selected CDR1, CDR2, and CDR3 are paired according to Table 4, further comprising FR1, FR2, FR3, and FR4 regions, the FR1, FR2, FR3, and FR4 regions being paired according to Table 14. In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, selected CDR1, CDR2, and CDR3 are paired according to Table 14. In one embodiment, the antibodies disclosed herein comprise CDR1, CDR2, and CDR3, selected CDR1, CDR2, and CDR3 are paired according to Table 14, further comprising FR1, FR2, FR3, and FR4 regions, the FR1, FR2, FR3, and FR4 regions being paired according to Table 14.

[0101] In some embodiments, the antibodies disclosed herein include one, two, three, or four framework regions (FRs) selected from the group consisting of FR1, FR2, FR3, and FR4 regions. In some embodiments, the antibodies disclosed herein include FR1, FR2, FR3, and FR4 regions, and the FR1, FR2, FR3, and FR4 regions are paired according to Table 14. In some embodiments, the FR1 region includes an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentage to any one of SEQ ID NOs: 19, 23, 163, 164, 175, 176, 177, or 230. In some embodiments, the FR1 region, having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of SEQ ID NOs. 19, 23, 163, 164, 175, 176, 177, or 230, includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, and this antibody possesses the ability to bind to an antigen (e.g., CCR8). In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of any one of SEQ ID NOs. 19, 23, 163, 164, 175, 176, 177, or 230.

[0102] In some embodiments, the FR2 region includes an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of sequence numbers 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, or 233. In some embodiments, the FR2 region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of SEQ ID NOs. 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, or 233 includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, and this antibody possesses the ability to bind to an antigen (e.g., CCR8). In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in one of the amino acid sequences of SEQ ID NOs. 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, or 233.

[0103] In some embodiments, the FR3 region includes an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of sequence numbers 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236. In some embodiments, the FR3 region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of SEQ ID NOs. 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236 includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, and this antibody possesses the ability to bind to an antigen (e.g., CCR8). In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in any one of the amino acid sequences of SEQ ID NOs. 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236.

[0104] In some embodiments, the FR4 region includes an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of SEQ ID NOs. In some embodiments, the FR4 region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical residue percentages to any one of SEQ ID NOs. 22, 26, 172, 173, 185, or 237 includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, and this antibody possesses the ability to bind to an antigen (e.g., CCR8). In some embodiments, a total of 1 to 11 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of any one of SEQ ID NOs. 22, 26, 172, 173, 185, or 237.

[0105] In some embodiments, the antibodies of this disclosure include VHH antibodies corresponding to SEQ ID NOs. 27-30. Figures 8A-8B show schematic diagrams of the amino acid sequences of exemplary VHH antibodies, including SEQ ID NOs. 27 and 28, where the CDR is identified using the IMGT method (underlined).

[0106] Therefore, using the IMGT method, the CDRs identified within each VHH antibody sequence (Figures 8A-8B) correspond to those in Table 1 below, as defined above:

[0107] [Table 1]

[0108] In some embodiments, the antibodies of this disclosure are SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, 210-214, 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, 215-224, 7, 8, 9, 16, 17, 18, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 243-246 and 225-2 The CDR comprises at least one, preferably at least two, most preferably three CDRs selected from the group consisting of 29, or at least one, preferably at least two, most preferably three amino acid sequences having at least 80% amino acid identity with respect to these CDR sequences, or at least one, preferably at least two, most preferably three amino acid sequences having three, two, or one amino acid sequence difference from these CDR sequences.

[0109] In some embodiments, the antibody of the present disclosure comprises an amino acid sequence selected from one of SEQ ID NOs: 7, 8, 9, 16, 17, 18, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229 or 243-246, and (c) SEQ ID NOs: 7, 8, 9, 16, 17, 18, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 15 (d) CDR3 comprises an amino acid sequence having at least 80% amino acid identical residue rate for any one of 2-162, 225-229, or 243-246, and an amino acid sequence having 3, 2, or 1 amino acid different from any one of sequence numbers 7, 8, 9, 16, 17, 18, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246. More preferably, CDR3 corresponds to sequence numbers 7, 8, 9, 16, 17, 18, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246.

[0110] In some embodiments, the antibody of the present disclosure comprises a CDR1 comprising (c) an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214; (c) an amino acid sequence having at least 80% amino acid identity with respect to any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214; and (d) an amino acid sequence having 3, 2, or 1 amino acids different from any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214. More preferably, CDR1 corresponds to sequence numbers 1, 2, 3, 10, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214.

[0111] In some embodiments, the antibody of the present disclosure comprises a CDR2 containing (c) an amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 215-224; (c) an amino acid sequence having at least 80% amino acid identity with respect to any one of SEQ ID NOs: 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 215-224; and (d) an amino acid sequence having 3, 2, or 1 amino acids different from any one of SEQ ID NOs: 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 215-224. More preferably, CDR2 corresponds to sequence numbers 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 215-224.

[0112] In certain embodiments, this disclosure provides an hCCR8-conjugated antibody comprising a combination of the CDR1, CDR2, and CDR3 sequences described herein (including the acceptable variations described for these CDR regions).

[0113] In some embodiments, the antibody of the present disclosure comprises a sequence containing three CDRs having sequences of SEQ ID NOs: 1, 4, and 7 or SEQ ID NOs: 3, 6, and 9, or comprises three CDRs having sequences of SEQ ID NOs: 12, 15, and 18 or SEQ ID NOs: 10, 13, and 16.

[0114] In some embodiments, the antibodies of this disclosure further comprise a sequence in which at least 85, 90, 95, 98, or 99% of the sequence is identified for at least one framework region (FR) of the single-domain antibody moiety described herein. In another embodiment of the invention, as detailed above, the single-domain antibody moiety further comprises an amino acid sequence having at least 85, 90, 95, 98, or 99% identical residue rates for four framework regions (FR) of the single-domain antibody moiety described herein. It is understood that the method used to determine the FR of the single-domain antibody moiety is the same as the method used to identify the CDR.

[0115] Therefore, using the IMGT method, the FRs identified within the single-domain antibody portion correspond to the following, as defined above:

[0116] Sequence ID 163: QVQLQESGGGLVQAGGSLRLSCVAS

[0117] Sequence ID 20: IGWYRQAPGKQRELVARF

[0118] Sequence ID 21:YADFVKGRFTISGDNAKNTMYLQMNSLKPEDTAVYYC

[0119] Sequence ID 172: WGQGTQVTVSS

[0120] Sequence ID 175: QVQLQESGGGLVQAGGSLRLTCAVS

[0121] Sequence ID 24:FAWYRQAPGKQRELVAAT

[0122] Sequence ID 25:YADSVKGRFRFSRDNARSAVYLQMDNLRPEDTAVYFC

[0123] Sequence ID 185: WGLGTQVTVSS

[0124] In some embodiments, the antibodies of the present disclosure include at least one, preferably at least two, more preferably at least three, and most preferably four amino acid sequences having at least 85%, preferably 90%, more preferably 95% identical residue rate to any one sequence selected from the group consisting of SEQ ID NOs: 19, 23, 163, 164, 175, 176, 177, 230, 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, 233, 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, 236, 22, 26, 172, 173, 185, or 237.

[0125] In some embodiments, the antibody of this disclosure comprises four framework regions (FRs) in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 has at least 85%, preferably 90%, more preferably 95% identical residues to the sequence of SEQ ID NOs. 19, 23, 163, 164, 175, 176, 177, or 230, and FR2 has at least 85%, preferably 90%, more preferably 95% identical residues to the sequence of SEQ ID NOs. 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, FR3 has at least 85%, preferably 90%, and more preferably 95% identical residues for sequence 232 or 233; FR4 has at least 85%, preferably 90%, and more preferably 95% identical residues for sequence numbers 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236; and FR4 has at least 85%, preferably 90%, and more preferably 95% identical residues for sequence number 22 or 26.

[0126] In some embodiments, the antibodies of this disclosure include an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 26, 172, 173, 185, or 237.

[0127] Sequence ID 27

[0128] QVQLQESGGGLVQAGGSLRLSCVASGSTSSIRRIGWYRQAPGKQRELVARFSSGGGTNYADFVKGRFTISGDNAKNTMYLQMNSLKPEDTAVYYCSARRLLGTSRVDYWGQGTQVTVSS

[0129] Sequence ID 28

[0130] QVQLQESGGGLVQAGGSLRLTCAVSGSTSTINRFAWYRQAPGKQRELVAATSSRTSPNYADSVKGRFRFSRDNARSAVYLQMDNLRPEDTAVYFCQARMAMGSSQVVYWGLGTQVTVSS

[0131] Sequence ID 29

[0132] QVQLQESGGGLVQAGGSLRLSCVASGSTSSIRRIGWYRQAPGKQRELVARFSSGGGTNYADFVKGRFTISGDNAKNTMYLQMNSLKPEDTAVYYCGARRLLGTSRVDYWGQGTQVTVSS

[0133] Sequence ID 30:

[0134] QVQLQESGGGLVQAGGSLRLTCAVSGSTSTINRFAWYRQAPGKQRELVAATSSRTSPNYADSVKGRFRFSRDNARSAVYLQMDNLRPEDTAVYFCQARMAMGASQVVYWGLGTQVTVSS [Table 2]

[0135] In some embodiments, the antibody described above is a full-length antibody. In other embodiments, the antibody is its conjugated fragment. In some cases, the antibody is a humanized antibody or its conjugated fragment, a chimeric antibody or its conjugated fragment, a monoclonal antibody or its conjugated fragment, a multispecific antibody or its conjugated fragment, or a bispecific antibody or its conjugated fragment. In some cases, the antibody is a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-strand variable fragment (scFv), bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its conjugated fragment, or a chemically modified derivative thereof.

[0136] In some embodiments, the antibody is a multispecific antibody. In some cases, the multispecific antibody comprises two or more target antigen-binding domains, each of which binds specifically to an antigen, and the two or more antigens are different. In some cases, the multispecific antibody comprises antigen-binding domains that bind specifically to three or more different antigens, four or more different antigens, or five or more different antigens.

[0137] In some embodiments, the antibodies disclosed herein are bispecific antibodies. In some cases, the bispecific antibody or binding fragment may be KiH (Knobs-into-Holes), ART-Ig (Asymmetric Re-engineering Technology-immunoglobulin), Triomab quadroma, bispecific monoclonal antibodies (BiMAb, BsmAb, BsAb, bsMab, BS-Mab, or Bi-MAb), FcΔAdp, XmAb, Azymetric, BEAT (Bispecific Engagement by Antibodies based on the T-cell receptor), BiTE (Bispecific T-cell Engager), Biclonics, Fab-scFv-Fc, or DAF (Two-in-one / Dual Action). This includes Fab (two-in-one / dual-action Fab), FinomAb, scFv-Fc-(Fab)-fusion, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), TandAb (Tandem diAbody), DART (Dual-affinity-ReTargeting), or nanobody.

[0138] In some cases, a bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, a bispecific antibody is a trifunctional antibody. In some cases, a trifunctional antibody is a full-length monoclonal antibody containing binding sites for two different antigens.

[0139] In some cases, bispecific antibodies are bispecific miniantibodies. In some cases, bispecific miniantibodies include bivalent Fab2, F(ab)'3 fragments, bis-scFv, (scFv)2, diabodies, minibodies, triabodies, tetrabodies, or bispecific T cell engagers (BiTEs). In some embodiments, a bispecific T cell engager is a fusion protein containing two single-strand variable fragments (scFv), where these two scFvs target epitopes of two different antigens.

[0140] In some cases, the antibodies disclosed herein are trispecific antibodies. In some cases, the trispecific antibodies include an F(ab)'3 fragment or a triabody. In some embodiments, the anti-hCCR8 antibody is a trispecific antibody as described in Dimas, et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells", Mol. Pharmaceuticals, 12(9):3490-3501 (2015).

[0141] In some cases, the antibodies disclosed herein include the antibody format shown in Figure 2 of Brinkmann and Kontermann, "The making of bispecific antibodies," MABS9(2):182-212 (2017).

[0142] In some embodiments, the antibodies described herein include an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the antibody includes an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the antibody includes an IgG1 framework. In some cases, the antibody includes an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the antibody includes an IgG2a framework. In some cases, the antibody includes an IgG2b framework. In some cases, the antibody includes an IgG3 framework. In some cases, the antibody includes an IgG4 framework.

[0143] In certain embodiments, the antibodies described herein consist of a fusion of a single-domain antibody (e.g., VHH) to the hinge region of an IgG1, IgG2, IgG3, and IgG4 antibody. In some embodiments, the Fc region is an IgG Fc domain (SEQ ID NO: 34) derived from a human IgG1 antibody. In some embodiments, the Fc region is an IgG Fc domain derived from a short hinge variant of a human IgG1 antibody. In some embodiments, the antibodies described herein are genetically engineered polypeptides comprising at least one single-domain antibody molecule (e.g., VHH) linked to an IgG1 Fc domain by direct binding. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, or 90% identical residue rates to SEQ ID NO: 34.

[0144] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0145] In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a manipulated antibody.

[0146] In some cases, the antibodies described herein contain one or more mutations within a framework region, for example, the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof.

[0147] In some embodiments, the antibodies described herein include humanized antibodies. With respect to full-size antibodies, a single variable domain, such as VHH, may undergo other optimization techniques, such as sequence optimization, i.e., increasing the degree of sequence identity to the nearest human germline sequence, and improving the physicochemical or other properties of the binder. In particular, a single variable domain of a humanized immunoglobulin, such as VHH, may be a single-domain antibody having at least one single amino acid residue (in particular, at least one framework residue) that is a humanization substitution (as further defined herein) and / or corresponding thereto.

[0148] Humanized antibodies, such as single-domain antibodies (VHH), may have several advantages compared to their corresponding naturally occurring antibodies, such as single-domain antibodies (VHH), including reduced immunogenicity. Humanization means that certain amino acids are mutated so that immunogenicity is minimal or absent when administered to human patients. Humanization substitutions must be selected so that the resulting humanized amino acid sequence and / or VHH continue to retain desirable properties of the VHH antibody, such as antigen-binding ability.

[0149] In some embodiments, the antibody, for example, the VHH antibody described herein, is an optimized humanized antibody, for example, a single-domain antibody, for example, a VHH antibody. In some embodiments, the antibody disclosed herein, for example, a single-domain antibody, for example, a VHH antibody, is optimized by introducing a mutation, for example, an amino acid substitution, at any one of positions 1, 5, 15, 24, 42, 49, 50, 52, 54, 55, 70, 80, 83, 87, 95, 96, and / or 123 of VHH-1 (SEQ ID NO: 27). In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, are mutations, e.g., wild-type antibodies, e.g., wild-type single-domain antibodies, e.g., wild-type VHH antibodies of SEQ ID NO: 27, numbered according to the IMGT scheme, comprising amino acid substitutions at one or more amino acid residues 1, 5, 15, 24, 42, 49, 50, 52, 54, 55, 70, 80, 83, 87, 95, 96, and / or 123.

[0150] In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, are optimized by introducing a mutation, e.g., an amino acid substitution, at any one of positions 1, 5, 15, 25, 42, 49, 50, 52, 54, 77, 78, 83, 87, 91, 92, 96, 103, 120, and / or 123 of, for example, VHH-2 (SEQ ID NO: 28). In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, include a mutation, e.g., an amino acid substitution at one or more amino acid residues 1, 5, 15, 25, 42, 49, 50, 52, 54, 77, 78, 83, 87, 91, 92, 96, 103, 120, and / or 123 of, for example, a wild-type antibody, e.g., a wild-type single-domain antibody, e.g., a wild-type VHH antibody of SEQ ID NO: 28, numbered according to the IMGT scheme.

[0151] These residues are indicated by asterisks in Figures 8A and 8B. The amino acid positions are numbered according to the IMGT (International ImmunoGeneTics Information System) (Lefranc, M.-P. et al., 2009, Nucleic Acids Research, D1006-1012, http: / / www.imgt.org).

[0152] In some embodiments, the antibody, for example, the VHH antibody described above, is an optimized humanized antibody, for example, a single-domain antibody, for example, a VHH antibody. In some embodiments, the antibody disclosed herein, for example, a single-domain antibody, for example, a VHH antibody, is optimized by introducing a mutation, for example, an amino acid substitution, at any one of positions 1, 5, 15, 24, 49, 50, 52, 70, 83, 95, 96 and / or 123 of, for example, VHH-1 (SEQ ID NO: 27). In some embodiments, the antibody disclosed herein, for example, a single-domain antibody, for example, a VHH antibody, includes a mutation, for example, an amino acid substitution at one or more amino acid residues 1, 5, 15, 24, 49, 50, 52, 70, 83, 95, 96 and / or 123 of the wild-type antibody, for example, a wild-type single-domain antibody, for example, a wild-type VHH antibody of SEQ ID NO: 27, numbered according to the IMGT scheme.

[0153] In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, are optimized by introducing a mutation, e.g., an amino acid substitution, at any one of positions 1, 5, 15, 49, 50, 52, 77, 83, 84, 85, 86, 91, 92, 96, 103, 120, and / or 123 of, for example, VHH-2 (SEQ ID NO: 28). In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, include a mutation, e.g., an amino acid substitution at one or more amino acid residues 1, 5, 15, 49, 50, 52, 77, 83, 84, 85, 86, 91, 92, 96, 103, 120, and / or 123 of, for example, a wild-type antibody, e.g., a wild-type single-domain antibody, e.g., a wild-type VHH antibody of SEQ ID NO: 28, numbered according to the IMGT scheme.

[0154] In some embodiments, the antibody, e.g., the VHH antibody described above, is an optimized humanized antibody, e.g., a single-domain antibody, e.g., a VHH antibody. In some embodiments, the antibody disclosed herein, e.g., a single-domain antibody, e.g., a VHH antibody, is optimized by introducing a mutation, e.g., an amino acid substitution, at any one of positions 1, 5, 15, 24, 49, 70, 83, 95, 96 and / or 123 of, for example, VHH-1 (SEQ ID NO: 27). In some embodiments, the antibody disclosed herein, e.g., a single-domain antibody, e.g., a VHH antibody, includes a mutation, e.g., an amino acid substitution at one or more amino acid residues 1, 5, 15, 24, 49, 70, 83, 95, 96 and / or 123 of the wild-type antibody, e.g., a wild-type single-domain antibody, e.g., a wild-type VHH antibody of SEQ ID NO: 27, numbered according to the IMGT scheme.

[0155] In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, are optimized by introducing a mutation, e.g., an amino acid substitution, at any one of positions 1, 5, 15, 49, 77, 83, 84, 85, 86, 91, 92, 96, 103, 120, and / or 123 of, for example, VHH-2 (SEQ ID NO: 28). In some embodiments, the antibodies disclosed herein, e.g., single-domain antibodies, e.g., VHH antibodies, include a mutation, e.g., an amino acid substitution at one or more amino acid residues 1, 5, 15, 49, 77, 83, 84, 85, 86, 91, 92, 96, 103, 120, and / or 123 of, for example, a wild-type antibody, e.g., a wild-type single-domain antibody, e.g., a wild-type VHH antibody of SEQ ID NO: 28, numbered according to the IMGT scheme.

[0156] In some cases, one or more mutations are intended to stabilize the antibody and / or extend its half-life. In some cases, one or more mutations are intended to modulate Fc receptor interactions to enhance Fc effector function, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In additional examples, one or more mutations are intended to modulate glycosylation.

[0157] In some embodiments, the antibody contains at least one modification within the Fc region that enhances cell killing. In some embodiments, the enhanced cell killing is enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC). In some embodiments, at least one modification is fucosylation. In some embodiments, at least one modification is a mutation in one or more heavy chain constant regions at one or more positions selected from L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, 1332, E333, K334, and P396. In some embodiments, one or more heavy chain constant region mutations are one or more mutations selected from S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L, A330M, I332E, E333A, K334A, K334E, and P396L. In some embodiments, one or more heavy chain constant region mutations are selected from F243L / R292P / Y300L / V305I / P396L, S239D / I332E, S239D / I332E / A330L, S298A / E333A / K334A, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and D270E / K326D / A330M / K334E.

[0158] In some cases, one or more mutations modulate Fc receptor interactions to reduce or eliminate Fc effector functions such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional examples, one or more mutations modulate glycosylation.

[0159] In some embodiments, the Fc region includes mutations or combinations of mutations at residue positions L235, L236, D265, N297, K322, L328, or P329. In some examples, the Fc region includes mutations at L235 and L236.

[0160] In some embodiments, the antibodies described herein have an improved serum half-life compared to a reference anti-hCCR8 antibody. In some cases, the improved serum half-life is at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer than that of the reference anti-hCCR8 antibody.

[0161] In some embodiments, the antibodies or antigen-binding fragments of the Disclosure are isolated. With respect to isolated antibodies, “isolated” means that the antibody polypeptide has been separated from at least some of the components of the cell from which it was produced (e.g., a host cell). If the polypeptide is secreted by a host cell after expression, physically separating the supernatant containing the polypeptide from the host cell that produced it is considered “isolated” the polypeptide. The term “isolated” means a protein (e.g., an antibody) that is substantially free of other cellular material and / or chemicals. In one embodiment, an isolated antibody is substantially free of other proteins from the same species. In one embodiment, an isolated antibody is expressed by cells from a different species and is substantially free of other proteins from this different species. Proteins may be brought to a state substantially free of naturally associated components (or components associated with the cell expression system used to produce the antibody) by isolation using protein purification techniques well known in the Art. As used herein, the term “isolated antibody” also includes antibodies that substantially do not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to CCR8 (e.g., human CCR8) substantially does not contain antibodies that specifically bind to antigens other than CCR8). Isolated antibodies bind specifically to an epitope but may cross-reactive to other CCR8 proteins from different species. However, antibodies continue to demonstrate specific binding to human CCR8 in the specific binding assays described herein. Furthermore, isolated antibodies typically substantially do not contain other cellular material and / or chemicals. In some embodiments, combinations of “isolated” antibodies with different CCR8 specificities are combined in a clearly defined composition. In some embodiments, the isolated antibodies are recombinant antibodies.

[0162] Those skilled in the art will understand that antibodies suitable for use in the methods disclosed herein may be modified to differ from the naturally occurring sequences from which they originate, while retaining the desired activity of the natural sequences. For example, nucleotide or amino acid substitutions may be made that result in conservative substitutions or changes in "non-essential" amino acid residues. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0163] Antibodies suitable for use in the methods disclosed herein may include conserved amino acid substitutions in one or more amino acid residues, for example, in essential or non-essential amino acid residues. A “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. The family of amino acid residues having similar side chains is defined in the art and includes basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of the binding polypeptide are preferably replaced with other amino acid residues from the same side-chain family. In certain embodiments, the amino acid chain can be replaced with structurally similar chains that differ in the order and / or composition of the members of the side-chain family. Alternatively, in certain embodiments, mutations may be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into the binding polypeptide of the present invention and screened for their ability to bind to the desired target.

[0164] As used herein, the terms “epitope” or “antigenic determinant” refer to a site on an antigen (e.g., CCR8) to which an immunoglobulin or antibody specifically binds. The term “epitope mapping” refers to a process or method for identifying the binding site or epitope of an antibody or its antigen-binding fragment on its target protein antigen. Epitope mapping methods and techniques are provided herein. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids paralleled by tertiary folding of the protein. Epitopes formed from adjacent amino acids are usually retained even when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound to a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which duplicate or adjacent peptides from CCR8 are tested for reactivity with a given anti-CCR8 antibody. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0165] Similarly, included in this disclosure are antibodies that bind to epitopes on CCR8, including all or part of the epitopes recognized by the specific antibodies described herein (e.g., the same region, overlapping regions, regions between these regions, or regions spanning these regions).

[0166] Similarly, antibodies that bind to the same epitope, and / or antibodies that compete for binding to human CCR8 with the antibodies described herein, can be identified using standard techniques. Such techniques include, for example, immunoassays, i.e., competitive binding assays, that demonstrate the ability of one antibody to block the binding of another antibody to a target antigen. Competitive binding is determined by assays in which the immunoglobulin being tested inhibits the specific binding of a reference antibody to a common antigen such as CCR8. Numerous types of competitive binding assays, e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (see Stahli et al., Methods in Enzymology 9:242 (1983)), solid-phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid-phase direct labeling RIA using I-125 labeling (see Morel et al., Mol.Immunol. 25(1):7 (1988)), solid-phase direct biotin-avidin EIA (see Cheung et al. The methods known are Moldenhauer et al., Virology 176:546 (1990), and the direct-labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assays involve the use of purified antigens bound to solid surfaces or cells, either unlabeled test immunoglobulin or labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of test immunoglobulin. Typically, test immunoglobulin is present in excess.Typically, when there is an excess of competing antibodies, the specific binding of the reference antibody to the common antigen is inhibited by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more.

[0167] Other techniques include epitope mapping methods, such as X-ray analysis of antigen:antibody complex crystals that provide atomic resolution for epitopes, and mass spectrometry combined with hydrogen / deuterium (H / D) exchange to study the conformation and dynamics of antigen:antibody interactions. Other methods monitor antibody binding to antigen fragments or mutated variants of antigens, and loss of binding due to modification of amino acid residues in the antigen sequence is often considered an indication of epitope components. Furthermore, computational combinatorial methods of epitope mapping can also be used. These methods depend on the ability of the antibody of interest to isolate specific short peptides by affinity from combinatorial phage display peptide libraries. The peptides are then considered leads for defining epitopes corresponding to antibodies used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that are known to map conformationally discontinuous epitopes.

[0168] Single-domain antibody In some embodiments, the antibodies of this disclosure are single-domain antibodies. As used herein, the terms “single-domain antibody,” “heavy chain variable region domain of a heavy chain antibody,” “single variable domain,” “VHH,” “nanobody,” “sdAb,” and “VHH domain” are used interchangeably and generally refer to a single variable region (e.g., VH) that specifically binds to a target antigen (e.g., CCR8). In other words, the single variable domain of a single-domain antibody does not need to recognize the target antigen (e.g., CCR8) by interacting with another variable region (e.g., VL). Examples of single-domain antibodies include those derived from antibodies of camelids (camels and llamas) and cartilaginous fish (e.g., nurse sharks). A single-domain antibody is an antibody whose complementarity-determining region is part of a single variable domain (e.g., CCR8-binding domain) polypeptide. A single-domain antibody therefore comprises a single variable domain (e.g., CCR8-binding domain) containing a single CDR1, a single CDR2, and / or a single CDR3. In some embodiments, a single-domain antibody comprises a single variable domain (e.g., a CCR8-binding domain) containing a single CDR1, a single CDR2, a single CDR3, or a combination thereof. A single-domain antibody may comprise only a variable domain of an immunoglobulin chain having CDR1, CDR2, CDR3, or a combination thereof, and in some embodiments further comprises one or more framework regions (e.g., one or more human framework regions). Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking a light chain, single-domain antibodies derived from conventional quadruple-chain antibodies, engineered antibodies, and single-domain scaffolds other than those derived from antibodies. A single-domain antibody may be any single-domain antibody in the art or any future single-domain antibody. A single-domain antibody may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cattle. According to one aspect of the present invention, the single-domain antibody used herein is a naturally occurring single-domain antibody known as a heavy-chain antibody lacking a light chain.Such single-domain antibodies are disclosed, for example, in WO9404678. For clarity, this variable domain lacking a light chain, or a variable domain derived from a heavy-chain antibody lacking a light chain, is known herein as VHH or nanobody to distinguish it from conventional VH of quadruple-chain immunoglobulins. In some embodiments, the variable domain is derived from a heavy-chain antibody that naturally lacks a light chain. Such VHH molecules may be derived from antibodies produced in camelid species, such as camels, llamas, dromedaries, alpacas, and guanacos. Thus, in some embodiments, the antibodies of this disclosure are Camillidae-derived VHH or llama-derived VHH (or llama-derived single-domain antibodies). Other non-camellid species may also produce heavy-chain antibodies that naturally lack a light chain, and such VHHs are within the scope of the present invention. VHH molecules are about 10 times smaller than IgG molecules. These are single polypeptides, highly stable, and tolerant of extreme pH and temperature conditions. Furthermore, they exhibit resistance to protease activity, which is not the case with conventional antibodies. In addition, in vitro expression of VHH produces well-folded functional VHH in high yield. Moreover, antibodies produced in camelids recognize epitopes other than those recognized by antibodies produced in vitro, either through the use of an antibody library or by immunization of non-camelid mammals (WO9749805). In some embodiments, the single-domain antibody is a VHH targeted at a specific target, and the VHH belongs to a class having a human-like sequence. This class is characterized in that the VHH has an amino acid at position 45 (e.g., L45) from the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine, according to Kabat numbering. Therefore, peptides belonging to this class exhibit high amino acid sequence homology to the human VH framework region, and these peptides can be administered directly to humans without expecting an undesirable immune response and without burdening further humanization.

[0169] In some embodiments, the single-domain antibody is the variable domain of a VNAR (Variable new antigen receptor), an immunoglobulin novel antigen receptor (IgNAR) antibody derived from cartilaginous fish such as sharks. In some embodiments, the VNAR antibody includes CRD1 and CDR2. In some embodiments, the VNAR antibody further includes two other hypervariable (HV) regions called HV2 and HV4 regions. In some embodiments, the VNAR antibody further includes one or more framework regions. The CDR and HV regions are surrounded by a framework (FW) region in the following N-terminus-C-terminus order: FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4. In some embodiments, the single-domain antibody is a chimeric, synthetic, or humanized antibody. In some embodiments, the single-domain antibody is multispecific. In some embodiments, the single-domain antibody is polyvalent.

[0170] In some embodiments, the antibodies of this disclosure comprise two or more single-domain antibodies. For example, the antibodies disclosed herein may include at least two single-domain antibodies. In some embodiments, the two single-domain antibodies target the same antigen (e.g., CCR8). Such multivalent polypeptide constructs have the advantage of unusually high functional affinity to the target and exhibit much higher inhibitory properties than expected compared to their monovalent counterparts. In some embodiments, the antibodies of this disclosure are bispecific antibodies comprising a first single-domain antibody containing a CCR8-binding domain and a second single-domain antibody containing an antigen-binding domain that specifically binds to a target other than CCR8. For example, the second single-domain antibody contains an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen).

[0171] In some embodiments, two or more single-domain antibodies are covalently linked. In some embodiments, the two or more single-domain antibodies may have identical or different sequences, but target the same target or antigen (e.g., CCR8). Depending on the number of linked single-domain antibodies, the polyvalent single-domain antibody may be bivalent (two single-domain antibodies), trivalent (three single-domain antibodies), tetravalent (four single-domain antibodies), or have molecules with higher valencies.

[0172] In some embodiments, two or more single-domain antibodies may have different sequences and target two different targets or antigens. For example, the antibody of this disclosure may include a first single-domain antibody containing a CCR8-binding domain (i.e., targeting CCR8) and a second single-domain antibody containing an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen). Depending on the number of linked single-domain antibodies, the multispecific single-domain antibody may be bispecific (two single-domain antibodies), triplicate (three single-domain antibodies), quadruplicate (four single-domain antibodies), or have molecules with higher specificity.

[0173] In some embodiments, two or more single-domain antibodies are linked using methods known in the art or any future methods. They may be linked non-covalently (e.g., using streptavidin / biotin combinations, antibody / tag combinations) or covalently. They may be fused by chemical crosslinking by reacting amino acid residues with organic derivatizers, as described by Blattler et al, Biochemistry 24, 1517-1524; EP294703. Alternatively, single-domain antibodies may be genetically fused at the DNA level, i.e., a polynucleotide construct encoding a complete polypeptide construct containing one or more single-domain antibodies may be formed. Methods for producing bivalent or polyvalent single-domain antibodies are disclosed in PCT patent application WO96 / 34103. One method for linking two or more single-domain antibodies is via a genetic pathway by linking single-domain antibody coding sequences either directly or via a peptide linker. For example, the C-terminus of one single-domain antibody may be linked to the N-terminus of the next single-domain antibody.

[0174] This linking mode can be extended to link additional single-domain antibodies for the construction and production of functional constructs such as trifunctional and tetrafunctional antibodies. According to one aspect of the present invention, single-domain antibodies are linked to one another via a peptide linker sequence. Such a linker sequence may be a naturally occurring sequence or a sequence that does not exist in nature. The linker sequence is expected to be non-immunogenic in the target to which the multivalent antitarget polypeptide is administered. The linker sequence may provide sufficient flexibility to the multivalent antitarget polypeptide while also being resistant to proteolysis. A non-limiting example of a linker sequence may be derived from the hinge region of VHH described in WO96 / 34103.

[0175] Polypeptide constructs disclosed herein can be prepared by those skilled in the art according to methods known in the art or any future methods. For example, VHH can be obtained using methods known in the art, for example, by immunizing camels and thereby obtaining hybridomas, or by cloning a library of single-domain antibodies using molecular biology techniques known in the art and then selecting them using phage display or yeast display.

[0176] In some embodiments, the antibodies disclosed herein further comprise an Fc region. Similarly, provided herein are fusion proteins comprising single-domain antibodies and heterologous proteins disclosed herein. In some embodiments, the heterologous protein is an Fc domain, such as a human Fc domain. Thus, in some embodiments, a single-domain antibody is fused to an Fc domain (e.g., a human Fc domain).

[0177] In some embodiments, the antibodies of this disclosure are heavy-chain-only antibodies. The term “heavy-chain-only antibody” refers to a single-domain antibody that lacks a light chain and further comprises at least a portion of the hinge region in the absence of the CH2, CH3 constant domains and / or the CH1 domain.

[0178] Species selectivity and interspecific cross-reactivity According to certain embodiments of this disclosure, an antibody may bind to human CCR8 but not to CCR8 derived from other species. Alternatively, in certain embodiments, the antibody may bind to human CCR8 and also to CCR8 derived from one or more non-human species. In some embodiments, the antibodies of this disclosure are cross-reactive. For example, an antibody or its antigen-binding fragment may bind to human CCR8 and, depending on the circumstances, to one or more CCR8s of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus macaque, marmoset, rhesus macaque, or chimpanzee, or may not bind. As used herein, the term “cross-reactive” refers to the ability of the antibodies of this disclosure to bind to CCR8 derived from different species. For example, an antibody of this disclosure that binds to human CCR8 may also bind to CCR8 of another species. As used herein, cross-reactivity is measured by detecting specific reactivity with a purified antigen in a binding assay (e.g., SPR, ELISA), or by binding to cells that physiologically express CCR8, or by functionally interacting with those cells in other ways. Methods for determining cross-reactivity include, for example, BIACORE® SPR analysis using a BIACORE® 2000 surface plasmon resonance (SPR) instrument (BIACORE AB, Uppsala, Sweden), or standard binding assays described herein by flow cytometry techniques.

[0179] Nucleic acids encoding antibodies in this disclosure In one embodiment, what is provided herein is an isolated nucleic acid molecule encoding an antibody disclosed herein. The nucleotide sequences of the present invention can be useful for several applications, including cloning, gene therapy, protein expression and purification, mutagenesis, antibody production for host DNA vaccination as needed, passive immunization, PCR, primer and probe production, etc.

[0180] The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may include, but are not limited to, natural and / or non-natural nucleotides, and include DNA, RNA, and PNA. “Nucleic acid sequence” refers to a linear sequence of nucleotides.

[0181] As used herein, an “isolated” nucleic acid molecule or “isolated” nucleic acid sequence is considered isolated if it is either (1) a nucleic acid molecule identified and separated from at least one contaminating nucleic acid molecule that normally associates with the natural source of the nucleic acid, or (2) a nucleic acid molecule that has been cloned, amplified, tagged, or otherwise distinguished from a background nucleic acid so that the sequence of the nucleic acid of interest can be determined. An isolated nucleic acid molecule is one that is other than the form or setting it is found in nature. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules that exist in native cells. However, isolated nucleic acid molecules include, for example, nucleic acid molecules contained in cells that normally express antibodies at chromosomal locations different from the chromosomal locations of native cells. As used herein, “isolated nucleic acid” is a nucleic acid that is substantially separated from proteins or complexes such as ribosomes and polymerases that naturally accompany the natural sequence, in addition to other genomic DNA sequences. The term encompasses nucleic acid sequences removed from their naturally occurring environment and includes recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs by heterologous systems. Substantially pure nucleic acids include isolated forms of nucleic acids. Naturally, this refers to the initially isolated nucleic acid and does not exclude genes or sequences subsequently added to nucleic acids isolated by human hands. In some embodiments, the isolated nucleic acid molecules of this disclosure include nucleic acids encoding antibodies or antibody moieties that bind to CCR8 (e.g., VH, CDR1, CDR2, CDR3) disclosed herein, and these nucleic acids encoding antibodies or antibody moieties do not include other nucleotide sequences encoding antibodies or antibody moieties that bind to antigens other than CCR8, and these other sequences may naturally be adjacent to nucleic acids in human genomic DNA.

[0182] Isolated nucleic acid molecules encoding antibodies, partials, or polypeptides of this disclosure can be recombined with vector DNA (e.g., an expression vector) according to conventional techniques, which include blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate termination, cohesive end packing as needed, alkaline phosphatase treatment to avoid undesirable ligation, and ligation with an appropriate ligase. Techniques for such operations are disclosed, for example, by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel, 1987, 1993, and can be used to construct nucleic acid sequences encoding antibody molecules. Accordingly, this disclosure provides vectors or expression vectors comprising the isolated nucleic acids described herein. Once isolated, nucleic acid molecules are placed in an expression vector, they can then be transfected into host cells, such as E. coli cells, monkey COS cells, human fetal kidney 293 cells (e.g., 293E cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not normally produce immunoglobulin proteins, to obtain the antibodies disclosed herein in recombinant host cells. Recombinant production of antibodies is well known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more selection marker genes, enhancer elements, promoters, and transcription termination sequences.

[0183] Desired antibody amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA or by peptide synthesis. Such variants include, for example, deletions from and / or insertions of residues within the antibody's amino acid sequence, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions is made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., binding to CCR8). Amino acid changes can also modify post-translational treatments of monoclonal, human, humanized, or variant antibodies, for example, by altering the number or location of glycosylation sites.

[0184] Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of pre-prepared variant or non-variant versions of antibodies.

[0185] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are produced in a cell-free system. Exemplary cell-free systems, not limited to these, are described, for example, Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); and Endo et al., Biotechnol. Adv. 21:695-713 (2003). For such purposes, the nucleic acid encoding the polypeptide needs to be modified to allow in vitro transcription to produce mRNA and to enable cell-free translation of the mRNA in the specific cell-free system used (e.g., eukaryotic cells such as mammalian or yeast cell-free translation systems, or prokaryotes such as bacterial cell-free translation systems). Antibody polypeptides can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to proteins can be brought about by chemical synthesis. In some embodiments, the antibodies or antigen-binding fragments of this disclosure are synthesized. The polypeptides of this disclosure can be purified by protein isolation / purification methods commonly known in the field of protein chemistry.

[0186] Antigen-CCR8 This specification provides antibodies that specifically bind to CCR8 or a portion thereof. As used herein, the terms “CCR8,” “CC chemokine receptor type 8,” or “CCR8 polypeptide” refer to a G protein-coupled receptor polypeptide. As used herein, the term “CCR8” refers to the human CCR8 polypeptide. The term “CCR8” encompasses all natural or variant (whether natural or synthetic) CCR8. The term “natural” CCR8 encompasses naturally occurring cleaved or secreted forms (e.g., extracellular domain sequences described in SEQ ID NO: 38, etc.), naturally occurring mutants (e.g., or spliced ​​forms), and naturally occurring allelic mutants. As used herein, the term “CCR8” refers to the 355-amino acid polypeptide represented in SEQ ID NO: 37, along with naturally occurring alleles and their processing forms. The term “CCR8” includes CCR8 variants. As used herein, the term “CCR8” refers to the full-length CCR8 polypeptide, or a portion or derivative thereof. The CCR8 polypeptide can be full-length human CCR8 and / or its functional fragment, an interspecies homolog and / or its functional fragment, or an ortholog of human CCR8 and / or its functional fragment. The CCR8 polypeptide can be a mammalian CCR8 polypeptide. CCR8 can also be a functional isoform of full-length CCR8 or a part thereof. In some embodiments, CCR8 comprises or is derived from the human CCR8 amino acid sequence shown in SEQ ID NO: 37.

[0187] The term “CCR8” also includes truncated human CCR8. In some embodiments, CCR8 is a truncated form of the 355-amino acid human CCR8 (e.g., SEQ ID NO: 37). As used herein, the term “CCR8 variant” refers to a CCR8 polypeptide containing one or more amino acid mutations in the native CCR8 sequence (e.g., SEQ ID NO: 37). Where applicable, one or more amino acid mutations may include amino acid substitutions. CCR8 is known to have at least four ligands, namely CCL1, CCL8, CCL16, and CCL18. CCL1 is thought to enhance human Treg cells by inducing CCR8, FOXp3, CD39, granzyme B, and IL-10 expression in a STAT3-dependent manner. See, for example, Barsheshet et al., PNAS 114(23):6086-91 (Jun. 6, 2017). CCR8 is primarily expressed in Treg cells and to a lesser extent in TH2 cells, monocytes, NK cells, and a small number of CD8+ cells. CCR8 is a transmembrane receptor having seven transmembrane domains, one extracellular N-terminal domain (SEQ ID NO: 38), and one intracellular C-terminal domain, and interacts with G proteins. In some embodiments, CCR8 is expressed intracellularly (e.g., on the cell surface). In some embodiments, the cells are regulatory T cells, tumor-infiltrating T cells, TH2 cells, NK cells, monocytes, CD8+ cells, or cancer cells. In some embodiments, CCR8 is expressed on the cell surface. In some embodiments, CCR8 is soluble. In some embodiments, CCR8 is recombinant. In some embodiments, the antibodies disclosed herein bind specifically to the transmembrane domains of CCR8 (e.g., human CCR8).

[0188] antibody complex In some embodiments, the antibodies described herein are further conjugated to a payload. In some examples, the payload comprises small molecules. In other examples, the payload comprises proteins or peptides. In additional examples, the payload comprises polynucleic acid molecules.

[0189] In some cases, the payload-to-antibody ratio (drug-to-antibody ratio or DAR ratio) is approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1.

[0190] In some cases, the anti-hCCR8 antibody conjugate, A-(X 1 -B) n Equation (I) During the ceremony, A contains an anti-hCCR8 antibody. B includes the payload, X 1 It consists of a linker or bond, n is the average value selected from 1 to 12.

[0191] In some cases, the B-to-A (anti-hCCR8 antibody) DAR ratio is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the B-to-A DAR ratio is approximately 1. In some cases, the B-to-A DAR ratio is approximately 2. In some cases, the B-to-A DAR ratio is approximately 3. In some cases, the B-to-A DAR ratio is approximately 4. In some cases, the B-to-A DAR ratio is approximately 6. In some cases, the B-to-A DAR ratio is approximately 8. In some cases, the B-to-A DAR ratio is approximately 10. In some cases, the B-to-A DAR ratio is approximately 12. In some cases, the B-to-A DAR ratio is approximately 16.

[0192] In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 1. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 2. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 3. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 4. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 5. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 6. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 7. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 8. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 9. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 10. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 11. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 12. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 13. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 14. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 15. In some cases, the DAR ratio between polynucleotide molecule (B) and anti-hCCR8 antibody A is approximately 16.

[0193] In some embodiments, B includes small molecules, peptides, or proteins.

[0194] In some embodiments, B comprises a polynucleic acid molecule. In some cases, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some cases, the passenger strand is AX 1 It is conjugated to AX. In some cases, AX 1is conjugated to the 5'-end of the passenger strand. In some cases, A-X 1 is conjugated to the 3'-end of the passenger strand.

[0195] In some cases, the anti-hCCR8 antibody conjugate is A-X 1 -(B-X 2 -C) n Formula (II) wherein A contains an anti-hCCR8 antibody, B contains a payload, C consists of a polymer, X 1 is composed of a bond or a first linker, X 2 consists of a bond or a second linker, n is an average value selected from 1 to 12.

[0196] In some embodiments, C is polyethylene glycol.

[0197] In some embodiments, B is a polynucleic acid molecule. In some cases, the polynucleic acid molecule contains a passenger strand and a guide strand. In some cases, the passenger strand is conjugated to A-X 1 and X 2 -C. In some cases, A-X 1 is conjugated to the 5'-end of the passenger strand, and X 2 -C is conjugated to the 3'-end of the passenger strand. In some cases, X 2 -C is conjugated to the 5'-end of the passenger strand, and A-X 1 is conjugated to the 3'-end of the passenger strand.

[0198] In some embodiments, X 1 and X 2 are each an independent non-polymeric linker.

[0199] In some embodiments, formula (II)AX 1 -(BX 2 -C) n The complex further includes D, an endosomal lytic moiety.

[0200] Conjugation Chemistry In some embodiments, B is conjugated to A by a chemical ligation process. In some cases, B is conjugated to A by natural ligation. In some cases, conjugation is referred to as Dawson, et al. "Synthesis of proteins by native chemical ligation", Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives", J.Am.Chem.Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology", Proc.Natl.Acad.Sci.USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation" This protocol is described in Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some cases, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleic acid molecules are conjugated to the binding site either site-specifically or non-specifically by natural ligation chemistry.

[0201] In some cases, B is conjugated to A by a site-specific method utilizing "traceless" coupling technology (Philochem). In some cases, "traceless" coupling technology utilizes the N-terminal 1,2-aminothiol group on the binding site and then conjugates with a polynucleic acid molecule containing an aldehyde group. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery", JACS134(13):5887-5892 (2012)).

[0202] In some cases, B is conjugated to A by a site-specific method using unnatural amino acids incorporated into the binding site. In some cases, the unnatural amino acid includes p-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPhe is selectively coupled to the alkoxy-amine derivative conjugate site to form an oxime bond. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids", PNAS 109(40):16101-16106 (2012)).

[0203] In some cases, B is conjugated to A by a site-specific method utilizing an enzyme-catalyzed process. In some cases, the site-specific method utilizes SMARTag® technology (Redwood). In some cases, SMARTag® technology includes generating a formylglycine (FGly) residue from cysteine ​​by formylglycine-producing enzyme (FGE) through an oxidation process in the presence of an aldehyde tag, and then conjugating FGly to an alkylhydraine-functionalized polynucleic acid molecule via HIPS (hydrazino-Pictet-Spengler) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51 (2013)).

[0204] In some embodiments, the enzyme-catalyzed process involves a microbial transglutaminase (mTG). In some cases, B is conjugated to A using a microbial transglutaminase-catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2)161-167 (2013)).

[0205] In some cases, B is conjugated to A by a method described in PCT Publication WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0206] In some cases, B is conjugated to A by the method described in U.S. Patent Publications 2015 / 0105539 and 2015 / 0105540.

[0207] The antibody conjugates described herein can be prepared using any of the following bifunctional protein coupling agents: N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyl HCl adipiimidoate), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., 238 Science 1098 (1987). Carbon-14-labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example chelating agent for the conjugation of radioactive nucleotides to antibodies. See WO94 / 11026.

[0208] Techniques for conjugating such payloads into antibodies are well known. For example, see Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987).

[0209] In some embodiments, the payload can attach to the hinge region of the reduced antibody component via disulfide bond formation. For example, a tetanus toxoid peptide can be conjugated with a single cysteine ​​residue used to attach this peptide to the antibody component.

[0210] In some embodiments, the payload can be conjugated to the Fc region of the antibody (for example, a therapeutic agent can be conjugated using a carbohydrate moiety within the Fc region of the antibody). In some embodiments, the payload can be conjugated to the variable region of the antibody (for example, a carbohydrate moiety can be introduced into the variable region of the antibody or antibody fragment; see, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Patent No. 5,443,953). The manipulated carbohydrate moiety is then used to attach the payload. Furthermore, those skilled in the art will recognize numerous possible variations of the conjugation method. For example, polyethylene glycol can be attached to the carbohydrate moiety to extend the half-life of an intact antibody or its antigen-binding fragment in blood, lymph, or other extracellular fluid. Furthermore, it is possible to construct a “divalent conjugate” by attaching a therapeutic agent to the carbohydrate moiety and to a free sulfhydryl group. Such a free sulfhydryl group may be located in the hinge region of the antibody component.

[0211] payload In some embodiments, the payload is a polynucleic acid molecule. In some cases, the polynucleic acid molecule hybridizes to the target region of the oncogene. Exemplary oncogenes include Ab1, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, and MAS1. This includes, but is not limited to, MDM2, MET, MLL(KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1(TAN1), NTRK1(TRK), OST(SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some cases, polynucleic acid molecules hybridize to target regions of KRAS, EGFR, AR, HPRT1, CNNTB1(β-catenin), or β-catenin-related genes.

[0212] Small molecules, proteins, and peptides In some embodiments, the payload is a small molecule. In some examples, the small molecule is a cytotoxic payload. Exemplary cytotoxic payloads include, but are not limited to, microtubule inhibitors, DNA modifiers, or Akt inhibitors.

[0213] In some cases, the payload may include immunomodulators. Useful immunomodulators include antihormone drugs that block hormonal effects on tumors, and immunosuppressants that suppress cytokine production, downregulate the expression of autoantigens, or mask MHC antigens. Representative antihormone drugs include anti-estrogens (e.g., tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapnstone, and toremifene), as well as anti-androgens (e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin), and anti-adrenal agents. Examples of immunosuppressants include, but are not limited to, 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde, anti-idiotype antibodies against MHC antigens and MHC fragments, cyclosporine A, steroids such as glucocorticosteroids, streptokinase, or rapamycin.

[0214] In some embodiments, the payload includes a protein or peptide toxin or a fragment thereof. Exemplary enzyme-active toxins and their fragments include, but are not limited to, diphtheria toxin A fragment, diphtheria toxin unbound active fragment, exotoxin A (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesin A chain, α-sacrin, certain A leurites fordii proteins, certain Dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Morodica charantia inhibitors, curcin, crotin, Saponaria officinalis inhibitors, geronin, mitogillin, restrictosin, phenomycin, enomycin, and trichothecenes.

[0215] In some embodiments, the payload is an immunomodulator. Exemplary immunomodulators include gancyclovier, etanercept, tacrolimus, sirolimus, voclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, and mycophenolate mofetil. This includes, but is not limited to, mofetil, methotrexate, glucocorticoids and their analogues, xanthines, stem cell growth factors, lymphotoxins, hematopoietic factors, tumor necrosis factor (TNF) (e.g., TNFα), interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (e.g., interferon-alpha, interferon-beta, interferon-gamma), stem cell growth factors known as "S1 factors," erythropoietin and thrombopoietin, or combinations thereof.

[0216] In some examples, the payload includes cytokines. In some embodiments, the cytokines include IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, interferons (e.g., IFNα, IFNβ), or TNFα.

[0217] This disclosure also intends to conjugate more than one payload to the antibodies of this disclosure. For example, in some embodiments, the antibodies of this disclosure can be conjugated to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 payloads. In one embodiment, the antibody is used as a radiosensitizer. In such embodiments, the antibody is conjugated to the radiosensitizer. Thus, in some embodiments, the payload includes the radiosensitizer. As used herein, the term “radiosensitizer” is defined as a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective dose to increase the sensitivity of cells to radiosensitization to electromagnetic radiation and / or to facilitate the treatment of diseases treatable with electromagnetic radiation. Diseases treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells. In another embodiment, the antibody may be conjugated to a receptor (such as streptavidin) for use in tumor pre-targeting, for example, the antibody may be used on a tumor site or tumor microenvironment (e.g., a tumor site or tumor microenvironment containing cells expressing CCR8 polypeptide), the antibody-receptor complex may be administered to the patient, followed by removal of the unbound complex from the blood circulation using a scavenging agent, and then administration of a ligand (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide). In some embodiments, the payload may contain biotin, and the biotin-conjugated antibody or antibody fragment may be further conjugated or linked to a streptavidin conjugate or coating agent, such as streptavidin-coated microbubbles, for use in molecular imaging of angiogenesis, for example.

[0218] This disclosure further provides the antibodies described above in a detectably labeled form. The antibodies can be conjugated with a detectable label. In some embodiments, the payload is a label, e.g., a radioisotope, an affinity label (e.g., biotin, avidin), an enzyme label (e.g., horseradish peroxidase, alkaline phosphatase), a fluorescent, luminescent, or bioluminescent label (e.g., FITC or rhodamine), a paramagnetic atom, etc. In some embodiments, the payload includes an enzyme, a fluorescent marker, a chemiluminescent marker, a bioluminescent substance, or a radioactive substance. Procedures for achieving such labeling are well known in the art. For example, see (Sternberger, LA et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, EA et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, JWJ Immunol. Meth. 13:215 (1976)).

[0219] The detectable label itself may be detectable by itself (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze the chemical denaturation of a detectable substrate compound or composition. Alternatively, the label may not be detectable by itself but may be an element conjugated by another detectable agent (e.g., an epitope tag, or one of a binding partner pair, e.g., biotin-avidin). Thus, an antibody may contain a label or tag that facilitates its isolation, and the present invention's method for identifying an antibody includes the step of isolating the antigen / antibody by interaction with the label or tag.

[0220] In some embodiments, the payload includes a chemotherapeutic agent, a toxin (e.g., an enzyme-active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radiocomplex).

[0221] In some embodiments, the payload is a therapeutic agent such as a chemotherapeutic cytotoxin, for example, a cell proliferation inhibitor or cytotoxic agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthracine dione). Anthracindione), mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepachlorambucil) This includes chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mitramycin, and anthramycin (AMC), as well as antimitotic substances, thrombotic or anti-angiogenic agents, or radiolabels. Examples of cytotoxic and chemotherapeutic agents suitable for forming immunoconjugates are known in the art; see, for example, WO05 / 103081).

[0222] In some embodiments, the payload includes toxins (e.g., enzyme-active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), small molecules, siRNA, nanoparticles, targeting agents (e.g., microbubbles), or radioisotopes (i.e., radioconjugates). Antibody conjugates can be used, for example, in diagnostic, theranostic, or targeting methods. Various radioisotopes are available for use as payloads with the antibodies of this disclosure. Examples include, but are not limited to, 212Bi, 131I, 131In, 90Y, and 186Re.

[0223] Linker In some embodiments, the linkers described herein are either detachable or non-detachable linkers. In some examples, the linker is a detachable linker. In other examples, the linker is a non-detachable linker.

[0224] In some embodiments, the linker is a nonpolymer linker. A nonpolymer linker refers to a linker that does not contain repeating units of monomers produced by the polymerization process. Exemplary nonpolymer linkers include, but are not limited to, C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-destructing linkers, maleimide linkers, or combinations thereof. In some cases, a nonpolymer linker includes C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-destructing linkers, maleimide linkers, or combinations thereof. In further cases, a nonpolymer linker does not contain more than two linkers of the same type, e.g., more than two homobifunctional crosslinkers or more than two peptide linkers. In further cases, the nonpolymer linker optionally includes one or more reactive functional groups.

[0225] In some embodiments, the non-polymer linker does not include the polymers described above. In some cases, the non-polymer linker does not include the polymer encompassed by polymer portion C. In some embodiments, the non-polymer linker does not include polyalkylene oxides (e.g., PEG). In some embodiments, the non-polymer linker does not include PEG.

[0226] In some embodiments, the linker includes a homobifunctional linker. Exemplary homobifunctional linkers include Lomant reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionic acid (DTSSP)), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycol bis( Succinimidyl succinate (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipinimide (DMA), dimethylpimerimidate (DMP), dimethylsperimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamide)butane This includes, but is not limited to, DPDPB, bismaleimide hexane (BMH), aryl halide-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0227] In some embodiments, the linker includes a heterobifunctional linker. Exemplary heterobifunctional linkers are amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionic acid (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionic acid (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionic acid (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl- α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimid ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimid ester (sulfo-MBs), N-succinimidyl (4-Yo Iodoacteyl aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacte) aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl) butylate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl) butylate (sulfo-sMPB), N-(γ-maleimidobutyloxy) succinimide ester (GMBs), N-(γ-maleimidobutyryloxy) sulfosuccinimidide ester (sulfo-GMBs), Succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), etc.Carbonyl-reactive and sulfhydryl-reactive crosslinkers, such as 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH); and amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA) and N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA). Sulfosuccinimidyl-(4-azidosalicylamide)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamide)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6 -(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl Sulfhydryl-reactive and photoreactive crosslinkers such as imidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyldiazopirubate (ρNPDP), and ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), for example,This includes, but is not limited to, 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive crosslinkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4-(ρ-azidosalicylamide)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers such as ρ-azidophenylglyoxal (APG).

[0228] In some embodiments, the linker contains a reactive functional group. In some cases, the reactive functional group contains a nucleophile that is reactive to an electrophile present in the bonding portion. Exemplary electrophiles include carbonyl groups, e.g., aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophiles include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0229] In some embodiments, the linker comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer. In some cases, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, for example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as described above.

[0230] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some cases, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide, thereby providing an intramolecular catalyst for thiosuccinimide ring hydrolysis, preventing the maleimide from being eliminated via a reverse Michael reaction. In some cases, the self-stabilizing maleimide is the maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some cases, the linker contains a self-stabilizing maleimide. In some cases, the linker is a self-stabilizing maleimide.

[0231] In some embodiments, the linker includes a peptide moiety. In some cases, the peptide moiety includes at least 2, 3, 4, 5, or 6 or more amino acid residues. In some cases, the peptide moiety includes at most 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some cases, the peptide moiety includes about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some cases, the peptide moiety is cleavable (e.g., enzymatically or chemically). In some cases, the peptide moiety is incleavable. In some cases, the peptide portion may include Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker contains peptide moieties, such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker contains Val-Cit. In some cases, the linker is Val-Cit.

[0232] In some embodiments, the linker includes a benzoic acid group or a derivative thereof. In some cases, the benzoic acid group or its derivative includes para-aminobenzoic acid (PABA). In some cases, the benzoic acid group or its derivative includes gamma-aminobutyric acid (GABA).

[0233] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some cases, the maleimide group is maleimidocaproyl (mc). In some cases, the peptide group is val-cit. In some cases, the benzoic acid group is PABA. In some cases, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-Cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0234] In some embodiments, the linker is a self-destructing linker or a self-destructing linker. In some cases, the linker is a self-destructing linker. In other cases, the linker is a self-destructing linker (e.g., a cyclized self-destructing linker). In some cases, the linker includes a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0235] In some embodiments, the linker is a dendritic linker. In some cases, the dendritic linker includes a branched, polyfunctional linker moiety. In some cases, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some cases, the dendritic linker includes a PAMAM dendrimer.

[0236] In some embodiments, the linker is a traceless linker or linker that leaves no linker portion (e.g., an atom or linker group) on the binding portion A, polynucleotide B, polymer C, or endosomal soluble portion D after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15):2493-2497 (2013). In some cases, the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102:2607-2024 (2002). In some cases, the linker is a traceless linker, as described in U.S. Patent No. 6,821,783.

[0237] In some cases, the linker is the linker described in U.S. Patent Nos. 6,884,869, 7,498,298, 8,288,352, 8,609,105, or 8,697,688; U.S. Patent Publications 2014 / 0127239, 2013 / 028919, 2014 / 286970, 2013 / 0309256, 2015 / 037360, or 2014 / 0294851; or PCT Publications WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0238] In some embodiments, X 1 and X 2 Each is an independent bond or a non-polymer linker. In some cases, X 1 and X2 These are independent connections. In some cases, X 1 and X 2 These are each independent non-polymer linkers.

[0239] In some cases, X 1 It is a bond or a non-polymer linker. In some cases, X 1 It is a combination. In some cases, X 1 It is a non-polymer linker. In some cases, the linker is a C1-C6 alkyl group. In some cases, X 1 These are C1-C6 alkyl groups, such as C5, C4, C3, C2, or C1 alkyl groups. In some cases, the C1-C6 alkyl groups are unsubstituted C1-C6 alkyl groups. Regarding linkers, in particular X 1 When used in relation to, alkyl refers to a saturated linear or branched hydrocarbon radical containing up to six carbon atoms. In some cases, X 1 This includes the homobifunctional or heterobifunctional linkers described above. In some cases, X 1 This includes heterobifunctional linkers. In some cases, X 1 This includes sMCC. In other examples, X 1 It contains a heterobifunctional linker that is optionally conjugated to a C1-C6 alkyl group. In other examples, X 1 It contains sMCCs optionally conjugated to C1-C6 alkyl groups. In further cases, X 1 This does not include the homobifunctional or heterobifunctional linkers mentioned above.

[0240] In some cases, X 2 is a linker or connector. In some cases, X 2 It is a combination. In other cases, X 2 is a linker. In further cases, X 2 X is a non-polymer linker. In some embodiments, X 2 These are C1-C6 alkyl groups. In some cases, X 2This is the homobifunctional or heterobifunctional linker described above. In some cases, X 2 This is the homobifunctional linker mentioned above. In some cases, X 2 This is the heterobifunctional linker described above. In some cases, X 2 This includes maleimide groups such as maleimidocaproyl (mc), or the self-stabilizing maleimide groups mentioned above. In some cases, X 2 It contains peptide portions such as Val-Cit. In some cases, X 2 It contains benzoic acid groups such as PABA. In further cases, X 2 This comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further cases, X 2 It contains an mc group. In further cases, X 2 It contains an mc-val-cit group. In further cases, X 2 It contains a val-cit-PABA group. In further cases, X 2 It contains an mc-val-cit-PABA group.

[0241] Pharmaceutical compositions and drugs In one embodiment, this specification provides a pharmaceutical composition comprising the antibody of this disclosure. The antibody or composition described herein can be used not only for the treatment of disease, prevention of disease, screening of disease, detection of the presence or severity of disease, provision of prognosis for disease, monitoring of disease progression or recurrence, but also for assessment of the therapeutic effectiveness and / or recurrence of disease, disorder or condition, as well as for the selection of therapies and / or treatments for disease, optimization of a given therapy for disease, monitoring of disease treatment, and / or prediction of the suitability of therapy for a specific patient or subpopulation, or for determining the appropriate administration of therapeutic agents in a patient or subpopulation. In some embodiments, the disease is cancer.

[0242] For clinical use of the methods described herein, the administration of the antibodies of this disclosure may include formulation into pharmaceutical compositions or pharmaceutical preparations, or drugs for administration via subcutaneous, intravenous, intradermal, intraperitoneal, oral, intramuscular, intracranial, or other routes of administration. In some embodiments, the antibodies described herein may be administered with any pharmaceutically acceptable carrier, excipient, or diluent to constitute an effective treatment for a target. Accordingly, in one embodiment, this disclosure provides a pharmaceutical composition comprising one or more antibodies described herein in combination with one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0243] The term "pharmaceutically acceptable" means a compound, material, composition and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of safe medical judgment, and commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, e.g., liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids (e.g., lubricants, magnesium talc, calcium, or zinc stearate, or steric acid), or solvent encapsulating materials that are involved in maintaining the stability, solubility, or activity of the antibody or antigen-binding fragment of this disclosure. Examples include, but are not limited to, any of several standard pharmaceutical carriers, such as sterile phosphate-buffered saline or bacteriostatic water. Various water-soluble carriers, such as water, buffer water, 0.4% saline, and 0.3% glycine, may be used, as may other proteins, such as albumin, lipoproteins, and globulins, that have undergone minor chemical modifications to enhance stability. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and must not be harmful to the patient. The terms "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. The compositions of this disclosure may further include one or more pharmaceutically acceptable carriers, excipients, and other agents incorporated into the formulation to provide improved mobility, delivery, tolerance, etc. (collectively referred to herein as "pharmaceutically acceptable carriers or diluents"). Numerous suitable formulations can be found in the prescription collection known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) - containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil - in - water and water - in - oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi - solid gels, and semi - solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA, 1998, J Pharm Sci Technol 52:238 - 311.

[0244] Acceptable carriers, excipients, or stabilizers are non - toxic to the recipient at the dosages and concentrations employed, and include buffering agents such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 - pentanol; and m - cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt - forming counterions such as sodium; metal complexes (e.g., Zn - protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0245] Although arbitrary, the pharmaceutical formulation containing the composition described in this specification contains a pharmaceutically acceptable salt, usually, for example, sodium chloride, preferably at about physiological concentration. Optionally, the pharmaceutical formulation of the present invention can contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1 to 2.0 (typically v / v)%. Suitable preservatives include those known in the pharmaceutical field. Benzyl alcohol, phenol, m-cresol, methyl paraben, and propyl paraben are examples of preservatives. Optionally, the pharmaceutical formulation of the present invention can contain a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

[0246] The composition described in this specification can be specially formulated for administration of an antibody or its antigen-binding fragment to a subject in solid, liquid or gel form, and the administration can be by the following: (1) parenteral administration, for example, sterile aqueous solution or suspension, or as a sustained-release formulation, for example, by subcutaneous, intramuscular, intravenous, or epidural injection; (2) topical application, for example, as a cream, ointment, or controlled-release patch or spray applied to the skin; (3) intravaginal or intrarectal, for example, as a pessary, cream, or foam; (4) ophthalmic administration; (5) transdermal administration; (6) transmucosal administration; or (7) nasal administration. Further, the antibody or its antigen-binding fragment, or composition of the present disclosure can be implanted in a patient or injected using a drug delivery system. See, for example, Urquhart et al., 24 Ann.Rev.Pharmacol.Toxicol. 199 (1984); Controlled Release of Pesticides&Pharmaceuticals (Lewis, ed., Plenum Press, New York, 1981); U.S. Patent Nos. 3,773,919 and 3,270,960.

[0247] The compositions disclosed herein, including the antibodies described herein, may also contain two or more active compounds necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, the compositions may further contain therapeutic agents, anticancer agents, cytotoxic agents, cytokines, proliferation inhibitors, and / or angiogenesis inhibitors such as VEGFR antagonists. Such molecules are preferably present in combination in amounts effective for the intended purpose. The active components of the antibody-containing compositions described herein may also be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, fine particles, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th ed, Osol, A.Ed. 1980). Furthermore, pharmaceutical compositions can be delivered in vesicles, particularly liposomes (see Langer 1990 Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327, generally ibid.). Liposomes, comprising emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers, etc., can function as vehicles for targeting M-CSF antibodies to specific tissues and for extending the half-life of compositions. Various methods for preparing liposomes are available, for example, as described in U.S. Patents 4,837,028 and 5,019,369, which are incorporated herein by reference.

[0248] In some embodiments, the antibodies of the present invention can be conjugated to liposomes via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem. 257:286-288 (1982).

[0249] In some embodiments, sustained-release formulations can be used. A preferred example of a sustained-release formulation is a semipermeable matrix of a solid hydrophobic polymer containing the antibody or antigen-binding fragment of the Disclosure, the matrix being in the form of a molded article, such as a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-glutamic acid and ethyl-L-glutamic acid copolymers, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT® (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow for molecular release over a period of 100 days, while certain hydrogels release proteins over a shorter period. When encapsulated antibodies remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, potentially leading to loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable measures for stabilization can be taken. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain situations, pharmaceutical compositions can be delivered using a release control system. In one embodiment, a pump may be used (see Langer, supra; Sefton 1987 CRC Crit.Ref.Biomed.Eng.14:201). In another embodiment, a polymer material may be used.In another embodiment, the release control system may be positioned close to the composition target and therefore may require only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, op. cit., vol. 2, pp. 115-138).

[0250] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously, for example, using standard needles and syringes. Furthermore, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable when delivering the pharmaceutical compositions of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. Disposable pen-type delivery devices do not have replaceable cartridges. Rather, disposable pen-type delivery devices are designed to retain the pharmaceutical composition by pre-filling a reservoir within the device. Once the reservoir is empty of pharmaceutical composition, the entire device is discarded. Numerous reusable pen-type and auto-injector delivery devices are applicable to the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, to name just a few, AUTOPEN (trademark) (Owen Mumford, Inc., Woodstock, UK), DISETRONIC (trademark) pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX75 / 25 (trademark) pen, HUMALOG (trademark) pen, HUMALIN70130 (trademark) pen (Eli Lilly and Co., Indianapolis, India), NOVOPEN (trademark) I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (trademark) (Novo Nordisk, Copenhagen, Denmark), BD (trademark) pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (trademark), OPTIPEN PRO (trademark), and OPTIPEN. This includes, but is not limited to, STARLET (trademark) and OPTICLIK (trademark) (Sanofi-Aventis, Frankfurt, Germany).Examples of disposable pen-type delivery devices for subcutaneous delivery of pharmaceutical compositions include, but are not limited to, the SOLOSTAR® pen (Sanofi-Aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (Eli Lilly).

[0251] Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, as well as intravenous infusion. These injectable formulations may be prepared by known methods. For example, an injectable formulation may be prepared by dissolving, suspending, or emulsifying the antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injection. As aqueous media for injection, there are isotonic solutions containing, for example, physiological saline, glucose, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As oily media, for example, sesame oil, soybean oil, etc., may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injectable drugs thus prepared are preferably filled into suitable ampoules.

[0252] The compositions of this disclosure may be in the form of, for example, granules, powders, tablets, capsules, syrups, suppositories, injections, emulsions, elixirs, suspensions, or solutions. The amount of the aforementioned antibody contained may be about 5 to about 500 mg per dosage form in a unit dose. Particularly in the form of injection, it is preferable that the aforementioned antibody is contained in about 5 to about 100 mg, and in other dosage forms in about 10 to about 250 mg.

[0253] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive, such as starch or other additives. Suitable additives include sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginic acid, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Optionally, oral dosage forms may contain other ingredients to aid administration, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens or sorbic acid, antioxidants such as ascorbic acid, tocopherol or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or fragrances. Tablets and pills may further be treated with suitable coating materials known in the art.

[0254] Liquid dosage forms for oral administration may be in the form of solutions that may contain pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and inert diluents such as water. In some embodiments, the pharmaceutical formulation may be prepared as a liquid suspension or aqueous solution using sterile liquids, such as, but not limited to, oils, water, alcohols, and combinations thereof. In some embodiments, the pharmaceutical composition may be prepared in lyophilized form. Lyophilized formulations may contain ice-crystal inhibitors known in the art. As used herein, the term “ice-crystal inhibitor” generally includes agents that provide stability to proteins from freeze-induced stress. Examples of ice-crystal inhibitors include polyols, such as mannitol, as well as saccharides, such as sucrose, and surfactants, such as polysorbates, poloxamers, or polyethylene glycol. Ice-crystal inhibitors also contribute to the tonicity of the formulation. Pharmaceutically suitable surfactants, suspending agents, and emulsifiers may be added for oral or parenteral administration.

[0255] As described above, the suspension may contain oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. The suspension formulation may also contain fatty acid esters, such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. The suspension formulation may contain, but is not limited to, alcohols, such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. The suspension formulation may also contain, but is not limited to, ethers such as poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water.

[0256] For intranasal administration, the pharmaceutical formulation and drug may be a spray or aerosol containing a suitable solvent(s) and, optionally, other compounds such as stabilizers, antibacterial agents, antioxidants, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof. The propellant for aerosol formulations may include compressed air, nitrogen, carbon dioxide, or a low-boiling point hydrocarbon solvent.

[0257] Injectable dosage forms generally include aqueous or oily suspensions that can be prepared using appropriate dispersants or wetting agents and suspending agents. The injectable form may be in the form of a solution phase or a suspension prepared with a solvent or diluent. Acceptable solvents or vehicles include sterile water, Ringer's solution, or isotonic saline solution. Alternatively, sterile oil may be used as a solvent or suspension. Preferably, the oil or fatty acid is non-volatile and includes natural or synthetic oils, fatty acids, monoglycerides, diglycerides, or triglycerides.

[0258] In the case of injection, the pharmaceutical formulation and / or drug may be a powder suitable for reconstitution with appropriate solutions as described above. Examples of these include, but are not limited to, lyophilized, tumble-dried, or spray-dried powders, amorphous powders, granules, precipitates, or fine particles. In the case of injection, the formulation may optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0259] For rectal administration, pharmaceutical formulations and drugs may be in the form of suppositories, ointments, enemas, tablets, or creams for releasing the compound in the intestine, sigmoid flexure, and / or rectum. Rectal suppositories are prepared by mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or tautomers thereof, with an acceptable vehicle, such as cocoa butter or polyethylene glycol, which exists as a solid phase at normal storage temperatures and as a liquid phase at temperatures suitable for drug release in the body, such as the rectum. Oils may also be used in the preparation of soft gelatin and suppository formulations. Water, saline, aqueous dextrose and related sugar solutions, and glycerol may be used in the preparation of suspension formulations that may contain not only suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylcellulose, or carboxymethylcellulose, but also buffers and preservatives.

[0260] The concentrations of antibodies or their antigen-binding fragments in these compositions can vary considerably, i.e., from less than about 10% by weight, typically at least about 25% by weight, to as high as 75% or 90% by weight, and will be selected primarily by fluid volume, viscosity, etc., according to the chosen specific mode of administration. Practical methods for preparing compositions for oral, topical, and parenteral administration are known or obvious to those skilled in the art and are described in detail, for example, in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co., Easton, Pa. (1995), which is incorporated herein by reference.

[0261] Another embodiment of the present invention provides a product containing a substance useful for treating the diseases, disorders, or conditions described above, including for the treatment of cancer. The product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective in treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). The active agent in the composition is the antibody of the present invention. A label on or associated with the container indicates that the composition is used to treat a selected condition. The product may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and glucose solution. The product may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. The pharmaceutical compositions and drugs described herein are useful for the treatment of cancerous diseases.

[0262] Production of antibodies or their conjugated fragments In some embodiments, the polypeptides described herein (e.g., antibodies and their conjugated fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), particularly by chemical synthesis or recombinant expression, and preferably by recombinant expression techniques.

[0263] In some cases, the antibody or its binding fragment is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., Kutmeier et al., 1994, BioTechniques 17:242), and is involved in the duplication synthesis of oligonucleotides containing a portion of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0264] Alternatively, the nucleic acid molecule encoding the antibody may be optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulin) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0265] In some cases, the antibody or its binding may be optionally produced by immunizing animals such as rabbits to generate polyclonal antibodies, or more preferably by producing monoclonal antibodies as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), or Kozbor et al. (1983, Immunology Today 4:72), or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of an antibody can be optionally obtained by a Fab expression library of clones of Fab fragments that bind a specific antigen (e.g., Huse et al., 1989, Science 246:1275-1281), or by screening an antibody library (e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0266] In some embodiments, techniques developed for the production of "chimeric antibodies" by splicing genes derived from mouse antibody molecules with appropriate antigen specificity together with genes derived from human antibody molecules with appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as having variable regions derived from mouse monoclonal antibodies and human immunoglobulin constant regions, like humanized antibodies.

[0267] In some embodiments, the techniques described for the production of single-chain antibodies (U.S. Patent No. 4,694,778, Bird, 1988, Science 242:423-42, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, and Ward et al., 1989, Nature 334:544-54) are adapted to produce single-chain antibodies. A single-chain antibody becomes a single-chain polypeptide when the heavy and light chain fragments of the Fv region are linked via an amino acid bridge. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0268] In some embodiments, an expression vector containing the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and then the introduced cells are cultured by conventional techniques to produce an antibody. In specific embodiments, the expression of the antibody is controlled by a constitutive, inducible, or tissue-specific promoter.

[0269] In some embodiments, various host expression vector systems are used to express the antibodies or their conjugated fragments described herein. Such host expression systems represent vehicles that are purified after the antibody coding sequence is generated, but also represent cells that express the antibody or its conjugated fragment in situ when transformed or transfected with a suitable nucleotide coding sequence. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibodies or their binding fragment coding sequences (e.g., E. coli and B. subtilis); yeast transformed with recombinant yeast expression vectors containing antibodies or their binding fragment coding sequences (e.g., Saccharomyces, Pichia); insect cell lines infected with recombinant virus expression vectors containing antibodies or their binding fragment coding sequences (e.g., baculovirus); plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors containing antibodies or their binding fragment coding sequences (e.g., Ti plasmid); or mammalian cell lines possessing recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian virus-derived promoters (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) (e.g., COS, CHO, BH, 293, 293T, 3T3 cells).

[0270] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express antibodies are manipulated by arbitrary selection. Instead of using expression vectors containing viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers. Following the introduction of the exogenous DNA, the manipulated cells are allowed to grow in enriched medium for 1-2 days, and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate the plasmid into their chromosomes and grow to form lesions, which are then cloned and expanded into cell lines. This method can be advantageously used to manipulate cell lines that express antibodies or their binding fragments.

[0271] In some cases, but not limited to, numerous selection systems including the genes for herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) are used for TK cells, HGPRT cells, or APRT cells, respectively. Furthermore, antimetabolite resistance is used as the basis for selecting the following genes: dhfr (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527) which confers resistance to methotrexate; gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072) which confers resistance to mycophenolic acid; and neo (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro (Santerre et al., 1984, Gene 30:147), which confers resistance to hygromycin.Commonly known recombinant DNA techniques that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J.Mol.Biol.150:1).

[0272] In some cases, antibody expression levels can be increased by vector amplification (see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987) for an overview). If a marker within the antibody-expressing vector system is amplified, the copy number of the marker gene increases due to an increase in the level of the inhibitor present in the host cell culture. Since the amplified region is associated with the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0273] In some cases, any method known in the art for the purification or analysis of antibodies or antibody complexes may be used, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity of specific antigens after protein A, and column sizing chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and high-performance protein liquid chromatography.

[0274] Method of Disclosure Certain aspects of this disclosure relate to methods for producing and / or using antibodies disclosed herein.

[0275] Certain aspects of this disclosure relate to methods for shrinking, reducing, depleting, or killing cells expressing CCR8 (e.g., Treg cells, e.g., tumor-infiltrating Tregs), which include administering the antibodies or compositions disclosed herein to a subject, or contacting a population of cells expressing CCR8 (e.g., Treg cells, e.g., tumor-infiltrating Tregs) with the antibodies or compositions disclosed herein. In one aspect, this specification provides a method for enhancing an immune response in a subject, which includes administering the antibodies or compositions disclosed herein to a subject. The terms “induce an immune response” and “enhance an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., passive or adaptive) to a particular antigen (e.g., cancer antigen). The term “induce” as used in relation to the induction of CDC or ADCC refers to the stimulation of a particular direct cell-killing mechanism. In some embodiments, enhancement is relative to a reference level. In some embodiments, enhancement is at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more relative to the reference level. In some embodiments, enhancement is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more relative to the reference level. In some embodiments, the immune response is an anti-cancer immune response. As used herein, the term “anti-cancer immune response” refers to an immune response induced by the presence of a tumor, cancer cells, or cancer antigen. In some embodiments, this response includes the proliferation of cancer antigen-specific lymphocytes. In some embodiments, this response includes the expression and upregulation of antibodies and T cell receptors, as well as the formation and release of lymphokines, chemokines, and cytokines. Both the innate and adaptive immune systems interact to initiate an antigenic response to tumors, cancer cells, or cancer antigens.In some embodiments, the immune response (e.g., an anti-cancer immune response) is a T-cell response. As used herein, the term “T-cell response” refers to any response mediated by T cells, including but not limited to effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). A T-cell response includes, for example, cytotoxicity and proliferation of T cells. In some embodiments, the immune response (e.g., an anti-cancer immune response) is a cytotoxic T lymphocyte (CTL) response. As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ T cells.

[0276] In one embodiment, the Specified herein provides a method for diagnosing or treating a condition associated with an antigen disclosed herein (e.g., CCR8). In some embodiments, the CCR8-associated condition results from increased expression or bioactivity of CCR8. In some embodiments, the CCR8-associated condition results from decreased expression or bioactivity of CCR8. In some embodiments, the method of the Disclosure comprises administering an effective amount of the antibody or composition of the Disclosure to a subject and / or contacting a population of cells (e.g., cells expressing CCR8, e.g., Treg cells) with an effective amount of the antibody or composition of the Disclosure. In some embodiments, the results of these administrations and / or contacts include (a) binding of antibodies specific to CCR8, (b) binding of antibodies specific to cells expressing CCR8 (e.g., Treg or cancer cells), (c) decreased expression of CCR8, (d) decreased biological activity of CCR8, (e) decreased CCR8-mediated signaling, (d) decreased or inhibited binding of CCR8 to CCL1 polypeptide, (e) induction of cytolysis of cells expressing CCR8 (e.g., cytolysis of Treg cells and / or cancer cells) (e.g., ADCC), (f) decreased levels of cells expressing CCR8 (e.g., Treg cells or cancer cells), (g) decreased biological activity of Treg cells, (h) induction of internal translocation of CCR8 in cells expressing CCR8 (e.g., internal translocation of CCR8 in Treg cells and / or cancer cells), (i) enhancement of the anti-cancer immune response, or (j) a combination thereof.

[0277] In some embodiments, the antibodies disclosed herein have the ability to (a) bind antibodies specifically to CCR8, (b) bind antibodies specifically to cells expressing CCR8 (e.g., Treg or cancer cells), (c) reduce CCR8 expression, (d) reduce the biological activity of CCR8, (e) reduce CCR8-mediated signaling, (d) reduce or inhibit the binding of CCR8 to CCL1 polypeptide, (e) induce cytolysis of CCR8-expressing cells (e.g., cytolysis of Treg cells and / or cancer cells) (e.g., ADCC), (f) reduce the level of CCR8-expressing cells (e.g., Treg cells or cancer cells), (g) reduce the biological activity of Treg cells, (h) induce internal translocation of CCR8 in CCR8-expressing cells (e.g., internal translocation of CCR8 in Treg cells and / or cancer cells), (i) enhance the anti-cancer immune response, or (j) a combination thereof.

[0278] In some embodiments, the reduction is relative to a reference level. In some embodiments, the reduction is at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more relative to the reference level. In some embodiments, the reduction is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more relative to the reference level. In some embodiments, the reference level is the level in the absence of the antibody disclosed herein. In some embodiments, the reference level is the level before administration of the antibody, contact with the antibody, and / or treatment with the antibody disclosed herein. In some embodiments, the reference level is the level in the absence of administration of the antibody, contact with the antibody, and / or treatment with the antibody disclosed herein.

[0279] As used herein, “expression” may mean transcription to a polynucleotide encoding CCR8, translation to a CCR8 polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide encoding CCR8, a translated polypeptide CCR8, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, regardless of whether they originate from a transcript produced by alternative splicing, a degraded transcript, or post-translational processing of a polypeptide by, for example, proteolysis.

[0280] "Decreased expression," "reduced expression level," "reduced level," or "decreased expression" can refer to a decrease in the level of the transcribed polynucleotide encoding CCR8 (e.g., the mRNA encoding CCR8) and / or the CCR8 polypeptide, or even further, the polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of the polypeptide). In some embodiments, decreased expression means little to no expression. Expression levels can be measured by methods known in the art, such as RT-PCR, Western blotting, and FACS.

[0281] In some embodiments, the antibodies disclosed herein reduce the biological activity of CCR8 and / or downstream pathways, i.e., CCR8-mediated signaling or other CCR8-mediated functions. In some embodiments, the antibody or its antigen-binding fragment can block, weaken, suppress, inhibit, or reduce the biological activity of CCR8. In some embodiments, this biological activity is the binding of CCR8 to a ligand (e.g., chemokine (CC motif) ligand, activation of G protein signaling, CCR-mediated signaling, or induction of a cellular response (e.g., immunosuppression).

[0282] In some embodiments, the antibodies disclosed herein reduce the activation of G protein signaling, including the binding of CCR8 to a ligand (e.g., CCL1), downstream pathways mediated by CCR8 signaling or function, such as the binding of receptors to CCR8 or its metabolites and / or the induction of cellular responses (e.g., immunosuppression). In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of human CCR8 and / or the binding of CCR8 to a ligand (e.g., CCL1). In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of human CCR8 and / or the interaction between CCR8 and G proteins. In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of CCR8 to CCL1. In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of CCR8 to CCL8. In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of CCR8 to CCL16. In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of CCR8 to CCL8. In some embodiments, the antibodies disclosed herein prevent, block, inhibit, or reduce the binding of CCR8 to CCL18.

[0283] In one aspect of this disclosure, the antibodies disclosed herein can initiate a potent immune response against cells and / or tumor cells expressing CCR8 by direct cytotoxicity. In this regard, the antibodies disclosed herein may induce cytolysis by either a complement-mediated mechanism or an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism, both of which require an intact Fc portion of an immunoglobulin molecule for interaction with effector cell Fc receptor sites or complement proteins. Furthermore, antibodies that exert a direct biological effect on tumor growth are useful in the implementation of this disclosure. Potential mechanisms by which such direct cytotoxic antibodies may act include inhibition of cell proliferation, regulation of cell differentiation, regulation of tumor angiogenic factor profiles, and induction of apoptosis. The mechanisms by which specific antibodies disclosed herein exert their cytotoxic effects can be evaluated using any number of in vitro assays designed to determine ADCC, ADMMC, complement-mediated cytotoxicity, etc., as is commonly known in the art.

[0284] This disclosure provides a method for treating or preventing any disease or disorder characterized by neoplasms, tumors, metastases, or uncontrolled cell proliferation, by administering an effective dose of the antibodies disclosed herein to a patient.

[0285] In some embodiments, cancer may be carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, or melanoma. In some embodiments, cancer may originate from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, testes, tongue, or uterus. In some embodiments, cancer may be neoplasm, malignant cancer, carcinoma, undifferentiated, giant and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pyromatrix carcinoma, transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma; gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma), funicular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyps, adenocarcinoma, familial adenomatous polyposis, solid carcinoma, carcinoid tumor, bronchioloalveolar adenocarcinoma, papillary chromophobe carcinoma, eosinophilic carcinoma, eosinophilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granulocytoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, non-encapsulated sclerosing carcinoma, adrenocortical carcinoma, endometrioid carcinoma, cutaneous adnexal carcinoma, Apoc Lymph gland carcinoma, sebaceous gland carcinoma, ceruminous gland carcinoma, mucoepidermoid carcinoma, cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, invasive ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease, acinar cell carcinoma of the mammary gland, adenosquamous cell carcinoma, adenocarcinoma with squamous metaplasia, thymoma, ovarian stromal tumor, theca cell tumor, granulosa cell tumor, androblastoma, Sertoli cell tumor, Leydig Cell tumor, lipid cell tumor, paraganglioma, extramammary paraganglioma, pheochromocytoma, glomus angiosarcoma, melanoma, lentigo malignant, melanoma derived from lentigo malignant, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, fibrous melanoma, cutaneous melanoma, apigmented melanoma, superficial spreading melanoma, melanoma in giant pigmented nevi, epithelioid cells Melanoma, blue nevus, sarcoma, fibrosarcoma, fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, mixed tumor, Müllerian mixed tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, mesenchymal tumor, Brenner tumor, phyllodes tumor, synovial sarcoma, mesothelioma, undifferentiated germ cell tumor, embryonal carcinoma, teratoma, ovarian goiter, choriocarcinoma, mesonephroma, angiosarcoma, hemangioendothelioma,Kaposi's sarcoma, hemangiopericytoma, lymphangiosarcoma, osteosarcoma, paraosteal osteosarcoma, chondrosarcoma, chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, odontogenic tumor, ameloblastic gingivoma, ameloblastoma, ameloblastofibrosarcoma, pineal glandoma, chordomatous glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendrocyte, primitive neuroectodermal tumor, cerebellar sarcoma, ganglioblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, meningioma, neurofibrosarcoma, schwannoma, granulocyte tumor, malignant Lymphoma, Hodgkin's disease, Hodgkin's, paragranuloma, lymphoma, microlymphocytic, malignant lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mycosis fungoides, other certain non-Hodgkin lymphomas, histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative bowel disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myelosarcoma, or hairy cell leukemia. In some embodiments, the cancer is skin cancer. In some embodiments, skin cancer is basal cell carcinoma, squamous cell carcinoma, cutaneous melanoma, Merkel cell carcinoma, atypical fibroxanthoma, cutaneous lymphoma, or cutaneous fibrosarcoma. In some embodiments, cutaneous melanoma is superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, subungual melanoma, lentigo malignant melanoma, fibroplastic melanoma, mucosal melanoma, or polypoid melanoma.

[0286] The antibodies disclosed herein may be administered to a subject on their own or in the form of pharmaceutical compositions disclosed herein for the treatment or prevention of a disease, such as cancer. In some embodiments, the antibodies or compositions described herein may be administered alone or in combination with a second therapeutic agent or therapy useful for the treatment of cancer. Examples of second therapies useful for the treatment of cancer include, but are not limited to, radiotherapy, cryotherapy, antibody therapy, chemotherapy, photodynamic therapy, surgery, hormone therapy, immunotherapy, cytokine therapy, or combination therapy with conventional drugs. In some embodiments, the second therapeutic agent may be cytotoxic drugs, tumor vaccines, peptides, peptide bodies, small molecules, cytotoxic drugs, cell division inhibitors, immunomodulators, interferons, interleukins, immunostimulant growth hormones, cytokines, vitamins, minerals, aromatase inhibitors, RNAi, histone deacetylase inhibitors, proteasome inhibitors, cancer chemotherapy agents, Treg targeting agents, other antibodies, immunostimulant antibodies, NSAIDs, corticosteroids, antioxidants, nutritional supplements, cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol. In some embodiments, the second therapeutic agent is a chemotherapeutic agent selected from the group consisting of platinum compounds, antibodies with anticancer activity, anthracyclines, anthracendions, alkylating agents, antimetabolites, mitotic inhibitors, taxanes, taxoids, microtubule inhibitors, vinca alkaloids, folic acid antagonists, topoisomerase inhibitors, antiestrogens, antiandrogens, aromatase inhibitors, GnRh analogs, and 5α-reductase inhibitors and bisphosphonates.

[0287] In some embodiments, the second therapeutic agent may be a PD-1 inhibitor, a histone deacetylase (HDAC) inhibitor, a proteasome inhibitor, an mTOR pathway inhibitor, a JAK2 inhibitor, a tyrosine kinase inhibitor (TKI), a PI3K inhibitor, a protein kinase inhibitor, a serine / threonine kinase inhibitor, an inhibitor of intracellular signaling, an inhibitor of Ras / Raf signaling, a MEK inhibitor, an AKT inhibitor, an inhibitor of survival signaling proteins, a cyclin-dependent kinase inhibitor, a therapeutic monoclonal antibody, a TRAIL pathway agonist, an anti-angiogenic agent, a metalloproteinase inhibitor, a cathepsin inhibitor, an inhibitor of urokinase-type plasminogen activator receptor function, an immune complex, an antibody-drug conjugate, an antibody fragment bispecific antibody, or a bispecific T cell engager (BiTE). In some embodiments, other antibodies include cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, rituximab, ofatumumab, bertuzumab, alemtuzumab, rabetuzumab, adecatumumab, olegobomab, onarutuzumab; apomab, mapatuzumab, lexatumumab, conatumumab, tigatuzumab, catumakisomab, blinatumomab, ibritumomab triuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, and clivatuzumab tetraxetan. The following are selected from tetraxetan, pemtumomab, trastuzumab emtansine, bevacizumab, etaracizumab, boroximab, ramucirumab, and aflibercept. In yet another embodiment, the second therapeutic agent may be an antibody currently used to treat cancer. Examples of such antibodies include, but are not limited to, Herceptin®, Retuxan®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath I / H, Smart MI95, LymphoCide, Smart I D10, and Oncolym.In some embodiments, the antibody is selected from antagonist antibodies targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and / or agonist antibodies targeting one or more of CD40, CD137, OX40, GITR, CD27, CD28, ICOS, or a combination thereof. In some embodiments, the second therapeutic agent targeting immunosuppressive cells Treg and / or MDSCs is a mitotic inhibitor, cyclophosphamide, gemcitabine, mitoxantrone, fludarabine, thalidomide, thalidomide derivatives, COX-2 inhibitors, anti-CD25 daclizumab, basiliximab, ligand-directed toxins, denileukin difutitox (Ontak) (a fusion protein of human IL-2 and diphtheria toxin), or LMB-2 (scF for CD25). The following are selected therapeutic agents: a fusion between v and Pseudomonas exotoxin, antibodies targeting Treg cell surface receptors, TLR modulators, agents that interfere with the adenosinergic pathway, ectonucleotidase inhibitors, or inhibitors of the A2A adenosine receptor, TGF-β inhibitors, chemokine receptor inhibitors, retinoic acid, all-trans retinoic acid (ATRA), vitamin D3, phosphodiesterase 5 inhibitors, sildenafil, ROS inhibitors, and nitroaspirin. In some embodiments, the second therapeutic agent is cytokine therapy selected from one or more cytokines such as IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, and IL-21, IL-23, IL-27, GM-CSF, IFNα (interferon alpha), IFNα-2b, IFNβ, IFNγ, and their different delivery strategies. In some embodiments, the second therapeutic agent is a cancer-treating vaccine selected from the group consisting of exogenous cancer vaccines containing proteins or peptides used to initiate an immunogenic response to tumor antigens, recombinant viruses and bacterial vectors encoding tumor antigens, DNA-based vaccines encoding tumor antigens, dendritic cell-based vaccines targeting proteins, whole tumor cell vaccines, genetically modified tumor cells expressing GM-CSF, ICOS and / or Flt3 ligands, and oncolytic virus vaccines.

[0288] In some embodiments, the second therapeutic agent is EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1-18, including variants and analogs; interferons or cytokines, such as interferon α,' and γ hormones, such as luteinizing hormone-releasing hormone (LHRH) and analogs, and gonadotropin-releasing hormone (GnRH); growth factors, such as transforming growth factor, fibroblast growth factor (FGF), and other neurotransmitters. This includes transgrowth factors (NGF), growth hormone-releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homolog (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor alpha (TNF-α); infiltration inhibitor 2 (IIF-2); bone morphogenetic proteins 1-7 (BMP1-7); somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD); complement factors; anti-angiogenic factors; antigenic substances; and prodrugs.

[0289] A prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug, and can be activated or converted by enzymes to an active or more active parent form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy,” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Examples of prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs, which can be converted into more active cytotoxic free drugs. Examples of cytotoxic agents that can be derivatized into prodrug forms for use herein include, but are not limited to, the chemotherapeutic agents described above.

[0290] In other embodiments, the antibodies disclosed herein are administered for the prevention or treatment of cancer before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concurrently with the second therapeutic agent. In other embodiments, the second therapeutic agent may be an antibody immunospecific to one or more cancer cell antigens. In some embodiments, the cancer is resistant to other anti-cancer treatments or the second therapeutic agent. In some embodiments, the cancer is in remission. In some embodiments, one or more antibodies disclosed herein are administered to animals, preferably mammals, most preferably humans. In some embodiments, the antibodies disclosed herein are administered after surgical resection of cancer. The methods and compositions disclosed herein intend not only single antibodies disclosed herein, but also combinations of more than one antibody disclosed herein, or “cocktails.” In some embodiments, more than one antibody includes at least two, at least three, at least four, or all of the antibodies disclosed herein. Such antibody cocktails may have certain advantages because they include antibodies that utilize different effector mechanisms or antibodies that directly combine cytotoxic antibodies with antibodies that depend on the function of an immunoeffector.

[0291] Antibody-based gene therapy In another aspect of this disclosure, nucleic acid molecules comprising sequences encoding antibodies of this disclosure are administered to treat, inhibit, or prevent a disease or disorder via gene therapy. In some embodiments, the disease is cancer. In some embodiments, the disease is skin cancer. In some embodiments, the disease is cutaneous melanoma. In some embodiments, the disease is associated with the abnormal expression and / or activity of an antigen to which the antibody binds. Gene therapy refers to therapy carried out by administering expressed or expressible nucleic acid molecules to a subject. In this embodiment of this disclosure, the nucleic acid molecules produce their encoded proteins (e.g., antibodies disclosed herein) that mediate the therapeutic effect. Any of the available methods for gene therapy can be used in accordance with the present invention. Exemplary methods are described below. For a general review of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991).

[0292] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an antibody, and this nucleic acid molecule is part of an expression vector that expresses the antibody or its fragments or chimeric protein or heavy or light chain in a suitable host. In particular, such a nucleic acid sequence has a promoter operably linked to the antibody-coding region, and this promoter is inducible or constitutive, and optionally tissue-specific.

[0293] In another specific embodiment, nucleic acids encoding antibodies are expressed within chromosomes by using nucleic acid molecules in which the antibody-coding sequence and any other desired sequences are adjacent to regions that promote homologous recombination at desired sites in the genome (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijistra et al., Nature 342:435-438 (1989). In some embodiments, the expressed antibody is a single-chain antibody, and alternatively, the nucleic acid sequence includes sequences encoding both the heavy and light chains of the antibody, or fragments thereof.

[0294] The delivery of nucleic acid molecules into a patient may be either direct, involving direct exposure of the patient to the nucleic acid or nucleic acid carrier vector, or indirect, involving the transplantation of cells into the patient after they have been transformed with the nucleic acid in vitro. These two approaches are known as in vivo or ex vivo gene therapy, respectively.

[0295] In specific embodiments, nucleic acid molecules are administered directly in vivo, where they are expressed and generate coding products. This can be achieved by any of the numerous methods known in the art, for example, by constructing them as part of a suitable nucleic acid expression vector and administering them to make them intracellular, for example by infection with a deficient or attenuated retrovirus or other viral vector (see U.S. Patent No. 4,980,286), or by using a microparticle gun (e.g., gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules, or by conjugating them to peptides known to enter the nucleus and administering them conjugated to ligands that undergo receptor-mediated endocytosis (which can be used to target cell types that specifically express the receptor) (see, for example, Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)). In another embodiment, nucleic acid-ligand complexes may be formed, the ligands of which include viral membrane fusion peptides for disrupting endosomes, thereby enabling the nucleic acids to evade lysosomal degradation. In yet another embodiment, nucleic acids may be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (see, e.g., PCT publications WO92 / 06180;WO92 / 22635;WO92 / 20316;WO93 / 14188, WO93 / 20221). Alternatively, nucleic acids can be introduced into cells and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989), Zjistra et al., Nature 342:435-438 (1989)).

[0296] In specific embodiments, a viral vector containing a nucleic acid sequence encoding the antibody of the present invention is used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and its integration into host cell DNA. Cloning nucleic acid molecules encoding antibodies used in gene therapy into one or more vectors facilitates gene delivery to patients. Further details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994).

[0297] Adenoviruses may also be used in this invention. Another approach to gene therapy involves introducing genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the introduction method involves introducing a selectable marker into the cells. These cells are then selectively placed to take up the introduced gene and isolate the cells expressing it. These cells are then delivered to the patient.

[0298] In this embodiment, the nucleic acid molecule is introduced into cells prior to in vivo administration of the resulting recombinant cells. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, micronucleus cell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for introducing foreign genes into cells (see, for example, Locffler and Behr, Meth. Enzymol. 217:599-618 (1993)) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not impaired. The technique needs to provide stable introduction of the nucleic acid into cells so that the nucleic acid is expressible by the cells, preferably heritable and expressible by the offspring of those cells. The resulting recombinant cells can be delivered to the patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem cells or progenitor cells) are preferably administered intravenously. The amount of cells to be used can be determined by those skilled in the art, depending on the desired effect, the patient's condition, and other factors.

[0299] Cells into which nucleic acids can be introduced for gene therapy purposes include, but are not limited to, any desired available cell type, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes; and various stem cells or progenitor cells, particularly hematopoietic stem cells or progenitor cells, such as those obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

[0300] In one embodiment, the cells used in gene therapy are the patient's own cells. The nucleic acid sequence encoding the antibody of the present invention is introduced into the cells so that it can be expressed by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In specific embodiments, stem cells or progenitor cells are used. Any stem cells and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this embodiment of the present invention (see, for example, PCT Publication WO94 / 08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0301] Dosage This specification provides compositions comprising antibodies disclosed herein for the treatment (including prevention) of cancer. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition is administered in an amount effective for the treatment (including prevention) of cancer. In some embodiments, the composition (e.g., an antibody or a nucleic acid molecule encoding an antibody) is administered in an amount effective for enhancing the immune response and / or increasing T cell activation in a subject. The composition is used for in vivo administration to a subject by any available means, such as parenteral administration. For administration to a subject, the antibody-containing composition or drug described herein can be sterile, and this sterility can be readily achieved by filtration through a sterile filtration membrane or by other methods known to those skilled in the art. In one embodiment, the composition or drug is treated to be pyrogen- or endotoxin-free. Testing a pharmaceutical composition or drug for pyrogen or endotoxin, and preparing a pharmaceutical composition or drug that is pyrogen- or endotoxin-free, or that has clinically acceptable levels of endotoxin, will be well understood to those skilled in the art. Commercially available kits are available to test pharmaceutical compositions or drugs for pyrogens or endotoxins.

[0302] In the methods described herein, compositions used for in vivo administration, such as parenteral administration, can be sterile, and this sterility can be readily achieved by filtration through a sterile filtration membrane or by other methods known to those skilled in the art.

[0303] The antibodies disclosed herein are formulated, administered, and given in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific target being treated, the clinical condition of the individual target, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to healthcare professionals. The “therapeutic effective dose” of a substance / molecule, agonist, or antagonist may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit the desired response in the individual. The therapeutic effective dose is also the amount in which the toxic or adverse effects of the substance / molecule, agonist, or antagonist outweigh the therapeutically beneficial effects. The therapeutic effective dose may be delivered in one or more doses. The therapeutic effective dose refers to the amount effective in the dose and duration required to achieve the desired therapeutic and / or preventive outcome. The “therapeutic effective dose” administered refers to the minimum amount necessary to induce remission, treat, or stabilize cancer, determined by considerations such as extending time to progression (progression-free survival), or treating or preventing the development or recurrence of tumors, dormant tumors, or micrometastases. The antibodies disclosed herein are optionally formulated with one or more additional therapeutic agents currently used to prevent or treat cancer or the risk of developing cancer. The effective dose of such other agents depends on the amount of antibody present in the formulation, the type of impairment or treatment, and other factors considered above. These are generally used at the same doses and routes of administration as previously used, or at approximately 1–99% of previously used doses.

[0304] The antibody dose may vary depending on the age and size of the recipient, the target disease, condition, and route of administration. Preferred doses are usually calculated based on body weight or body surface area. When the antibodies disclosed herein are used to treat a condition or disease in adult patients, it may be advantageous to administer the antibodies in single intravenous doses of approximately 0.01 to approximately 20 mg / kg body weight, more preferably approximately 0.02 to approximately 7, approximately 0.03 to approximately 5, or approximately 0.05 to approximately 3 mg / kg, approximately 5 mg / kg, approximately 7.5 mg / kg, approximately 10 mg / kg, or approximately 15 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and administration schedules may be determined empirically, for example, by monitoring the patient's progress through regular assessments and adjusting the dose accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0305] In some embodiments, the compositions herein may include, for example, a prophylactic effective dose for administration to subjects at risk of cancer or subjects in an earlier stage of the disease. “Prophylactic effective dose” refers to an amount effective in achieving the desired prophylactic outcome at the required dose and duration of administration. Typically, prophylactic doses are less than therapeutic doses, as they are used in subjects before or during the early stages of the disease.

[0306] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared into dosage forms in unit doses appropriate to the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like.

[0307] The administration may be by one or more separate doses or by continuous infusion. In the case of repeated administration over several days or longer, the treatment may be continued, depending on the condition, until the cancer is cured, for example, by methods known in the art. However, other administration regimens may also be useful. In one non-limiting example, the antibody disclosed herein is administered once a week, once every two weeks, or once every three weeks in a dose range of about 5 mg / kg to about 15 mg / kg, for example, but not limited to 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. Progress using the methods described herein can be readily monitored by conventional art and assays. The duration of therapy using the methods described herein may be continued for as long as medically required or until the desired therapeutic effect (e.g., as described herein) is achieved. In certain embodiments, administration of one or more antibodies or compositions described herein is continued for a period of one month, two months, four months, six months, eight months, ten months, one year, two years, three years, four years, five years, ten years, twenty years, or the lifespan of the subject.

[0308] Treatment effectiveness The effectiveness of a method of treating cancer, comprising administering an antibody or composition of the Disclosure (e.g., a pharmaceutical composition), can be measured by a variety of endpoints commonly used in the evaluation of cancer treatment, including but not limited to tumor regression, reduction in tumor weight or size, tumor arrest time, survival time, progression-free survival time, overall response rate, duration of response, and quality of life. The antibodies disclosed herein may require a unique measure and definition of the clinical response to the drug. In the case of cancer, a therapeutically effective amount of an antibody or composition comprising the same disclosed herein may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow, preferably stop) the invasion of cancer cells into peripheral organs; inhibit (i.e., slow, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that the antibodies disclosed herein inhibit the growth of existing cancer cells and / or kill them, the drugs may be cell proliferation inhibitory and / or cytotoxic. In cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0309] These embodiments relate to the treatment or prevention of skin cancer (e.g., cutaneous melanoma). Symptoms of melanoma include, but are not limited to, changes in the shape or color of existing moles, or, in the case of nodular melanoma, the appearance of new lumps anywhere on the skin. In later stages, moles may be itchy, ulcerate, or bleed. Early signs of melanoma are summarized by asymmetry, borders (with irregular edges and angles), color (patchy), diameter (greater than 6 mm (0.24 in), roughly the size of a pencil eraser), progression over time, and an odd appearance. Nodular melanoma is raised above the skin surface, hard to the touch, and appears to be growing. Metastatic melanoma may cause nonspecific paraneoplastic symptoms, including loss of appetite, nausea, vomiting, and fatigue. Metastasis of early melanoma is possible but relatively rare: less than one-fifth of melanomas diagnosed early become metastatic. Brain metastases are particularly common in patients with metastatic melanoma. It may also spread to the liver, bones, abdomen, or distant lymph nodes. In some embodiments, one or more symptoms of skin cancer (e.g., cutaneous melanoma) are inhibited or treated using the compositions and methods described herein.

[0310] In other embodiments, this specification describes methods for extending progression-free survival in human subjects who are susceptible to or diagnosed with cancer, such as skin cancers like cutaneous melanoma. Time to disease progression is defined as the time from administration of a drug to disease progression or death. In preferred embodiments, combination therapy of the present invention using the antibodies disclosed herein and one or more chemotherapeutic agents can significantly extend progression-free survival by at least about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, or 3.5 months, for example, about 1 to about 5 months, compared to chemotherapy alone. In other embodiments, the methods described herein can significantly increase the response rate in a group of human subjects who are susceptible to or diagnosed with cancer and treated with a variety of therapeutic agents. Response rate is defined as the percentage of treated subjects who responded to the treatment. In one embodiment, combination therapy described herein using the antibodies disclosed herein, such as recombinant antibodies, and one or more chemotherapeutic agents significantly increases the response rate in a group of treated subjects compared to a group treated with chemotherapy alone.

[0311] As used herein, the terms “to treat,” “treatment,” “to treat,” or “remission” refer to therapeutic treatments whose purpose is to reverse, alleviate, induce remission, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder. The term “to treat” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder, including but not limited to chronic infections or cancer. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is “effective” if the progression of the disease is reduced or halted. In other words, “treatment” includes not only improvement of symptoms or markers but also halt, which is at least a slowing of the progression or worsening of symptoms that would be expected without treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, whether detectable or undetectable; reduction of the severity of the disease; stabilization of the condition (i.e., no worsening); delay or slowing of disease progression; improvement or alleviation of the condition; and remission (whether partial or in whole). The term “treatment” of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

[0312] For example, in some embodiments, the methods described herein include administering an effective dose of the antibody described herein to a target in order to alleviate the symptoms of cancer. As used herein, “alleviate the symptoms of cancer” means to remit or reduce any condition or symptom associated with cancer. Compared to an equivalent untreated control, the reduction or degree of such prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique. Ideally, the cancer is considered treated if it has completely disappeared as detectable by any standard method known in the art. A patient receiving treatment for cancer is a patient diagnosed by a physician as having such a condition. The diagnosis can be made by any appropriate means. Diagnosis and monitoring may include, for example, detecting levels of cancer cells in a biological sample (e.g., tissue or lymph node biopsy, blood test, or urine test), detecting levels of surrogate markers for cancer in a biological sample, detecting specific cancer-related symptoms, or detecting immune cells involved in an immune response typical of such cancer.

[0313] As used herein, the term “effective dose” refers to the amount of antibody or a composition containing the antibody necessary to achieve a desired outcome, including but not limited to a reduction in the level of Treg cells, a decrease in the biological activity of Tregs, an enhancement of the immune response, and / or a reduction in at least one symptom of a disease or disorder, and relates to an amount of a pharmaceutical composition sufficient to provide the desired effect. Accordingly, the term “therapeutic effective dose” refers to the amount of the antibody disclosed herein sufficient to produce a particular effect when administered to a typical subject. The effective dose as used herein also includes an amount sufficient to delay the onset of disease symptoms, alter the course of a symptomatic disease (e.g., slowing the progression of disease symptoms, but not limited to the following), or reverse the symptoms of a disease. Accordingly, an exact “effective dose” cannot be specified. However, in any given case, a suitable “effective dose” can be determined by a person skilled in the art using ordinary experiments.

[0314] The effective dose, toxicity, and therapeutic efficacy can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (50% lethal dose) and ED50 (50% therapeutically effective dose). The dosage may vary depending on the dosage form used and the route of administration utilized. The dose-to-toxicity ratio is the therapeutic index, which can be expressed as the LD50 / ED50 ratio, and compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can first be estimated from a cell culture assay. The dose can also be formulated in animal models to achieve a circulating plasma concentration range, including the IC50 (i.e., antibody concentration), that achieves 50% inhibition of symptoms determined in cell culture or in a suitable animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay. The dosage is determined by a physician and can be adjusted as needed to suit the observed therapeutic effect.

[0315] Treatment and / or prevention of cancer includes, but is not limited to, alleviating cancer-related symptoms, inhibiting cancer progression, promoting cancer regression, promoting immune responses, inhibiting tumor growth, inhibiting tumor size, inhibiting metastasis, inhibiting cancer cell growth, inhibiting cancer cell proliferation, or inducing cancer cell death.

[0316] Method of administration The antibodies described herein may be administered to the target in question via any suitable route that provides an effective treatment to the target. As used herein, the terms “administer” and “introduce” are interchangeable and refer to positioning an antibody or an antibody portion of an antibody to a target by a method or route that localizes such agent at least partially to a desired site, such as a site of infection, inflammation, or cancer, in order to produce the desired effect(s).

[0317] In some embodiments, the antibodies described herein, or compositions comprising them, are administered to a subject with cancer, inhibited by any method of administration that delivers the drug systemically or to a desired surface or target, the method of administration may include, but is not limited to, injection, infusion, drip infusion, and inhalation. Oral administration forms are also contemplated herein. "Injection" includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intracranial, intraspinal, intracerebrospinal, and intrasternal injections and infusions.

[0318] As used herein, the terms "parenteral administration" and "administered parenterally" refer to methods of administration other than enteral and topical administration, typically by injection. As used herein, the terms "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to the administration of a bispecific or multispecific polypeptide agent to undergo metabolism and other similar processes by entering the target circulatory system, rather than directly administering it to a target site, tissue, or organ, such as a tumor site.

[0319] In some embodiments, the antibodies described herein, or compositions comprising the same, may be administered intramuscularly, intraperitoneally, intrathecally, subcutaneously, intraarticularly, intrabursally, intrathecally, orally, topically, or by inhalation, either by intravenous administration as a bolus or by continuous infusion over a period of time. If the use of the antibodies described herein, or compositions comprising the same, involves widespread side effects or toxicity, topical administration to a tumor or cancer site where angiogenesis is occurring is particularly desirable. In some embodiments, an ex vivo strategy may also be used for therapeutic purposes. An ex vivo strategy involves transfecting or transducing cells obtained from a subject with a nucleic acid sequence disclosed herein. The transfected or transduced cells are then returned to the subject. These cells may be any of a broad range of types, including, without limitation, hematopoietic cells (e.g., myeloid cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or myocytes.

[0320] In some embodiments, the antibodies disclosed herein, or compositions containing the same, are administered by any preferred means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and nasal administration (intra-lesional administration if topical immunosuppressive therapy is desired).

[0321] Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies or compositions of the Disclosure are preferably administered by pulse infusion (particularly with reduced doses of antibodies). Preferably, the administration is carried out by injection, most preferably intravenous or subcutaneous injection, depending on whether the administration is short-term or long-term. In some embodiments, the antibodies or compositions of the Disclosure are administered locally, e.g., by direct injection, if the location of the lesion or tumor allows, and the injection may be repeated periodically. In some embodiments, the antibodies or compositions of the Disclosure may also be delivered systemically to a subject or directly to tumor cells, e.g., tumor or tumor bed, following surgical incision of the tumor, to prevent or reduce local recurrence or metastasis, such as dormant tumors or micrometastases.

[0322] Antibody-targeted sonoporation methods are intended for use in some embodiments of the tumor-inhibiting methods described herein to enhance the efficacy and potency of the antibody-containing therapeutic compositions provided herein. As used herein, “sonoporation” refers to the interaction of ultrasound with a contrast agent (e.g., stabilized microbubbles) to enable the uptake of large molecules, such as therapeutic agents, by using sound of an ultrasonic frequency or by temporarily altering the permeability of the cell membrane. The membrane permeability induced by sonoporation is transient, leaving the drug trapped inside the cell after ultrasound exposure. Sonoporation utilizes the acoustic cavitation of microbubbles to facilitate the delivery of large molecules.

[0323] Accordingly, in some embodiments of the method, the antibodies described herein, mixed with an ultrasound contrast agent such as microbubbles, can be injected locally or systemically into a subject requiring cancer treatment, and ultrasound can be coupled to a limited area, e.g., a tumor site, and further focused in order to achieve targeted delivery. In some embodiments, the method uses focused ultrasound to achieve targeted delivery. As used herein, HIFU or “high-intensity focused ultrasound” refers to a non-invasive therapy that uses high-intensity ultrasound to heat and destroy malignant or pathogenic tissue without damaging healthy tissue over or around it. As described in Khaibullin...

Claims

1. An isolated antibody comprising a chemokine receptor 8 (CCR8) binding domain containing one or more complementarity-determining regions (CDRs), The one or more CDRs are selected from the group consisting of CDR1 region, CDR2 region, and CDR3 region. The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of sequence numbers 2, 3, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214. The CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of sequence numbers 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 216-224. The isolated antibody wherein the CDR3 region contains an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 8, 9, 17, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, and 243-246.

2. The isolated antibody according to claim 1, wherein the CCR8 binding domain includes the CDR1 region, the CDR2 region, and the CDR3 region.

3. The isolated antibody according to any one of claims 1 to 2, wherein the CDR1 region comprises an amino acid sequence in which the percentage of identical residues to a sequence selected from SEQ ID NOs: 2, 3, 11, 12, 47, 54, 57, 58, 112-119, 139-145, or 210-214 is at least 85%, 90%, 95%, 99%, or 100%.

4. The isolated antibody according to any one of claims 1 to 3, wherein the CDR2 region comprises an amino acid sequence in which the percentage of identical residues to a sequence selected from SEQ ID NOs: 4, 5, 6, 13, 14, 15, 48, 50, 51, 55, 59, 60, 120-125, 146-151, or 216-224 is at least 85%, 90%, 95%, 99%, or 100%.

5. The isolated antibody according to any one of claims 1 to 4, wherein the CDR3 region comprises an amino acid sequence in which the percentage of identical residues to a sequence selected from SEQ ID NOs: 8, 9, 17, 49, 52, 53, 56, 61, 62, 63, 64, 65, 66, 67, 68, 69, 126-137, 152-162, 225-229, or 243-246 is at least 85%, 90%, 95%, 99%, or 100%.

6. (a) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (b) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 13; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

56. (c) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 6; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

8. (d) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 13; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

157. (e) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (f) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

52. (g) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

53. (h) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 50; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

52. (i) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 51; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

243. (j) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 50; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

53. (k) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 51; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

244. (l) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 50; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

245. (m) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 50; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

246. (n) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of the sequence numbers 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of the sequence numbers 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of the sequence numbers 56. (o) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

61. (p) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 59; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

62. (q) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 60; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

63. (r) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 60; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

64. (s) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

65. (t) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 57; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

66. (u) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 58; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

67. (v) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

68. (w) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 60; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

69. (x) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 51; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (y) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

227. (z) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (aa) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 210; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

226. (bb) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 216; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

227. (cc) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 217; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

227. (dd) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 218; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

227. (ee) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 219; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (ff) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 211; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 220; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

228. (gg) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 212; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 221; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (hh) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 47; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 222; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

227. (ii) The CDR1 region includes an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 47 is at least 80%, the CDR2 region includes an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 223 is at least 80%, and the CDR3 region includes an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 49 is at least 80%. (jj) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 213; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 51; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

226. (kk) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 214; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

226. (ll) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 212; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 221; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (mm) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 212; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 48; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (nn) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 212; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 221; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

226. (oo) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 212; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs: 221; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs:

49. (pp) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 54; the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55; and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs.

56. (qq) The CDR1 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 54, the CDR2 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 55, and the CDR3 region includes an amino acid sequence in which at least 80% of the residues are identical to any one of SEQ ID NOs. 229, or (rr) The isolated antibody according to any one of claims 1 to 5, wherein the CDR1 region comprises an amino acid sequence having at least 80% identical residues to any one of sequence numbers 54, the CDR2 region comprises an amino acid sequence having at least 80% identical residues to any one of sequence numbers 224, and the CDR3 region comprises an amino acid sequence having at least 80% identity to any one of sequence numbers 56.

7. The isolated antibody according to any one of claims 1 to 6, wherein the CCR8-binding domain further comprises one or more framework regions (FRs), and the one or more FRs are selected from the group consisting of FR1 region, FR2 region, FR3 region and FR4 region.

8. The isolated antibody according to claim 7, comprising the FR1, FR2, FR3, and FR4 regions.

9. The isolated antibody according to claim 7, wherein the FR1, FR2, FR3, and FR4 regions are human.

10. The isolated antibody according to any one of claims 7 to 9, wherein the FR1 region comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 19, 23, 163, 164, 175, 176, or 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

11. The isolated antibody according to any one of claims 7 to 10, wherein the FR2 region comprises an amino acid sequence in which the percentage of identical residues for any one of SEQ ID NOs: 20, 24, 165, 166, 167, 168, 178, 179, 180, 181, 232, or 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

12. The isolated antibody according to any one of claims 7 to 11, wherein the FR3 region comprises an amino acid sequence in which the percentage of identical residues for any one of SEQ ID NOs: 21, 25, 169, 170, 171, 182, 183, 184, 234, 235, or 236 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

13. The isolated antibody according to any one of claims 7 to 12, wherein the FR4 region comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 22, 26, 172, 173, or 185 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

14. (a) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 163 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 165 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 169 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 172 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (b) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 166 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 170 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%, (c) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 167 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (d) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (e) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 175 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 178 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 182 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 185 is at least 80%, 85%, 90%, 95%, 99%, or 100%, (f) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 176 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 179 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 183 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (g) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 180 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 184 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (h) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 181 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 184 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%, (i) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (j) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 231 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (k) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%, (l) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 232 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (m) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (n) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%, (o) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (p) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (q) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (r) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 164 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (s) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (t) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (u) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 233 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 234 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (v) The FR1 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 171 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (w) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 168 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 235 is at least 80%, 85%, 90%, 95%, 99%, or 100%; and the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (x) The FR1 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR2 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 181 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR3 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 236 is at least 80%, 85%, 90%, 95%, 99%, or 100%; the FR4 region includes an amino acid sequence in which the percentage of identical residues relative to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%. (y) The FR1 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 177 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR2 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 181 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR3 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 236 is at least 80%, 85%, 90%, 95%, 99%, or 100%, the FR4 region includes an amino acid sequence in which the percentage of identical residues to SEQ ID NO: 173 is at least 80%, 85%, 90%, 95%, 99%, or 100%, or (z) The isolated antibody according to any one of claims 7 to 13, wherein the FR1 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 177, the FR2 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 181, the FR3 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO: 184, and the FR4 region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identical residues to SEQ ID NO:

173.

15. The isolated antibody according to any one of claims 7 to 14, wherein the FR1 comprises an IgG1, IgG2, IgG3, or IgG4 framework region.

16. The isolated antibody according to any one of claims 7 to 15, wherein the FR2 comprises an IgG1, IgG2, IgG3, or IgG4 framework region.

17. The isolated antibody according to any one of claims 7 to 16, wherein the FR3 comprises an IgG1, IgG2, IgG3, or IgG4 framework region.

18. The isolated antibody according to any one of claims 7 to 17, wherein the CCR8 binding domain comprises an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 27-30, 70-86, 106-111, or 186-209 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

19. The isolated antibody according to any one of claims 1 to 18, wherein the CCR8 binding domain comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 27 to 30 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

20. The isolated antibody according to any one of claims 1 to 18, wherein the CCR8 binding domain comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs. 70 to 86 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

21. The isolated antibody according to any one of claims 1 to 18, wherein the CCR8 binding domain comprises an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 106 to 111 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

22. The isolated antibody according to any one of claims 1 to 18, wherein the CCR8 binding domain comprises an amino acid sequence in which the percentage of identical residues to any one of sequence numbers 186 to 209 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

23. The isolated antibody according to any one of claims 1 to 22, wherein the percentage of identical residues is determined by BLASTp.

24. The isolated antibody according to any one of claims 1 to 23, wherein the isolated antibody further comprises an Fc region.

25. The isolated antibody according to claim 24, wherein the Fc region is a human Fc region.

26. The isolated antibody according to claim 24 or 25, wherein the Fc region is the Fc region of IgG1, IgG2, IgG3, IgG4, IgGGA1, or IgGGA2.

27. The isolated antibody according to any one of claims 24 to 26, wherein the isolated antibody further comprises one or more mutations within the Fc region.

28. The isolated antibody according to claim 27, wherein the one or more mutations modulate the effector function of the isolated antibody compared to a corresponding antibody lacking the at least one mutation.

29. The isolated antibody according to claim 28, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, and / or complement-dependent cell-mediated cytotoxicity (CDC).

30. The isolated antibody according to any one of claims 27 to 29, wherein the one or more mutations reduce the Fc-γ receptor binding of the isolated antibody compared to a corresponding antibody lacking the at least one mutation.

31. The isolated antibody according to any one of claims 27 to 30, wherein the one or more mutations increase the cell-lytic activity of the isolated antibody compared to a corresponding antibody lacking the at least one mutation (for example, increasing ADCC activity and / or CDC activity).

32. The isolated antibody according to any one of claims 27 to 31, wherein the one or more mutations are located in an amino acid residue selected from the group consisting of L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, 1332, E333, K334, and P396.

33. The isolated antibody according to any one of claims 27 to 32, wherein the one or more mutations are fucosylation.

34. The isolated antibody according to any one of claims 24 to 33, wherein the Fc region comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 34, 44, 238, or 239 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

35. The isolated antibody according to any one of claims 24 to 34, wherein the Fc region comprises one or more mutations in the wild-type Fc region, which comprises the amino acid sequence of SEQ ID NO: 34 or 237.

36. The isolated antibody according to any one of claims 27 to 35, wherein the one or more mutations include amino acid substitutions, deletions, insertions, or combinations thereof.

37. The one or more mutations described above are, in relation to the wild-type Fc region containing the amino acid sequence of SEQ ID NO: 34, numbered according to the kabat scheme, (a) Amino acid substitution of S252, (b) Amino acid substitution of I351, or (c) combinations of those, An isolated antibody according to any one of claims 27 to 36, comprising:

38. The isolated antibody according to claim 37, wherein the amino acid substitution of S252 is the amino acid substitution of S252D.

39. The isolated antibody according to claim 38, wherein the amino acid substitution of I351 is the amino acid substitution of I351E.

40. The isolated antibody according to any one of claims 24 to 39, wherein the Fc region comprises an amino acid sequence in which the percentage of identical residues to any one of SEQ ID NOs: 34, 44, 238, or 239 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

41. The isolated antibody according to any one of claims 1 to 40, wherein the isolated antibody comprises an amino acid sequence in which the percentage of identical residues for any one of SEQ ID NOs. 31-33, 45-46, or 87-103 is at least 80%, 85%, 90%, 95%, 99%, or 100%.

42. An isolated antibody containing a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain is numbered according to the IMGT scheme and contains the amino acid sequence of SEQ ID NO: 108 relative to a wild-type CCR8 binding domain. Amino acid substitution at G64, amino acid substitution at L110, amino acid substitution at I39, amino acid substitution at G40, amino acid substitution at R55, amino acid substitution at R38, amino acid substitution at R108, amino acid substitution at R55, amino acid substitution at A25, amino acid substitution at N82, amino acid substitution at G63, amino acid substitution at S58, amino acid substitution at G64, amino acid substitution at S28, amino acid substitution at T65, amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at S57, amino acid substitution at S58, amino acid substitution at S35, amino acid substitution at R37, amino acid substitution at R108, Amino acid substitutions at R55, N83, A25, S57, S58, S35, G64, R108, R37, R55, R108, N83, A25, R38, S57, N83, R55, S57, R108, G81, S28, or combinations thereof. The isolated antibody, including the above.

43. The aforementioned CCR8 binding domain is (a) Amino acid substitution in G64, (b) Amino acid substitution at L110, (c) Amino acid substitution at I39, amino acid substitution at G40, and amino acid substitution at R55, (d) Amino acid substitution at R38 and amino acid substitution at R108, (e) Amino acid substitution at R55, (f) Amino acid substitution at A25, amino acid substitution at R55, and amino acid substitution at N82, (g) Amino acid substitution at G63 and amino acid substitution at L110, (h) Amino acid substitution at T65 and amino acid substitution at L110, (i) Amino acid substitution in G64, (j) Amino acid substitution in S28, amino acid substitution in G64, amino acid substitution in L110, (k) Amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at S57, (l) Amino acid substitution at G64, Amino acid substitution at L110, (m) Amino acid substitution in S58 above, (n) Amino acid substitution in S35, amino acid substitution in R37, amino acid substitution in G64, amino acid substitution in R108, (o) Amino acid substitution at R37, amino acid substitution at R55, amino acid substitution at R108, amino acid substitution at N83, (p) Amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at S57, amino acid substitution at N83, (q) Amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at R55, amino acid substitution at N83, (r) Amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at S57, amino acid substitution at R108, (s) Amino acid substitution at A25, amino acid substitution at R28, amino acid substitution at S57, amino acid substitution at R108, or (t) Amino acid substitution at A25, amino acid substitution at R38, amino acid substitution at S571, amino acid substitution at G81, The isolated antibody according to claim 42, comprising

44. The amino acid substitution at G64 is G64S or G64T, the amino acid substitution at L110 is L110V or L110Y, the amino acid substitution at I39 is I39Y, the amino acid substitution at G40 is G40S, the amino acid substitution at R55 is R55W, R55H, or R55G, the amino acid substitution at R38 is R38T, the amino acid substitution at R108 is R108I, the amino acid substitution at A25 is A25V, the amino acid substitution at N82 is N82K, the amino acid substitution at G63 is G63I, the amino acid substitution at S58 is S58G, the amino acid substitution at G64 is G64S or G64T, the amino acid substitution at T65 is T65I, and the amino acid substitution at A25 is A The isolated antibody according to claim 42 or claim 43, wherein the amino acid substitution at R38 is R38G, the amino acid substitution at S57 is S57I, the amino acid substitution at S58 is S58G, the amino acid substitution at S35 is S35T, the amino acid substitution at R37 is R37V, the amino acid substitution at R108 is R108I, the amino acid substitution at R55 is R55H, the amino acid substitution at N83 is N83K, the amino acid substitution at A25 is A25V, the amino acid substitution at S57 is S57I, the amino acid substitution at R37 is R37V, the amino acid substitution at R108 is R108I, the amino acid substitution at G81 is G81A, and the amino acid substitution at S28 is S28F.

45. An isolated antibody comprising a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain comprises an amino acid substitution at D81, an amino acid substitution at M108, an amino acid substitution at P65, or a combination thereof, relative to the wild-type CCR8 binding region comprising the amino acid sequence of SEQ ID NO: 111, numbered according to the IMGT scheme.

46. The aforementioned CCR8 binding domain is (u) Amino acid substitution in D81, (v) Amino acid substitutions in D81 and M108, or (w) Amino acid substitution at P65, The isolated antibody according to claim 45, comprising

47. The isolated antibody according to claim 45 or claim 46, wherein the amino acid substitution at D81 is D81G, the amino acid substitution at M108 is M108K, and the amino acid substitution at P65 is P65L.

48. The isolated antibody according to any one of claims 1 to 47, wherein the isolated antibody comprises a full-length antibody, a chimeric antibody, a single-domain antibody, a single-chain antibody (scFv), a heavy-chain-only antibody (HcAb), (for example, a heavy-chain-only antibody derived from a camelid or a heavy-chain-only antibody derived from a shark), or a functional fragment thereof.

49. The isolated antibody according to claim 48, wherein the functional fragment comprises Fab, Fab', Fab'-SH, Fv, or F(ab')2.

50. The isolated antibody according to any one of claims 1 to 49, wherein the isolated antibody comprises Fab, Fab', Fab'-SH, Fv, or F(ab')2.

51. The isolated antibody according to any one of claims 1 to 50, wherein the isolated antibody is a single-domain antibody.

52. The isolated antibody according to any one of claims 48 to 51, wherein the single-domain antibody is a VHH antibody or a VNAR antibody.

53. The isolated antibody according to any one of claims 48 to 52, wherein the single-domain antibody is a VHH derived from a camelid animal.

54. The isolated antibody according to any one of claims 48 to 53, wherein the single-domain antibody is a llama-derived single-domain antibody.

55. The isolated antibody according to any one of claims 1 to 54, wherein the isolated antibody comprises a first single-domain antibody and a second single-domain antibody, the first single-domain antibody comprising the CCR8 binding domain and the second single-domain antibody comprising the antigen binding domain.

56. The isolated antibody according to claim 55, wherein the antigen-binding domain is a CCR8 antigen-binding domain.

57. The isolated antibody according to claim 55, wherein the antigen-binding domain is a cancer antigen-binding domain.

58. The isolated antibody according to any one of claims 1 to 57, wherein the isolated antibody is a bispecific antibody, the bispecific antibody comprises a first single-domain antibody and a second single-domain antibody, the first single-domain antibody is ligated to the second single-domain antibody, the first single-domain antibody comprises the CCR8 binding domain, and the second single-domain antibody comprises an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen).

59. The isolated antibody according to any one of claims 1 to 58, wherein the isolated antibody is a bispecific antibody, and the bispecific antibody comprises the CCR8 binding domain and further comprises an antigen-binding domain that binds to a target antigen (e.g., a cancer antigen).

60. The isolated antibody according to any one of claims 1 to 59, wherein the isolated antibody is a single-chain antibody.

61. The isolated antibody according to any one of claims 1 to 60, wherein the isolated antibody is humanized, monoclonal, deimmunized, bispecific, multispecific, polyvalent, or a combination thereof.

62. The isolated antibody according to any one of claims 1 to 61, wherein the isolated antibody is humanized.

63. The isolated antibody according to any one of claims 1 to 33, wherein the isolated antibody comprises IgG-scFv, nanobody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2.scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody.

64. The isolated antibody according to any one of claims 1 to 63, wherein the isolated antibody specifically binds to the CCR8 polypeptide or a portion thereof.

65. The isolated antibody according to any one of claims 1 to 64, wherein the isolated antibody specifically binds to a human CCR8 polypeptide or a portion thereof.

66. The isolated antibody according to any one of claims 1 to 65, wherein the isolated antibody specifically binds to the extracellular domain of a CCR8 polypeptide (e.g., human CCR8 polypeptide) or a portion thereof.

67. The isolated antibody according to any one of claims 1 to 66, wherein the isolated antibody specifically binds to the transmembrane domain of a CCR8 polypeptide (e.g., human CCR8) or a portion thereof.

68. The isolated antibody according to any one of claims 1 to 67, wherein the isolated antibody specifically binds to the cytoplasmic domain of a CCR8 polypeptide (e.g., human CCR8) or a portion thereof.

69. The isolated antibody according to any one of claims 1 to 68, further comprising a payload, the payload being conjugated or coupled to the isolated antibody.

70. The isolated antibody according to claim 69, wherein the payload is selected from the group consisting of therapeutic agents, small molecules, polypeptides, polynucleotides, enzymes, substrates, cofactors, fluorescent markers, chemiluminescent markers, peptide tags, magnetic particles, drugs, therapeutic agents, siRNA, antisense oligonucleotides, toxins, radionuclides, secondary antibody binding sites, metal-binding domains, or combinations thereof.

71. The isolated antibody according to any one of claims 1 to 70, wherein the isolated antibody is cell-lytic.

72. The isolated antibody according to any one of claims 1 to 71, wherein the isolated antibody exhibits ADCC activity.

73. The isolated antibody according to any one of claims 1 to 72, wherein the isolated antibody is cytolytic against regulatory T cells (Treg).

74. The isolated antibody according to any one of claims 1 to 73, wherein the isolated antibody is cytolytic against tumor-infiltrating regulatory T cells.

75. The isolated antibody according to any one of claims 1 to 74, wherein the isolated antibody induces ADCC of regulatory T cells (Treg), for example, tumor-infiltrating regulatory T cells.

76. The isolated antibody is (a) To enhance the immune response against tumors, (b) Reducing, depleting, or killing Treg cells such as tumor-infiltrating regulatory T ("Treg") cells. (c) Inducing the internal migration of the CCR8 polypeptide or a portion thereof into Treg cells, which are tumor-invasion-regulating T ("Treg") cells. (d) To bind to the CCR8 polypeptide or a portion thereof (for example, the human CCR8 polypeptide or a portion thereof), (e) Binding to the CCR8 polypeptide or a portion thereof (for example, human CCR8 polypeptide or a portion thereof) by a KD of 10 nM or less as measured by BIACORE (trademark), (f) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing the CCR8 polypeptide or a portion thereof, or (g) Any combination of them, An isolated antibody according to any one of claims 1 to 75, having the ability to do so.

77. An antibody conjugate comprising an isolated antibody according to claims 1 to 76 and a payload.

78. The antibody conjugate according to claim 77, wherein the payload is a therapeutic agent.

79. The antibody conjugate according to claim 77 or claim 78, wherein the payload (e.g., therapeutic agent) comprises a small molecule, peptide, protein, or polynucleotide (e.g., DNA, RNA, mRNA).

80. The antibody conjugate according to any one of claims 77 to 79, wherein the payload (e.g., therapeutic agent) comprises an immunomodulator, an anticancer agent, a cytotoxic drug, an NSAID, siRNA, an oligonucleotide (e.g., an antisense oligonucleotide), a corticosteroid, an antioxidant or other nutritional supplement, or a chemotherapy agent.

81. The antibody conjugate according to claim 80, wherein the immunomodulatory agent comprises a cytokine, a chemokine, or an interferon.

82. The antibody conjugate according to any one of claims 77 to 81, wherein the payload is covalently or noncovalently conjugated to the isolated antibody.

83. The antibody conjugate according to any one of claims 77 to 82, wherein the payload is conjugated to the isolated antibody by a linker.

84. The antibody complex is A-(X １ -B) ｎ Equation (I) During the ceremony, A comprises an isolated antibody according to any one of claims 1 to 61. B includes the aforementioned payload, X １ This includes a bond or linker, The antibody conjugate according to any one of claims 77 to 83, wherein n is an average value selected from 1 to 12.

85. An isolated nucleic acid molecule encoding an isolated antibody according to any one of claims 1 to 76, or an antibody complex according to any one of claims 77 to 84.

86. A vector comprising the nucleic acid molecule described in claim 85.

87. In vitro cells, (a) an isolated antibody according to any one of claims 1 to 76, or an antibody complex according to any one of claims 77 to 84, expressing and / or secreting (b) comprising and / or an isolated nucleic acid molecule as described in claim 85 (c) The in vitro cell comprising the vector according to claim 86.

88. (a) an isolated antibody according to any one of claims 1 to 76, or The antibody complex according to any one of claims 77 to 84, and (b) A pharmaceutically acceptable carrier, adjuvant, or diluent, A pharmaceutical composition containing the following:

89. The pharmaceutical composition according to claim 88, further comprising at least one additional therapeutic agent.

90. A pharmaceutical composition according to any one of claims 88 to 89, formulated for administration via a subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intraventricular, intracranial, intracavitary, or intracerebellar route of administration.

91. A pharmaceutical composition according to any one of claims 88 to 90, which is in a water-soluble or freeze-dried form.

92. A pharmaceutical composition according to any one of claims 88 to 91, which is housed in a delivery device selected from the group consisting of a syringe, a blunt tip syringe, a catheter, and an implantable pump.

93. Use of an isolated antibody according to any one of claims 1 to 76, or an antibody conjugate according to any one of claims 77 to 84, for the treatment of cancer or an infectious disease (e.g., a pathogenic infectious disease such as a microbial infection or a viral infection).

94. Use of an isolated antibody according to any one of claims 1 to 76, or an antibody conjugate according to claims 77 to 84, in the manufacture of a pharmaceutical product.

95. The use according to claim 94, wherein the agent is for a disease, disorder, or condition involving CCR8 polypeptide-expressing cells (e.g., Treg cells, e.g., tumor-infiltrating Treg cells).

96. The use of the drug according to claim 94 or claim 95, wherein the drug is for the treatment of cancer or an infectious disease (for example, a pathogenic infection such as a microbial infection or a viral infection).

97. Use of the antibody conjugate according to any one of claims 93 to 96 for delivering the payload to a target site.

98. The use of the antibody conjugate according to claim 97, wherein the target site is a tumor site.

99. A fusion protein comprising an isolated antibody according to any one of claims 1 to 76.

100. A chimeric antigen receptor (CAR) comprising an isolated antibody according to any one of claims 1 to 76.

101. A T cell receptor (TCR) comprising an isolated antibody according to any one of claims 1 to 76.

102. A method for delivering a payload to a target site within the target, The method comprising delivering the payload to the target site by administering an effective amount of the antibody conjugate described in any one of claims 77 to 84 to the subject.

103. The method according to claim 102, wherein the target site is an immune cell or cell expressing a portion of the CCR8 polypeptide.

104. The method according to claim 102 or claim 103, wherein the target site is a tumor site.

105. A treatment method for a subject requiring treatment, Effective amount (a) an isolated antibody according to any one of claims 1 to 76, (b) A pharmaceutical composition according to any one of claims 88 to 92, or (c) The antibody complex according to any one of claims 77 to 84, The method for treating the subject by administering the substance.

106. A method for reducing the level of Treg cells or inhibiting Treg cells, an effective amount of the target that is needed (a) an isolated antibody according to any one of claims 1 to 76, (b) A pharmaceutical composition according to any one of claims 88 to 92, or (c) The antibody complex according to any one of claims 77 to 84, Administering, or A population of Treg cells (for example, a population infiltrated by Treg cells) (a) an isolated antibody according to any one of claims 1 to 76, (b) A pharmaceutical composition according to any one of claims 88 to 92, or (c) The antibody complex according to any one of claims 77 to 84, By bringing it into contact with the Treg cells, the level of the Treg cells is reduced and / or the Treg cells are inhibited. The method, including the method described above.

107. A method for enhancing the immune response in the target area, Effective amount (a) an isolated antibody according to any one of claims 1 to 76, (b) A pharmaceutical composition according to any one of claims 88 to 92, or (c) The antibody complex according to any one of claims 77 to 84, The method comprising treating the subject by administering the following.

108. The subject is a person suffering from or at risk of having cancer or an infectious disease, according to any one of claims 102 to 107.

109. The method according to claim 108, wherein the cancer is a solid tumor.

110. The method according to claim 108, wherein the cancer is a hematological malignancy.

111. The method according to any one of claims 102 to 107, wherein the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

112. The method according to any one of claims 102 to 111, wherein the subject is a human.

113. The method according to any one of claims 102 to 111, further comprising administering an effective amount of a second therapeutic agent.

114. As a result of the aforementioned administration or contact, (a) To enhance the immune response (e.g., the immune response against tumors), (b) Reducing, depleting, or killing Treg cells (e.g., tumor-invasion regulatory T ("Treg") cells) (c) Inducing the internal translocation of CCR8 polypeptide within Treg cells (e.g., tumor-invasion-regulating T ("Treg") cells), (d) To bind to the CCR8 polypeptide or a portion thereof (for example, human CCR8), (e) Binding to the CCR8 polypeptide or a portion thereof (e.g., human CCR8) by a KD of 10 nM or less as measured by BIACORE (trademark), (f) Inducing antibody-dependent cell-mediated cytotoxicity (ADCC) in cells expressing the CCR8 polypeptide or a portion thereof (for example, Treg cells such as tumor-infiltrating Treg cells), or (g) Any combination of them, The method according to any one of claims 102 to 113, which results in the following:

115. The method according to any one of claims 102 to 114, wherein the administration is via a subcutaneous, intravenous, intradermal, intraperitoneal, intramuscular, intraventricular, intracranial, intracavitary, or intracerebellar route of administration.

116. The method according to any one of claims 102 to 115, wherein the contact is in vivo or in vitro.

117. The aforementioned method, (a) Culturing the in vitro cells according to claim 87 in a culture medium under conditions that enable the expression of polypeptide encoded by the isolated nucleic acid molecule and the assembly of the isolated antibody, (b) Purifying the isolated antibody from the culture medium of the cultured cells or the in vitro cells, A method for producing an isolated antibody according to any one of claims 1 to 76, comprising:

118. An isolated antibody according to any one of claims 1 to 76, The antibody complex according to any one of claims 77 to 84, The isolated nucleic acid molecule according to claim 85, or The vector according to claim 86, A kit that includes this.

119. The kit according to claim 118, further comprising an effective amount of one or more further therapeutic agents.

120. The kit according to claim 119, wherein the one or more further therapeutic agents include anticancer agents, cytotoxic drugs, NSAIDs, corticosteroids, nutritional supplements such as antioxidants, siRNA, small molecules, oligonucleotides, chemotherapeutic agents, or combinations thereof.

121. An isolated antibody comprising a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain comprises a wild-type CCR8 binding domain containing the amino acid sequence of SEQ ID NO: 27, numbered according to the IMGT scheme. Amino acid substitutions at Q1, Q5, A15, V24, Q49, R50, L52, F70, A83, K95, P96, Q123, or combinations thereof. The isolated antibody, including the above.

122. The aforementioned CCR8 binding domain is The amino acid substitutions in Q1, Q5, A15, V24, Q49, R50, L52, F70, A83, K95, P96, and Q123, The isolated antibody according to claim 121, comprising

123. The aforementioned CCR8 binding domain is The amino acid substitution in Q1, the amino acid substitution in Q5, the amino acid substitution in A15, the amino acid substitution in V24, the amino acid substitution in Q49, the amino acid substitution in A83, the amino acid substitution in K95, and the amino acid substitution in Q123, The isolated antibody according to claim 121 or claim 122, comprising

124. An isolated antibody according to any one of claims 121 to 123, wherein the amino acid substitution in Q1 is Q1V, the amino acid substitution in Q5 is Q5V, the amino acid substitution in A15 is A15P, the amino acid substitution in V24 is V24A, the amino acid substitution in Q49 is Q49G, the amino acid substitution in R50 is R50L, the amino acid substitution in L52 is L52W, the amino acid substitution in F70 is F70S, the amino acid substitution in A83 is A83S, the amino acid substitution in K95 is K95R, the amino acid substitution in P96 is P96A, and the amino acid substitution in Q123 is Q123T.

125. An isolated antibody containing a chemokine receptor 8 (CCR8) binding domain, wherein the CCR8 binding domain is numbered according to the IMGT scheme and contains the amino acid sequence of SEQ ID NO: 28 relative to a wild-type CCR8 binding domain. Amino acid substitutions at Q1, Q5, A15, Q49, R50, L52, R77, A83, R84, S85, A86, L120, Q123, or combinations thereof. The isolated antibody, including the above.

126. The aforementioned CCR8 binding domain is The amino acid substitution in Q1, the amino acid substitution in Q5, the amino acid substitution in A15, the amino acid substitution in Q49, the amino acid substitution in R50, the amino acid substitution in L52, the amino acid substitution in R77, the amino acid substitution in A83, the amino acid substitution in R84, the amino acid substitution in S85, the amino acid substitution in A86, the amino acid substitution in L120, and the amino acid substitution in Q123, The isolated antibody according to claim 125, comprising

127. The aforementioned CCR8 binding domain is The amino acid substitution in Q1, the amino acid substitution in Q5, the amino acid substitution in A15, the amino acid substitution in Q49, the amino acid substitution in R77, the amino acid substitution in A83, the amino acid substitution in R84, the amino acid substitution in S85, the amino acid substitution in A86, the amino acid substitution in L120, and the amino acid substitution in Q123, The isolated antibody according to claim 125 or claim 126, comprising

128. The isolated antibody according to any one of claims 125 to 127, wherein the amino acid substitution in Q1 is Q1V, the amino acid substitution in Q5 is Q5V, the amino acid substitution in A15 is A15P, the amino acid substitution in Q49 is Q49G, the amino acid substitution in R50 is R50L, the amino acid substitution in L52 is L52W, the amino acid substitution in R77 is R77T, the amino acid substitution in A83 is A83S, the amino acid substitution in R84 is R84K, the amino acid substitution in S85 is S85N, the amino acid substitution in A86 is A86Y, the amino acid substitution in L120 is L120Q, and the amino acid substitution in Q123 is Q123T.

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