anti-CCR8 antibodies

Abstract

The present invention relates to antigen binding proteins and related fragments thereof that bind to CCR8, the production of said antigen binding proteins and fragments, and the use of said antigen binding proteins and fragments for the detection and treatment of various disease states.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to antigen-binding proteins and related fragments thereof that bind to CCR8, the production of said antigen-binding proteins and fragments, and the use of said antigen-binding proteins and fragments for the detection and treatment of various pathological conditions.

[0002] Related Applications

[0002] This application claims priority to Australian Provisional Application No. 2022902742, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Background of the Invention Immunotherapy is a rapidly evolving and very promising treatment for various forms of cancer, with many recent successes. However, some patients respond poorly or not at all to current immunotherapies, and others experience relapse after an initial response.

[0004]

[0004] The human immune system is equipped with checks and balances that act to prevent an overactive immune system from harming the body. Regulatory T cells ("Tregs") play a crucial role in maintaining a functional immune system by suppressing immune responses. In particular, Tregs are a major immune cell population that play a key role in maintaining self-tolerance and resolving immune responses using multifaceted immunoregulatory mechanisms. However, Tregs readily infiltrate the tumor microenvironment (TME) and suppress anti-tumor immune responses, thereby posing a barrier to effective cancer immunotherapy. Treg modulation strategies have been shown to enhance anti-tumor immunity and reduce tumor burden in both preclinical and clinical settings.

[0005] CC chemokine receptor 8 (CCR8) is a chemokine receptor selectively expressed on a subset of intratumoral Tregs with the highest levels of suppressive markers, and its expression correlates with poor prognosis in multiple tumor types. This subset of CCR8-expressing Tregs (CCR8+ Tregs) has been shown to be a key factor promoting immunosuppression and is important for Treg function and suppression.

[0006]

[0006] Therefore, there remains a need in the art to develop CCR8-targeted therapeutic agents, such as anti-CCR8 antibodies, that can be used for therapeutic purposes in the treatment of cancer.

[0007]

[0007] Reference to prior art in this specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, be considered relevant, and / or could reasonably be expected to be combined with other prior art by a person skilled in the art. Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention In one aspect, the present invention provides an antigen binding protein that binds to or specifically binds to extracellular loop 2 of CCR8, preferably which antigen binding protein does not block the binding of CCR8 to CCL1.

[0009] In any embodiment, the antigen binding protein binds to residues 172-202 of CCR8 (wherein the amino acid sequence of CCR8 is as set forth in SEQ ID NO: 55). In any embodiment, the antigen binding protein binds to residues 172-190 of CCR8 (wherein the amino acid sequence of CCR8 is as set forth in SEQ ID NO: 55). In any embodiment, the antigen binding protein binds to residues 176-179 of CCR8 (wherein the amino acid sequence of CCR8 is as set forth in SEQ ID NO: 55).

[0010] In one aspect, the invention provides an antigen binding protein comprising CDRH1, CDRH2 and / or CDRH3 of an antibody having a variable heavy chain as defined in SEQ ID NO:1.

[0011] In another aspect, the present invention provides an antigen binding protein comprising CDRL1, CDRL2 and / or CDRL3 of an antibody having a variable light chain as defined in SEQ ID NO:2.

[0012]

[0012] In another aspect, the present invention provides an antigen binding protein comprising CDRH1, CDRH2 and / or CDRH3 of an antibody having a variable heavy chain defined in SEQ ID NO: 1 and CDRL1, CDRL2 and / or CDRL3 of an antibody having a variable light chain defined in SEQ ID NO: 2.

[0013] In another aspect, the present invention provides an antigen binding protein for binding to CCR8, comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a (where, FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are complementarity determining regions; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a, and CDR3a are complementarity determining regions. and wherein the sequence of any of the framework regions or complementarity determining regions is as described herein.

[0014] In another aspect, the present invention provides an antigen binding protein for binding to CCR8, comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a (where, FR1, FR2, FR3 and FR4 are framework regions; CDR1, CDR2 and CDR3 are complementarity determining regions; FR1a, FR2a, FR3a and FR4a are framework regions, respectively; CDR1a, CDR2a, and CDR3a are complementarity determining regions. Including, Antigen-binding proteins are provided in which the sequence of any of the complementarity-determining regions has the amino acid sequence set forth in Table 1 below. Preferably, the framework regions have the amino acid sequence also set forth in Table 1 below, with amino acid mutations at specific residues that can be determined by aligning various framework regions from each antibody. The present invention also includes cases in which CDR1, CDR2, and CDR3 are sequences derived from the variable heavy chain (VH) of an antibody and CDR1a, CDR2a, and CDR3a are sequences derived from the variable light chain (VL) of an antibody, or CDR1, CDR2, and CDR3 are sequences derived from the VL and CDR1a, CDR2a, and CDR3a are sequences derived from the VH.

[0015]

[0015] In any embodiment, the antigen binding protein described herein comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a.

[0016]

[0016] As defined herein, a linker can be a chemical compound formed between two cysteine ​​residues, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0017]

[0017] In certain preferred embodiments, the present invention provides antigen-binding proteins comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NOs: 1 and 2 (in N-terminal to C-terminal or C-terminal to N-terminal order).

[0018] Optionally, the antigen binding protein comprises SEQ ID NO:2(VL)-linker-SEQ ID NO:1(VH) or SEQ ID NO:1(VH)-linker-SEQ ID NO:2(VL).

[0019]

[0019] In certain preferred embodiments, the present invention provides antigen-binding proteins comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NOs: 83 and 84 (in N-terminal to C-terminal or C-terminal to N-terminal order).

[0020] Optionally, the antigen binding protein comprises SEQ ID NO:84(VL)-linker-SEQ ID NO:83(VH), or SEQ ID NO:83(VH)-linker-SEQ ID NO:84(VL).

[0021]

[0021] In any embodiment, the present invention provides an antigen binding protein that binds to or specifically binds to CCR8 and competitively inhibits binding of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO:1 and a VL comprising the sequence set forth in SEQ ID NO:2.

[0022]

[0022] In any embodiment, the present invention provides an antigen binding protein that binds to or specifically binds to CCR8 and competitively inhibits binding of an antibody comprising a VH comprising the sequence set forth in SEQ ID NO: 83 and a VL comprising the sequence set forth in SEQ ID NO: 84.

[0023] In one aspect, the present invention also provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds to or specifically binds to CCR8, and the antigen binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:6, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:7; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:1; (iii) a VL comprising a CDR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:8, a CDR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:9, and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:2; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; (vi) a VH comprising the sequence set forth in SEQ ID NO: 1; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 2; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; or (x) VH comprising the sequence set forth in SEQ ID NO: 1 and VL comprising the sequence set forth in SEQ ID NO: 2.

[0024] In one aspect, the present invention also provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds to or specifically binds to CCR8, and the antigen binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:6, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:7; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 83; (iii) a VL comprising a CDR1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:8, a CDR2 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:9, and a CDR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 84; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; (vi) a VH comprising the sequence set forth in SEQ ID NO: 83; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 84; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; or (x) A VH comprising the sequence set forth in SEQ ID NO: 83, and a VL comprising the sequence set forth in SEQ ID NO: 84.

[0025] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a framework region (FR) 1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 11; a FR comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 12; 2. A VH comprising an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 13, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 14; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 15; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 18; (iii) a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 11, FR2 comprising the sequence set forth in SEQ ID NO: 12, FR3 comprising the sequence set forth in SEQ ID NO: 13, and FR4 comprising the sequence set forth in SEQ ID NO: 14; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 15, FR2 comprising the sequence set forth in SEQ ID NO: 16, FR3 comprising the sequence set forth in SEQ ID NO: 17, and FR4 comprising the sequence set forth in SEQ ID NO: 18; (v) A VH comprising an FR1 having the sequence set forth in SEQ ID NO: 11, an FR2 having the sequence set forth in SEQ ID NO: 12, an FR3 having the sequence set forth in SEQ ID NO: 13, and an FR4 having the sequence set forth in SEQ ID NO: 14; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 15, an FR2 having the sequence set forth in SEQ ID NO: 16, an FR3 having the sequence set forth in SEQ ID NO: 17, and an FR4 having the sequence set forth in SEQ ID NO: 18.

[0026] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 60; a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 61; 2. A VH comprising an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 62, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 63; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 64; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 65; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:66, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:67; (iii) a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 60, FR2 comprising the sequence set forth in SEQ ID NO: 61, FR3 comprising the sequence set forth in SEQ ID NO: 62, and FR4 comprising the sequence set forth in SEQ ID NO: 63; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 64, FR2 comprising the sequence set forth in SEQ ID NO: 65, FR3 comprising the sequence set forth in SEQ ID NO: 66, and FR4 comprising the sequence set forth in SEQ ID NO: 67; (v) A VH comprising an FR1 having the sequence set forth in SEQ ID NO: 60, an FR2 having the sequence set forth in SEQ ID NO: 61, an FR3 having the sequence set forth in SEQ ID NO: 62, and an FR4 having the sequence set forth in SEQ ID NO: 63; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 64, an FR2 having the sequence set forth in SEQ ID NO: 65, an FR3 having the sequence set forth in SEQ ID NO: 66, and an FR4 having the sequence set forth in SEQ ID NO: 67.

[0027] In one aspect, the present invention also provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds to or specifically binds to CCR8, and the antigen binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 21; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:1; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 22, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:2; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; (vi) a VH comprising the sequence set forth in SEQ ID NO: 1; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 2; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; or (x) VH comprising the sequence set forth in SEQ ID NO: 1, and VL comprising the sequence set forth in SEQ ID NO: 2.

[0028] In one aspect, the present invention also provides an antigen binding protein comprising an antigen binding domain of an antibody, wherein the antigen binding domain binds to or specifically binds to CCR8, and the antigen binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 21; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 83; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 22, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 84; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; (vi) a VH comprising the sequence set forth in SEQ ID NO: 83; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 84; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (x) A VH comprising the sequence set forth in SEQ ID NO: 83, and a VL comprising the sequence set forth in SEQ ID NO: 84.

[0029] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a framework region (FR) 1 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 24; a FR comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 25; 2. A VH comprising an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:26, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:14; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 27; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 28; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:29, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:18; (iii) a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 24, FR2 comprising the sequence set forth in SEQ ID NO: 25, FR3 comprising the sequence set forth in SEQ ID NO: 26, and FR4 comprising the sequence set forth in SEQ ID NO: 14; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 27, FR2 comprising the sequence set forth in SEQ ID NO: 28, FR3 comprising the sequence set forth in SEQ ID NO: 29, and FR4 comprising the sequence set forth in SEQ ID NO: 18; (v) A VH comprising an FR1 having the sequence set forth in SEQ ID NO: 24, an FR2 having the sequence set forth in SEQ ID NO: 25, an FR3 having the sequence set forth in SEQ ID NO: 26, and an FR4 having the sequence set forth in SEQ ID NO: 14; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 27, an FR2 having the sequence set forth in SEQ ID NO: 28, an FR3 having the sequence set forth in SEQ ID NO: 29, and an FR4 having the sequence set forth in SEQ ID NO: 18.

[0030] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 68; a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 69; 2. A VH comprising an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 70, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 63; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 71; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 72; a VL comprising an FR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:73, and an FR4 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:67; (iii) a VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 68, FR2 comprising the sequence set forth in SEQ ID NO: 69, FR3 comprising the sequence set forth in SEQ ID NO: 70, and FR4 comprising the sequence set forth in SEQ ID NO: 63; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 71, FR2 comprising the sequence set forth in SEQ ID NO: 72, FR3 comprising the sequence set forth in SEQ ID NO: 73, and FR4 comprising the sequence set forth in SEQ ID NO: 67; (v) A VH comprising an FR1 having the sequence set forth in SEQ ID NO: 68, an FR2 having the sequence set forth in SEQ ID NO: 69, an FR3 having the sequence set forth in SEQ ID NO: 70, and an FR4 having the sequence set forth in SEQ ID NO: 63; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 71, an FR2 having the sequence set forth in SEQ ID NO: 72, an FR3 having the sequence set forth in SEQ ID NO: 73, and an FR4 having the sequence set forth in SEQ ID NO: 67.

[0031] As described herein, the antigen binding protein may be in the form of: (i) single chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); or (iii) one of (i) or (ii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0032] Furthermore, as described herein, the antigen binding protein may be in the form of: (i) diabody; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab')2; (vi) Fv; (vii) bispecific or other forms of multispecific antibodies; or (viii) One of (i) to (vii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of an antibody.

[0033]

[0033] The above-mentioned antigen-binding protein may also be referred to as an antigen-binding domain of an antibody.

[0034] In certain embodiments, the complementarity determining region sequences (CDRs) of the antigen binding proteins of the present invention may be defined according to the IMGT numbering system, the Kabat system, or the Chothia system.

[0035] In any aspect or embodiment, the antigen binding protein may comprise a human constant region.

[0036]

[0036] References herein to a protein or antibody that "binds" to CCR8 provide literal support for the protein or antibody "binds specifically to" or "specifically binds to" CCR8.

[0037]

[0037] Preferably, the antigen-binding protein described herein is an antibody or an antigen-binding fragment thereof. Typically, the antigen-binding protein is an antibody, for example a monoclonal antibody. The antigen-binding protein may be in the form of a recombinant antibody or an engineered antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a primatized antibody, a deimmunized antibody, a synthetic humanized antibody, a half antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody). The antibody may further comprise a chemical modification such as an active drug or a radiolabel, or conjugation to an agent to improve solubility, or other modification as described herein.

[0038] In any aspect of the invention, the antigen binding protein further comprises one or more human constant regions. In one embodiment, the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 58 and / or 59.

[0039] In another aspect, the invention also provides a method for producing an antibody comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region CDR H1 as set forth in SEQ ID NO: 5, CDR H2 as set forth in SEQ ID NO: 6, and CDR H3 as set forth in SEQ ID NO: 7 Includes; The light chain variable region CDR L1 as set forth in SEQ ID NO: 8, CDR L2 as set forth in SEQ ID NO: 9, and CDR L3 as set forth in SEQ ID NO: 10 Contains Anti-CCR8 antibodies or antigen-binding fragments thereof are provided.

[0040]

[0040] In any embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises a light chain variable region comprising FR L1 set forth in SEQ ID NO: 15, FR L2 set forth in SEQ ID NO: 16, FR L3 set forth in SEQ ID NO: 17, and FR L4 set forth in SEQ ID NO: 18.

[0041]

[0041] In any embodiment of the present invention, the anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising FR H1 set forth in SEQ ID NO: 11, FR H2 set forth in SEQ ID NO: 12, FR H3 set forth in SEQ ID NO: 13, and FR H4 set forth in SEQ ID NO: 14.

[0042]

[0042] In any embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises a light chain variable region comprising FR L1 set forth in SEQ ID NO: 64, FR L2 set forth in SEQ ID NO: 65, FR L3 set forth in SEQ ID NO: 66, and FR L4 set forth in SEQ ID NO: 67.

[0043]

[0043] In any embodiment of the present invention, the anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising FR H1 set forth in SEQ ID NO: 60, FR H2 set forth in SEQ ID NO: 61, FR H3 set forth in SEQ ID NO: 62, and FR H4 set forth in SEQ ID NO: 63.

[0044] In another aspect, the invention also provides a method for producing an antibody comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region CDR H1 as set forth in SEQ ID NO: 19, CDR H2 as set forth in SEQ ID NO: 20, and CDR H3 as set forth in SEQ ID NO: 21 Includes; The light chain variable region CDR L1 as set forth in SEQ ID NO: 22, CDR L2 as set forth in SEQ ID NO: 23, and CDR L3 as set forth in SEQ ID NO: 10 Contains Anti-CCR8 antibodies or antigen-binding fragments thereof are provided.

[0045]

[0045] In any embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises a light chain variable region comprising FR L1 set forth in SEQ ID NO: 27, FR L2 set forth in SEQ ID NO: 28, FR L3 set forth in SEQ ID NO: 29, and FR L4 set forth in SEQ ID NO: 18.

[0046]

[0046] In any embodiment of the present invention, the anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising FR H1 set forth in SEQ ID NO: 24, FR H2 set forth in SEQ ID NO: 25, FR H3 set forth in SEQ ID NO: 26, and FR H4 set forth in SEQ ID NO: 14.

[0047]

[0047] In any embodiment, the anti-CCR8 antibody or its antigen-binding fragment comprises a light chain variable region comprising FR L1 set forth in SEQ ID NO: 71, FR L2 set forth in SEQ ID NO: 72, FR L3 set forth in SEQ ID NO: 73, and FR L4 set forth in SEQ ID NO: 67.

[0048]

[0048] In any embodiment of the present invention, the anti-CCR8 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising FR H1 set forth in SEQ ID NO: 68, FR H2 set forth in SEQ ID NO: 69, FR H3 set forth in SEQ ID NO: 70, and FR H4 set forth in SEQ ID NO: 63.

[0049]

[0049] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:2.

[0050]

[0050] In any embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:1.

[0051]

[0051] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:84.

[0052]

[0052] In any embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:83.

[0053]

[0053] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:2 and a heavy chain variable region comprising the sequence of SEQ ID NO:1.

[0054]

[0054] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:84 and a heavy chain variable region comprising the sequence of SEQ ID NO:83.

[0055] In any aspect or embodiment, the antigen-binding protein or anti-CCR8 receptor antibody or antigen-binding fragment thereof may further comprise up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 0, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the amino acid sequence of the indicated SEQ ID NO. Preferably, the amino acid insertions, deletions, substitutions, or a combination thereof are not present in the CDRs.

[0056] In any aspect of the invention and any antigen binding protein described herein, the antibody further comprises an Fc region that has been engineered to have a reduced or enhanced ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the reduced or enhanced ability to induce ADCC is conferred by mutation, deletion or modification of amino acids in the Fc region that interact with Fc receptors.

[0057]

[0057] In another aspect, the present invention provides an anti-CCR8 antigen binding protein, an immunoglobulin variable domain, an antibody or antigen-binding fragment thereof, an scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or a multispecific antibody comprising an antigen binding protein having a sequence described herein or comprising a CDR and / or FR sequence described herein.

[0058] In any embodiment, the antigen binding protein is an antibody of the IgG2b isotype.

[0059] The antigen binding proteins described herein may comprise a human constant region, for example, an IgG constant region, such as an IgG1, IgG2, IgG3, or IgG4 constant region, or a mixture thereof.H and V L In the case of an antibody or protein containing V H can be linked to a heavy chain constant region, and V L can be linked to a light chain constant region.

[0060] In one example, the antigen binding protein described herein comprises the constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one example, the protein or antibody comprises an IgG4 constant region having a proline at position 241 (according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991)).

[0061] In one example, the antigen binding protein comprises a VLK antibody as disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as described above). H Including V L is linked or fused to a kappa light chain constant region.

[0062] In any embodiment of the present invention, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and has not been adapted to form a conjugate.

[0063] In any embodiment of the invention, the antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the antibody exhibits the same or similar level of ADCC as 19D7 (anti-CCR8 Shionogi antibody). ADCC can be measured by any method known in the art or any method described herein (including Example 11).

[0064] In any aspect of the invention, the antigen binding protein depletes or reduces tumor-infiltrating T regulatory (Treg) cells in the tumor (optionally, the antigen binding protein does not deplete or reduce Tregs in the spleen).

[0065] In any aspect of the invention, the antigen binding protein increases tumor-specific CD8+ T cells and / or decreases CD4+ T cells or tumor-specific CD4+ T cells.

[0066] In another aspect, the present invention also provides a conjugate in the form of an antigen-binding protein, immunoglobulin variable domain, antibody or antigen-binding fragment thereof, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody or fusion protein as described herein, linked to a label or a cytotoxic agent.

[0067]

[0067] In aspects of the invention directed to multiple polypeptide chains forming an antigen-binding protein, the expression construct comprises, for example, a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter, and a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0068] In another example, the expression construct is a bicistronic expression construct, for example, comprising the following operably linked components in 5' to 3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide; (wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa).

[0069] In another aspect, the present invention also contemplates separate expression constructs, one of which encodes a first polypeptide comprising a VH and another of which encodes a second polypeptide comprising a VL. For example, the present invention also provides a composition comprising: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter.

[0070] In another aspect, the invention provides a cell comprising the vector or nucleic acid described herein. Preferably, the cell is isolated, substantially purified, or recombinant. In one example, the cell comprises an expression construct of the invention, or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL operably linked to a promoter; (wherein the first and second polypeptides associate to form the antigen binding protein of the invention).

[0071]

[0071] Examples of cells of the present invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0072]

[0072] In another aspect, the present invention provides an antigen-binding protein, immunoglobulin variable domain, antibody or antigen-binding fragment thereof, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate as described herein.

[0073]

[0073] In another aspect, the present invention provides a vector comprising a nucleic acid described herein.

[0074]

[0074] In another aspect, the present invention provides a cell comprising a vector or nucleic acid described herein.

[0075]

[0075] In any embodiment, the nucleic acid may comprise a nucleotide sequence set forth in any of SEQ ID NOs: 3, 4 or 30-54, preferably SEQ ID NOs: 3 and / or 4, or a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

[0076]

[0076] In another aspect, the present invention provides a pharmaceutical composition comprising an antigen binding protein, or the CDR and / or FR sequences described herein, or an immunoglobulin variable domain, antibody or antigen-binding fragment thereof, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0077]

[0077] In another aspect, the present invention provides a kit or article of manufacture comprising an antigen binding protein, or comprising the CDR and / or FR sequences described herein, or the immunoglobulin variable domain, antibody or antigen-binding fragment thereof, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein or conjugate described herein.

[0078]

[0078] In another aspect, the present invention provides the use of a sequence comprising one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 described herein for generating an antigen binding protein that binds to CCR8.

[0079]

[0079] In another aspect, the present invention provides a method for producing an antigen-binding protein, antibody or antigen-binding fragment that binds to CCR8 as described herein, the method comprising expressing a nucleic acid as described herein in a cell or animal as described herein.

[0080]

[0080] The functional characteristics of the antigen-binding proteins of the invention are understood to apply mutatis mutandis to the antibodies or antigen-binding fragments of the invention.

[0081]

[0081] The antigen binding proteins described herein may be purified, substantially purified, isolated, and / or recombinant.

[0082]

[0082] The antigen binding protein of the present invention may be part of the supernatant taken from the culture in which a hybridoma expressing the antigen binding protein of the present invention is growing.

[0083] In another aspect, the present invention provides a method of preventing or treating a condition or disease associated with expression of CCR8 in an individual, the method comprising the step of providing to an individual in need of treatment for said condition or disease an antigen-binding protein, immunoglobulin variable domain, antibody or antigen-binding fragment thereof, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein, conjugate, or pharmaceutical composition as described herein. The disease or condition associated with expression of CCR8 is preferably cancer.

[0084] In another aspect, the present invention also provides a method of treating or preventing cancer in a subject, comprising administering to the subject an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the invention, thereby treating or preventing cancer in the subject. As used herein, a method of treating cancer includes a method of inhibiting, preventing, or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

[0085] In another aspect, the present invention also provides a method for reducing or depleting T regulatory cells in a tumor, the method comprising administering to a subject an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the present invention, thereby reducing or depleting T regulatory cells in the tumor.

[0086]

[0086] In another aspect, the present invention also provides a method for increasing tumor-specific CD8+ T cells and / or decreasing CD4+ T cells in a tumor, the method comprising administering to a subject an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the present invention, thereby increasing tumor-specific CD8+ T cells and / or decreasing CD4+ T cells in the tumor.

[0087] In another aspect, the present invention also provides the use of an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment or prevention of cancer in a subject.

[0088] In another aspect, the present invention also provides the use of an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the invention in the manufacture of a medicament for: Reducing or depleting T regulatory cells in tumors; or Increasing tumor-specific CD8+ T cells and / or decreasing CD4+ T cells in the tumor.

[0089]

[0089] In another aspect, the present invention provides an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the present invention for use in treating or preventing cancer in a subject.

[0090] In another aspect, the present invention provides an antigen-binding protein, antibody or antigen-binding fragment, or pharmaceutical composition of the present invention for use in: Reducing or depleting T regulatory cells in tumors; or Increasing tumor-specific CD8+ T cells and / or decreasing CD4+ T cells in the tumor.

[0091]

[0091] As used in this specification, unless the context otherwise requires, the term "comprise" and variations of that term, such as "comprising," "comprises," and "comprised," are not intended to exclude further additives, components, integers, or steps.

[0092]

[0092] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0093] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Reactivity of 2H8 antibody to both human and mouse transfected CHO-Flpn cell lines. 2H8 reactivity with mouse CCR8 (blue) or human CCR8 (orange) CHO-Flpn cells and non-transfected CHO Flpn (red) compared with (A) anti-hCCR8#1 antibody or (B) anti-hCCR8 (clone #L263G8 Biolegend). [Figure 2]

[0094] Reactivity of 2H8 antibody and other anti-hCCR8 antibodies to Cyno CCR8. HEK293T cells were transfected with a plasmid encoding cynomolgus monkey CCR8 and stained with various anti-hCCR8 antibodies (blue) or isotype control (red). [Figure 3]

[0095] Reactivity of the 2H8 antibody to other CCR chemokine receptors in transfected L1.2 cells. 2H8-B4 does not cross-react with non-transfected cells (purple); human CCR3 (pink); human CCR6 (dark green); human CCR7 (lime); or human CCR10 (blue) (order of non-binding histogram from top to bottom). 2H8-B4 binds to both human CCR8 (orange) and mouse CCR8 (red) transfected L1.2 cells (order of binding histogram from top to bottom). [Figure 4]

[0096] 2H8 binds to human tumor-infiltrating Tregs (TiTregs). Single-cell suspensions were isolated using fresh colon patient biopsies. Cells were stained with anti-human CD3, anti-human CD4, anti-human FoxP3, and 2H8-B4 or an isotype control. The histogram shows that 2H8-B4 (red) binds to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gates), but the isotype control does not (gray). [Figure 5]

[0097] Binding of hCCR8 antibodies to hCCR8-L1.2 transfected cells. The chimeric 2H8-B4 antibody was compared to chimeric 19D7 (Shionogi antibody) and chimeric 4A19 (BMS antibody) by flow cytometry in hCCR8-L1.2 transfected cells using increasing concentrations of purified mAb. [Figure 6]

[0098] Binding of humanized 2H8 to hCCR8-L1.2 transfected cells. Humanized 2H8-B4 was compared to 2H8 B4 (parent) and 19D7 (Shionogi antibody) by flow cytometry in hCCR8-L1.2 transfected cells using increasing concentrations of purified mAb. [Figure 7]

[0099] Single-cell suspensions were isolated using fresh colon patient biopsies. Cells were stained with anti-human CD3, anti-human CD4, anti-human FoxP3, and 2H8-B4 (right) or an isotype control (left). 2H8-B4 (right) binds to tumor-infiltrating Tregs (CD3+, CD4+, FoxP3+ gates), whereas the isotype control (left) does not. [Figure 8]

[0100] Chimeric 2H8 induces chemotaxis without inhibiting CCL1. Two hundred thousand hCCR8 L1.2 cells per well were pre-incubated with different concentrations of antibody at 37°C for 15 minutes. The cells were then transferred to a chemotaxis plate and incubated overnight (4 hours) with the same concentration gradient of antibody for chemotaxis to EC90 (hCCL1 - 5.84 nM). 100 μl of the bottom well was then taken, spun down, and resuspended in 50 μl of FACS buffer. 15 μl was then counted using an LSRIIb machine. [Figure 9]

[0101] Chimeric 2H8 exhibits ADCC potential with efficacy and potency comparable to that of 19D7. Dose-response curves of ADCC assays were performed to analyze the cytotoxicity-inducing potential of both chimeric 2H8 and chimeric 19D7 using L1.2-hCCR8 cells incubated with various antibody concentrations. The highest antibody concentration was 13.33 nM, and four-fold dilutions were performed to generate dose-response curves. Antibody concentrations were transformed to a logarithmic scale and plotted on the x-axis. Statistical analysis was performed using a sigmoidal line of best fit to calculate the IC50 and R2 for each antibody. [Figure 10]

[0102] 2H8 inhibits tumor growth in the MC38 model of colon cancer. Individual volume plots of MC38 tumors implanted subcutaneously in C57BL / 6 mice at 5x10 cells / injection. Animals were treated with 2H8 mIgG2a (n=9), anti-mPD1 (n=10), and isotype control (n=6). After the first antibody injection, tumor volume (mm3) was measured daily using digital calipers and compared between groups. Treatment was at a dose of 5 mg / kg, twice weekly for up to 3 weeks. [Figure 11]

[0103] 2H8 depleted Tregs in tumors but not in spleens. At endpoint, tumor and spleen tissues were processed, stained, and analyzed by flow cytometry. The percentages of CD4+FOXP3+CD25+ cells in the TCR-β+ cells (parent population) were collected and compared among the three groups. All graphs show mean and individual values, as well as histograms, scatter plots, and error bars showing SEM. (A) Representative flow cytometry scatter plots of the three groups, showing the mean ± SEM of the percentage of CD4+FOXP3+CD25+ cells. (B) Percentage of Tregs in tumors of C57BL / 6 wild-type mice. (C) Percentage of Tregs in spleens of C57BL / 6 wild-type mice. 2H8 group; n = 9, anti-PD-1 group; n = 10, control group; n = 6. Normality test using Shapiro-Wilk method. (b) Kruskal-Wallis with Dunn's multiple comparisons. (c) Ordinary one-way ANOVA with Tukey's multiple comparisons. *p<0.05, **p<0.005. [Figure 12]

[0104] Treatment with 2H8 increased tumor-specific CD8+ T cells and decreased CD4+ T cells. At endpoint, tumor and spleen tissues from MC38 tumors in C57BL / 6 mice were processed, stained, and analyzed by flow cytometry. The percentages of CD4+ and CD8+ cells in the TCR-β+ cells (parent population) were collected and compared among the three groups. All graphs show mean and individual values, as well as histograms, scatter plots, and error bars showing SEM. (A) Representative flow cytometry scatter plots of tumors from three different groups, showing the mean ± SEM of CD8+ T cells. (B) Percentage of CD8+ T cells in tumors. (C) CD8 / T-reg ratio normalized to tumor weight. (D) Percentage of CD4+ T cells in tumors. (E) Representative flow cytometry scatter plots of CD62L and CD44 markers in tumors from three different groups. (F) Percentage of CD62LloCD44hiCD8+ T cells in tumors. (G) Percentage of CD62LhiCD44hiCD8+ T cells in the tumor. (H) Percentage of CD62LloCD44loCD8+ T cells in the tumor. (I) Percentage of CD62LhiCD44loCD8+ T cells in the tumor. 2H8 group; n=9, anti-PD-1 group; n=10, control group; n=6. Normality test using the Shapiro-Wilk method (b, d, e, g, h). Ordinary one-way ANOVA with Tukey's multiple comparisons. (c) Kruskal-Wallis with Dunn's multiple comparisons. *p<0.05, **p<0.005, ***p=0.0001, ****p<0.0001). DETAILED DESCRIPTION OF THE INVENTION

[0094] Array Description

[0095] [Table 1]

[0096] [Table 2]

[0097] [Table 3]

[0098] [Table 4]

[0099] [Table 5]

[0100] [Table 6]

[0101] Detailed Description of the Embodiments

[0069] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which:

[0102]

[0070] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all modifications, variations, and equivalents included within the scope of the present invention as defined by the claims.

[0103] Various antigen-binding proteins that bind to CCR8 are known, but lack one or more properties that would make them suitable for preclinical or clinical development. The CCR8-binding antigen-binding proteins described herein exhibit the advantageous property of binding not only to human CCR8, but also to mouse CCR8 and cynomolgus monkey (cyno) CCR8. This provides an opportunity to explore the properties of the antigen-binding proteins in preclinical models of both mouse and cynomolgus monkeys. Furthermore, the antigen-binding proteins also exhibit specificity for CCR8, as they do not bind to many closely related receptors (as shown herein). Surprisingly, the CCR8 antigen-binding proteins described in the Examples that bind to human, mouse, and cynomolgus monkey CCR8 were generated by immunizing mice with mouse CCR8.

[0104]

[0072] Furthermore, the CCR8 antibodies generated were then humanized and, surprisingly, the binding affinity to human CCR8 was retained, as was the ability to bind to live cells.

[0105] General Terms and Definitions Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition, group of steps or compositions shall be construed as including one and a plurality (i.e., one or more) of those steps, compositions, group of steps or compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly dictates otherwise. For example, reference to "a" includes the singular and more than one, reference to "an" includes the singular and more than one, reference to "the" includes the singular and more than one, etc.

[0106]

[0074] Those skilled in the art will understand that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all steps, features, compositions and compounds referred to or shown in this specification, individually or collectively, and any combination of said steps or features, or any two or more thereof.

[0107]

[0075] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0108]

[0076] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0109]

[0077] The present invention is not limited in scope by the specific examples described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention.

[0110] For the purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that a stated definition conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0111]

[0079] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0112]

[0080] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors), Current Protocols in It is described and explained throughout the literature in sources such as Immunology, John Wiley & Sons (including all updates to date).

[0113]

[0081] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by a discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., J. Mol. Biol. 242, 309-320, 1994, Chothia and Lesk J. Mol. Biol. 196:901-917, 1987, Chothia et al. Nature 342, 877-883, 1989, Martin (“enhanced Chothia”; Mol. Immunol. (2008) 45:3832-9, and / or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0114]

[0082] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y", and is interpreted as clearly endorsing both meanings or either meaning.

[0115]

[0083] As used herein, the term "derived from" is intended to indicate that a particular integer is not necessarily obtained directly from a particular source, but may be obtained from that source.

[0116] As used herein, the term "CCR8" or "CC chemokine receptor type 8" refers to a G protein-coupled receptor. CCR8 is known to have at least four ligands: 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 on Treg cells, with low expression on a small proportion of TH2 cells, monocytic cells, NK cells, and CD8+ cells. CCR8 is a transmembrane receptor with seven transmembrane domains, an extracellular N-terminal domain, and an intracellular C-terminal domain that interacts with G proteins. Exemplary amino acid sequences of human CCR8 (SEQ ID NO: 55), mouse CCR8 (SEQ ID NO: 56), and cynomolgus monkey CCR8 (SEQ ID NO: 57) are shown in Table 1.

[0117] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or source of derivation, is not associated with naturally associated components which accompany it in its natural state and is substantially free of other proteins from the same source. A protein can be rendered substantially free of naturally associated components or substantially purified by isolation using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free from contaminants, e.g., at least about 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% free of contaminant substances.

[0118] The term "recombinant" is understood to mean the product of artificial genetic recombination. Thus, in the context of a recombinant protein containing an antibody antigen-binding domain, this term does not encompass naturally occurring antibodies in a subject's body that are the product of natural recombination that occurs during B-cell maturation. However, when such an antibody is isolated, it is considered an isolated protein containing an antibody antigen-binding domain. Similarly, when a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant protein also encompasses proteins expressed by artificial recombinant means, for example, when they are present in the cell, tissue, or subject in which they were expressed.

[0119] The term "protein" is intended to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains linked to each other covalently or non-covalently (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. A protein may also include one or more unnatural amino acids.

[0120]

[0088] The term "polypeptide" or "polypeptide chain" is understood from the preceding paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0121] As used herein, the term "antigen-binding domain" is intended to mean a region of an antibody capable of specifically binding to an antigen, i.e., a VH or VL, or an Fv comprising both a VH and a VL. The antigen-binding domain need not be in the context of a whole antibody, but may be in another form, such as, for example, an scFv, as described herein.

[0122]

[0090] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or a small number of closely related antigens via an antigen-binding domain contained within the Fv. The term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or engineered antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies).

[0123]

[0091] Antibodies generally contain a constant domain, which may be arranged in a constant region, or constant fragment, or fragment crystallizable (Fc). An exemplary form of an antibody contains a four-chain structure as a basic unit. A full-length antibody contains two covalently linked heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa each). The light chains generally contain a variable region (if present) and a constant domain, and in mammals are kappa or lambda light chains. The heavy chains generally contain a variable region and one or two constant domains linked to additional constant domains by a hinge region. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, two heavy chains and heavy and light chains are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary among different antibody types. Each chain contains an N-terminal variable region (V H or V L (wherein each is about 110 amino acids in length)) and one or more constant domains at the C-terminus. L ) is the first constant domain of the heavy chain (C, which is 330-440 amino acids long). H1 ) and are disulfide bonded. The light chain variable region is aligned with the heavy chain variable region. An antibody heavy chain consists of two or more additional C H Domain (e.g., C H2 , C H3 ) and C H1 Constant domain and C H2A hinge region may be included between the constant domains. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as a human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is humanized, synthetically humanized, chimeric, CDR-grafted, or deimmunized.

[0124] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0125] As used herein, the term "variable region" refers to the portion of the light chain and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen, and includes the amino acid sequences of the complementarity-determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region includes three CDRs as well as three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). V H refers to the variable region of the heavy chain. L refers to the variable region of the light chain.

[0126] As used herein, the term "complementarity determining region" (synonymous CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region whose presence is a major contributor to specific antigen binding. Each variable region domain (V H or V L ) typically have three CDRs identified as CDR1, CDR2, and CDR3. HThe CDRs of are also referred to herein as CDR H1, CDR H2 and CDR H3, respectively, and CDR H1 is V H CDR1 corresponds to V H CDR2 corresponds to V, and CDR H3 corresponds to V H Similarly, V L The CDRs of are referred to herein as CDR L1, CDR L2 and CDR L3, respectively, and CDR L1 is V L CDR1 corresponds to V L CDR2 corresponds to V L In one example, the amino acid positions assigned to the CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another embodiment, the amino acid positions assigned to the CDRs and FRs are defined according to the extended Chothia numbering scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but includes any numbering system, including the standard numbering systems of Chothia and Lesk J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Pluekthun J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211, 1997.

[0127] "Framework regions" (FR) are those variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively; FR H1 is the V H FR1 corresponds to V, and FR H2 corresponds to V H FR2 corresponds to V, FR H3 corresponds to V H FR3 corresponds to V, FR H4 corresponds to V H Similarly, V L The FRs are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, and FR L1 is V L FR1 corresponds to V, and FR L2 corresponds to V L FR2 corresponds to V, and FR L3 corresponds to V L FR3 corresponds to V, FR L4 corresponds to V L Compatible with FR4.

[0128] As used herein, the term "Fv" refers to a V, whether composed of multiple polypeptides or a single polypeptide. L and V H The term "antigen-binding domain" is understood to mean any protein in which V associates with V to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. H and V L The V may be a single polypeptide chain or different polypeptide chains. Furthermore, an Fv of the invention (and any protein of the invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term is understood to encompass fragments derived directly from antibodies, as well as proteins corresponding to such fragments produced using recombinant means. In some instances, the V H is the heavy chain constant domain (C H ) 1 and / or V Lis not linked to a light chain constant domain (CL). Exemplary Fv-containing polypeptides or proteins include a Fab fragment, a Fab' fragment, a F(ab') fragment, an scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., a CH2 or CH3 domain, e.g., a minibody.

[0129] A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of whole antibodies with the enzyme papain to yield fragments consisting of an intact light chain and a portion of the heavy chain, or can be produced by recombinant means. An "Fab' fragment" of an antibody can be obtained by treating whole antibodies with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain, including the VH and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds and can be obtained by treating whole antibody molecules with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked, for example, using a leucine zipper or CH3 domain. A "single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

[0130] As used herein, the term "bind" in reference to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies generally recognize and bind to a particular protein structure, rather than the protein itself. If an antibody binds to epitope "A," then in a reaction containing labeled "A" and the protein, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody.

[0131] As used herein, the terms "specifically binds" or "binds specifically" are intended to mean that an antigen-binding protein of the invention reacts or associates with a particular antigen or a cell expressing it more frequently, more rapidly, for a longer duration, and / or with higher affinity than with another antigen or cell. For example, the antigen-binding protein binds to CCR8 with extremely high affinity (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold, or 200-fold) compared to other related molecules, such as other receptors described herein, including CCR3 (preferably human CCR3), CCR6 (preferably human CCR6), and / or CCR7 (preferably human CCR7). In examples of the present invention, the antigen binding protein "specifically binds" to CCR8 with at least 1.5-fold or 2-fold or more (e.g., 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold) affinity compared to the receptors described herein, including CCR3 (preferably human CCR3), CCR6 (preferably human CCR6), and / or CCR7 (preferably human CCR7). Generally, but not necessarily, reference to binding refers to specific binding, and it is understood that each term explicitly supports the other.

[0132] As used herein, the term "does not detectably bind" is understood to mean that the antigen-binding protein, e.g., antibody, binds to the candidate antigen at a level less than 10%, or less than 8%, or less than 6%, or less than 5% above background. Background can be the level of binding signal detected in the absence of protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0133] As used herein, the term "does not bind significantly" is understood to mean that the level of binding of an antigen-binding protein of the invention to a polypeptide is not statistically significantly higher than the background, e.g., the level of binding signal detected in the absence of the antigen-binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the binding level detected in the presence of a negative control polypeptide. The binding level is detected using a biosensor assay (e.g., Biacore or Blitz) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0134] As used herein, the term "epitope" (synonym "antigenic determinant") is understood to mean the region of CCR8 to which an antigen-binding protein, including the antigen-binding domain of an antibody, binds. Unless otherwise defined, the term is not necessarily limited to the specific residues or structures that the antigen-binding protein contacts. For example, the term includes the amino acid region that the antigen-binding protein contacts, as well as 5-10 (or more), or 2-5, or 1-3 amino acids outside of this region. In some instances, an epitope includes a series of discontinuous amino acids that are located in close proximity to each other when the antigen-binding protein is folded, i.e., a "conformational epitope." One of skill in the art will also recognize that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes a collection of chemically active molecular surfaces, such as sugar, phosphoryl, or sulfonyl side chains, and in certain instances, may have particular three-dimensional structural characteristics, and / or particular charge characteristics.

[0135] As used herein, the terms "preventing," "prevent" or "prevention" include administering an antigen binding protein of the invention, thereby halting or preventing the onset of at least one symptom of a condition. The term also encompasses treatment that prevents or prevents recurrence in a subject in remission.

[0136]

[0104] As used herein, the terms "treating," "treat," or "treatment" include administering an antigen binding protein described herein, thereby reducing or eliminating at least one symptom of a particular disease or condition.

[0137]

[0105] As used herein, the term "subject" is intended to mean any animal, e.g., a mammal, including a human. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0138] As used herein, the terms "genetically engineered cell" and "genetically modified cell" can be used interchangeably to refer to cells that contain and / or express exogenous genes or nucleic acid sequences that, in turn, modify the genotype or phenotype of the cell or its progeny.

[0139] antibody

[0107] In one example, the antigen binding protein according to any example described herein is an antibody.

[0140] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, such methods involve administering CCR8 or a region thereof (e.g., the extracellular region), an immunogenic fragment or epitope thereof, or a cell expressing and presenting it (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0141]

[0109] The production of polyclonal antibodies can be monitored by collecting blood from the immunized animal at various time points after immunization. One or more further immunizations may be given if necessary to achieve the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, the immunized animal is bled and the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0142] Monoclonal antibodies are one exemplary form of antibody contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen, e.g., the same epitope within an antigen. The term is not limited with regard to the source of the antibody or the method by which it is made.

[0143]

[0111] Any of a number of known techniques can be used to produce mAbs, for example, procedures exemplified in US Pat. No. 4,196,265 or Harlow and Lane (1988) (supra).

[0144] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody-producing cells. Rodents such as rabbits, mice, and rats are exemplary animals. For example, mice genetically engineered to express human antibodies but not mouse antibodies (e.g., as described in WO 2002 / 066630) can also be used to generate antibodies of the present invention.

[0145] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal immunized with the immunogen.

[0146]

[0114] The hybrids are amplified by culturing in a selective medium containing drugs that block de novo synthesis of nucleotides in tissue culture media. Exemplary drugs are aminopterin, methotrexate, and azaserine.

[0147]

[0115] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.).

[0148] Alternatively, mAb-secreting cell lines are produced using ABL-MYC technology (NeoClone, Madison WI 53713, USA) (for example, as described in Largaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

[0149]

[0117] Antibodies can also be generated or isolated by screening display libraries, eg, phage display libraries, such as those described in US Pat. No. 6,300,064 and / or US Pat. No. 5,885,793.

[0150]

[0118] The antibody of the present invention may be a synthetic antibody. For example, the antibody is a chimeric antibody, a humanized antibody, a synthetic humanized antibody, a primatized antibody or a deimmunized antibody.

[0151] Several in vitro methods exist for determining the effectiveness of an antibody in inducing ADCC. These include the chromium-51 [Cr51] release assay, europium [Eu] release assay, and sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a particular surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are typically incubated with the antibody-labeled target cells. Target cell lysis is then typically measured by the release of intracellular label, e.g., by scintillation counting or spectrophotometry. Preferred tests are detailed in the Examples.

[0152] Antibody-binding domain-containing proteins Diabodies, triabodies, and tetrabodies

[0120] In some examples, the proteins of the present invention are or comprise diabodies, triabodies, tetrabodies, or higher order protein complexes such as those described in WO 98 / 044001 and / or WO 94 / 007921.

[0153] For example, a diabody is a protein that includes two related polypeptide chains, each of which has a V L -XV H Structure or V H -XV L structure (where V L is the antibody light chain variable region, and V H is an antibody heavy chain variable region, and X is a V H and V L a linker that contains insufficient residues for the VH of one polypeptide chain to associate (or form an Fv) with the VH of the other polypeptide chain, or is absent L to form an antigen-binding domain, i.e., an Fv molecule that can specifically bind to one or more antigens. L and V H can be the same in each polypeptide chain or can be different to form bispecific diabodies (ie, comprising two Fvs with different specificities).

[0154] Single chain Fc (scFv) Those skilled in the art will appreciate that scFvs contain V H and V L The V domain, as well as the scFv, form the desired structure for antigen binding (i.e., the V domain of a single polypeptide chain). H and V L V that allows V to associate with each other to form Fv H and V LIt will be appreciated that the present invention also includes a polypeptide linker between the scFv and the nucleotide sequence of the present invention. For example, the linker comprises more than 12 amino acid residues, and (Gly4Ser)3 is one preferred linker for scFvs.

[0155] The present invention also provides a method for treating a cancer cell comprising administering to a patient a cancer cell line ... H FR and V L Disulfide-stabilized Fvs (or diFvs or dsFvs) are contemplated in which cysteine ​​residues are introduced into the FR of the Fv and linked by disulfide bonds to generate a stable Fv.

[0156] Alternatively, or in addition, the present invention encompasses dimeric scFvs, i.e., proteins comprising two scFv molecules non-covalently or covalently linked, for example, by a leucine zipper domain (e.g., from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to antigen, as described, for example, in U.S. Patent Application Publication No. 2006 / 0263367.

[0157] Other antibodies and proteins containing their antigen-binding domains The present invention also relates to other antibodies and proteins comprising antigen-binding domains thereof, such as: (i) "key and hole" bispecific proteins, as described in U.S. Pat. No. 5,731,168; (ii) heteroconjugate proteins, such as those described in U.S. Pat. No. 4,676,980; (iii) heteroconjugate proteins produced using chemical cross-linkers, such as those described in U.S. Pat. No. 4,676,980; (iv) Fab3 (e.g., as described in EP19930302894) is also planned.

[0158] In any of the aforementioned antibody structures, the binding domains of the proteins may be linked via a linker. For example, in the context of an scFv, the linker between VH and VL may be a combination of one or more amino acid residues to provide a flexible linker. Those skilled in the art will be familiar with suitable linker sequences to employ. In a typical example, the linker is composed of one or more glycine and serine residues. In one example, the linker may include the sequence G4S (i.e., GGGGS). The linker may also include repeats of glycine and serine residues such as (G4S)3, although it will be understood that any variation thereof, such as (G4S)3T, may also be suitable.

[0159] Protein mutations The present invention also provides antigen binding proteins or nucleic acids encoding same which have at least 80% identity to the sequences disclosed herein. In one example, the antigen binding proteins or nucleic acids of the invention comprise sequences which are at least about 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences disclosed herein.

[0160] Alternatively, or in addition, the antigen binding protein may comprise a V according to any of the examples described herein. H or V L or 97% or 98% or 99% identical to the CDRs of (e.g., three CDRs).

[0161] In another example, a nucleic acid of the invention comprises a sequence that is at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence encoding an antigen binding protein having a function according to any example described herein, according to any example. The invention also encompasses nucleic acids encoding antigen binding proteins of the invention that differ from the sequences exemplified herein as a result of the degeneracy of the genetic code.

[0162]

[0130] The percent identity of a nucleic acid or polypeptide is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire lengths.

[0163] The present invention also contemplates nucleic acids that hybridize to nucleic acids encoding antigen-binding sites described herein under stringent hybridization conditions. "Moderate stringency" is defined herein as hybridization and / or washing performed in 2x SSC buffer, 0.1% (w / v) SDS, at a temperature ranging from 45°C to 65°C, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing performed in 0.1x SSC buffer, 0.1% (w / v) SDS, or a lower salt concentration, at a temperature of at least 65°C, or equivalent conditions. References herein to a particular level of stringency encompass equivalent conditions using washing / hybridization solutions other than SSC, as known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as the melting temperature or Tm) are known in the art. A temperature similar to (e.g., within 5°C or within 10°C) or equal to the Tm of the nucleic acid is considered to be high stringency. Moderate stringency is considered to be within 10°C to 20°C or 10°C to 15°C of the calculated Tm of the nucleic acid.

[0164] The present invention also contemplates variants of the antigen binding proteins of the present invention which comprise one or more conservative amino acid substitutions compared to the sequences set forth herein. In some examples, the antigen binding protein comprises 10 or fewer conservative amino acid substitutions, for example 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathic and / or hydrophilic properties.

[0165] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Hydropathic indexes are described, for example, in Kyte and Doolittle J. Mol. Biol., 157: 105-132, 1982, and hydrophilicity indices are described, for example, in U.S. Pat. No. 4,554,101.

[0166] The present invention also contemplates non-conservative amino acid changes. For example, of particular interest are substitutions of a charged amino acid with another charged amino acid, and substitutions with neutral or positively charged amino acids. In some examples, the antigen-binding protein contains 10 or fewer non-conservative amino acid substitutions, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1.

[0167] In one example, the mutations occur within the FRs of the antigen-binding domain of the antigen-binding protein of the invention. In another example, the mutations occur within the CDRs of the antigen-binding protein of the invention.

[0168] Exemplary methods for generating variants of antigen binding proteins include: mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525: 309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7: 18, 2007; and WO 1999 / 058661); introduction of nucleic acids encoding the polypeptides into mutagenized cells, such as XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling, as disclosed, for example, in Stemmer, Nature 370: 389-91, 1994; and Site-directed mutagenesis, as described, for example, in Dieffenbach (ed.) and Dveksler (ed.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995).

[0169] Exemplary methods for determining the biological activity of mutant antigen binding proteins of the invention will be apparent to those of skill in the art and / or are described herein (e.g., antigen binding). For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic efficacy are described herein.

[0170] constant region The present invention encompasses antigen binding proteins and / or antibodies described herein comprising an antibody constant region, including an antigen-binding fragment of an antibody fused to Fc.

[0171]

[0139] Constant region sequences useful for generating the proteins of the invention can be obtained from many different sources. In some examples, the protein's constant region, or a portion thereof, is derived from a human antibody. The constant region, or a portion thereof, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any antibody isotype, including IgG1, IgG2, IgG3, and IgG4.

[0172] In one example, the Fc region of this constant region has an increased ability to induce effector function, for example, as compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region-containing protein are known in the art and / or described herein.

[0173] Further modifications

[0141] The present invention also contemplates further modifications to antibodies or antigen binding proteins comprising the Fc region or constant region.

[0174] For example, the antibody contains one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody contains an Fc region containing one or more amino acid substitutions that increase the affinity of the Fc region for neonatal Fc region (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to promote Fc / FcRn binding in endosomes. In one example, the Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which promotes re-release of Fc into the blood after cellular recycling. These amino acid substitutions are useful for extending the half-life of the protein by reducing clearance from the blood.

[0175] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in U.S. Patent Application Publication No. 2007 / 0135620 or U.S. Patent No. 7,083,784.

[0176] Protein production In one example, an antigen binding protein according to any example described herein is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or known in the art.

[0177] Recombinant expression In another example, the antigen binding protein according to any example described herein is recombinant.

[0178] In the case of recombinant proteins, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells (e.g., monkey COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein). Exemplary cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and are described, for example, in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US Pat. No. 4,816,567 or US Pat. No. 5,530,101.

[0179]

[0147] After isolation, the nucleic acid is inserted in operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0180] As used herein, the term "promoter" should be interpreted in its broadest context and includes, for example, the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element required for correct transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter expression of the nucleic acid in response to developmental and / or external stimuli or in a tissue-specific manner. In this context, the term "promoter" is also used to describe a recombinant, synthetic, or fusion nucleic acid, or derivative, that confers, activates, or enhances expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression of the nucleic acid and / or alter spatial and / or temporal expression.

[0181]

[0149] As used herein, the term "operably linked" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0182]

[0150] Many vectors are available for expression in cells. Vector components generally include one or more of the following, but are not limited to: a signal sequence, a protein-coding sequence (e.g., obtained from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will know sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., the invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., the herpes simplex gD signal).

[0183] Examples of promoters active in mammalian cells include the cytomegalovirus immediate-early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), small nuclear RNA promoters (U1a and U1b), the α-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter; a hybrid regulatory element comprising the CMV enhancer / β-actin promoter, or an immunoglobulin promoter or an active fragment thereof. Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0184]

[0152] Typical promoters suitable for expression in yeast cells, such as yeast cells selected from the group including Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0185]

[0153] Means for introducing isolated nucleic acids or expression constructs containing the same into cells for expression are known to those skilled in the art. The technique used for the cells will depend on known successful techniques. Means for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and particle bombardment using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0186]

[0154] Host cells used to produce proteins may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing the other cell types discussed herein are known in the art.

[0187] Protein isolation

[0155] Methods for isolating proteins are known in the art and / or described herein.

[0188]

[0156] If the antigen-binding protein is secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF can be included in any of the foregoing steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants. Alternatively, or in addition, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0189] Antigen-binding proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the foregoing. These methods are known in the art and are described, for example, in WO 99 / 57134 or in Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0190] Those skilled in the art will also recognize that proteins can be modified to include tags for ease of purification or detection, such as a polyhistidine tag, e.g., a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-his tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA), which specifically binds to the hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0191] Assay of antigen-binding protein binding It will be clear to those skilled in the art that the antigen-binding proteins of the present invention bind to CCR8. Methods for assessing protein binding are known in the art, for example, as described in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing the antigen-binding protein and contacting it with labeled antigen (CCR8). After washing to remove non-specifically bound proteins, the amount of label and, consequently, bound antigen are detected. It is of course also possible to label the antigen-binding protein and immobilize the antigen. Panning-type assays can also be used. Alternatively, or in addition, surface plasmon resonance assays can be used.

[0192] Optionally, the dissociation constant (Kd), binding constant (Ka), and / or affinity constant (KD) of the immobilized antigen-binding protein for CCR8 or its epitope are determined. The "Kd" or "Ka" or "KD" of a CCR8-binding protein is measured, in one example, by a radiolabeled or fluorescently labeled CCR8 ligand binding assay. For "Kd," the assay equilibrates the antigen-binding protein with a minimum concentration of labeled CCR8 or its epitope in the presence of a titration series of unlabeled CCR8. After washing to remove unbound CCR8 or its epitope, the amount of label (which indicates the Kd of the protein) is determined.

[0193] According to another example, K d , K. a or K D is measured using a surface plasmon resonance assay, for example, using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) using immobilized dysfunctional P2X7 receptor or a region thereof or immobilized antigen binding protein.

[0194] Conditions to be treated The antigen binding proteins of the present invention have particular utility in the manufacture of medicaments (eg, antibodies, antibody-drug conjugates) for the treatment of cancer.

[0195]

[0163] Examples of cancers that can be treated according to the methods of the present invention include preneoplastic diseases and neoplastic diseases. Broad examples include breast cancer, colon tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinoma / cholangiocarcinoma, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumor.

[0196]

[0164] Examples of specific cancers include adenocarcinoma, adenoma, adenofibroma, adenolymphoma, adenoma, AIDS-related cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical carcinoma, primary myelofibrosis, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, angiolymph node hyperplasia with eosinophilia, hemangiosclerosis, hemangiomatosis, apodoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, colorectal cancer, brain stem glioma, brain tumor and CNS tumor, breast cancer, branchiomas, CNS tumors, carcinoid tumors, cervical cancer, pediatric Brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinoma (e.g., Walker, basal cell, basosquamous cell, Brown-Pierce, ductal, Ehrlich tumor, Krebs type 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, sclerotic, bronchial, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes, cementoma, chordoma, schistoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cystoma, cystadenoma, protuberance Dermatofibrosarcoma, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial carcinoma, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct carcinoma, eye cancer, ocular melanoma, retinoblastoma, fallopian tube carcinoma, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus angiomas, granulosa cell tumor, male germinoma, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus , hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, histiocytic diseases, malignant histiocytoma, histiocytoma, liver cancer, hidradenoma, chondrosarcoma, immunoproliferative small cell carcinoma, opoma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leiomyosarcoma, leukemia (e.g., B-cell, mixed cell, null cell, T-cell,Chronic T-cell, HTLV-II-associated, lymphangiosarcoma, acute lymphocytic, chronic lymphocytic, mast cell and myeloid), leukosarcoma, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangiomyofibroma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, Merkel cell carcinoma, mesothelioma, metastatic carcinoma, oral cancer, multiple endocrine adenoma, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorders, malignant carcinoid syndrome Carcinoid heart disease, medulloblastoma, meningioma, melanoma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma, fibroid, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen rupture syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, tumors (bone, breast) , digestive system, colon, liver, etc.), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer, pancreatic cancer, sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, non-chromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogene, rare cancer and related diseases, including, but not limited to, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), sarcoma, gastric cancer, synovial sarcoma, sarcomas (e.g., Ewing's experimental, Kaposi's sarcoma, and mast cell sarcoma), Sertoli cell tumor, synovioma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), choriocarcinoma, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, urothelioma, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0197] Other diseases and conditions include various inflammatory conditions. Examples may include proliferative components. Specific examples include acne, angina, arthritis, aspiration pneumonia, disease, empyema, gastroenteritis, inflammation, intestinal flu, knee, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pelvic inflammatory disease (PID), pleurisy, sore throat, redness, rubor, sore throat, stomach flu and urinary tract infection, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculopathy, chronic inflammatory demyelinating polyneuropathy, or chronic inflammatory demyelinating polyradiculopathy.

[0198] composition In some examples, the antigen-binding proteins described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, intravaginally, intracerebroventricularly, via an implanted reservoir in a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or any other convenient form of administration. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, substernal, and intracranial injection or infusion techniques.

[0199] Methods for preparing antigen-binding proteins in a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0200] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, e.g., intravenous administration, or administration into a body cavity or lumen of an organ or joint. Compositions for administration generally comprise a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. The compositions may contain pH adjusting and buffering agents, toxicity adjusting agents (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.), and pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions. The concentration of the antigen-binding protein of the present invention in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., in accordance with the particular method of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0201] The antigen-binding proteins of the invention may be formulated for topical administration, for example, for topical application to the skin or tissue in need of treatment. Formulations for topical administration typically comprise a topical vehicle in combination with an active agent, with or without additional optional ingredients. Pharmaceutical compositions of the invention may be in the form of a spray, cream, gel, lotion, etc. for topical administration.

[0202] Suitable topical vehicles and additional ingredients are well known in the art, and it will be apparent that the vehicle will be selected depending on the particular physical form and delivery method. Topical vehicles include alcohols (e.g., ethanol, isopropyl alcohol, or glycerin), glycols such as butylene, isoprene, or propylene glycol, fatty alcohols such as lanolin, organic solvents such as mixtures of water and organic solvents, and mixtures of alcohol and organic solvents such as glycerin, lipid-based materials such as fatty acids, oils such as mineral oil, and acylglycerols, including fats of natural or synthetic origin, phosphoglycerides, sphingolipids, and waxes, protein-based materials such as collagen and gelatin, silicone-based materials (both non-volatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

[0203] The composition may further comprise one or more ingredients adapted to improve the stability or effectiveness of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained-release substances. Examples of such ingredients are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. The formulation may comprise microcapsules, such as hydroxymethylcellulose or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

[0204] Topical formulations can be prepared in a variety of physical forms, including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays, and skin patches. The physical appearance and viscosity of such forms can be controlled by the presence and amount of emulsifiers and viscosity modifiers present in the formulation. Solids are generally firm and non-pourable and are generally formulated as bars or sticks or in particulate form. Solids can be opaque or transparent and can optionally contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are very similar but differ primarily in their viscosity. Both lotions and creams can be opaque, translucent, or transparent and often contain emulsifiers, solvents, and viscosity modifiers, as well as moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product.

[0205] Gels can be prepared in a range of viscosities, from thick or high to thin or low. These formulations, similar to lotion and cream formulations, may contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product.

[0206] Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers, nonionic emulsifiers such as cetearyl alcohol, polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate, and glyceryl stearate. Suitable viscosity modifiers include, but are not limited to, protective colloids or nonionic gums, such as hydroxyethyl cellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. Gel compositions can be produced by adding gelling agents such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaternium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, carbomer, or ammonium glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic, and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate can be used in the topical formulation.

[0207] Preservatives include, but are not limited to, antimicrobial agents such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate / ascorbic acid, and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids, their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate, and mineral oil. Suitable fragrances and colorants include FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that can be included in topical formulations include, but are not limited to, abrasives, absorbents, anti-caking agents, anti-foaming agents, anti-static agents, astringents (such as witch hazel), alcohols and herbal extracts such as chamomile extract, binders / excipients, buffers, chelating agents, film-forming agents, conditioning agents, propellants, opacifiers, pH adjusters, and protectants.

[0208] Typical delivery methods for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick, or brush, spraying, including mist, aerosol, or foam spray, application by dropper, sprinkling, dipping, and washing. Controlled release vehicles can also be used, and the compositions can be formulated for transdermal administration (e.g., as a transdermal patch).

[0209]

[0177] Once formulated, the antigen-binding proteins of the present invention are administered in a manner compatible with the dosage formulation and in an amount that will be therapeutically / prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although other pharmaceutically acceptable dosage forms are also contemplated, such as tablets, pills, capsules, or other solid forms for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomes, and the like. Pharmaceutical "sustained-release" capsules or compositions can also be used. Sustained-release formulations are generally designed to provide a constant drug level over an extended period of time and may be used to deliver the antigen-binding proteins of the present invention.

[0210]

[0178] WO 2002 / 080967 describes compositions and methods for administering aerosolized compositions containing antibodies for the treatment of asthma, for example, which are also suitable for administering the antigen-binding proteins of the present invention.

[0211] Dosage and timing of administration Suitable dosages of antigen binding proteins of the present invention will vary depending on the particular antigen binding protein, the condition being treated, and / or the subject being treated. It is within the ability of a skilled physician to determine suitable dosages, for example, by starting with a suboptimal dosage and gradually increasing the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies can be used to determine appropriate dosages for treatment / prophylaxis; suitable doses may be determined based on the ED of the active compound with little or no toxicity. 50 The therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. The dose can be determined based on the IC50 determined in cell culture. 50The compound can be formulated in animal models to achieve a circulating plasma concentration range that includes the compound (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0212]

[0199] In some examples, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0213] The term "therapeutically effective amount" refers to an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed and accepted as a clinical diagnosis or clinical feature of that condition. The amount administered to a subject will depend on the specific features of the condition being treated, the type and stage of the condition being treated, the method of administration, and characteristics of the subject, such as their general health, other diseases, age, sex, genotype, and weight. One of skill in the art will be able to determine the appropriate dosage depending on these and other factors. Thus, this term should not be construed to limit the invention to a specific amount, e.g., weight or amount of protein; rather, the invention encompasses any amount of antigen-binding protein sufficient to achieve a desired result in a subject.

[0214] As used herein, the term "prophylactically effective amount" is intended to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One of skill in the art will recognize that such an amount will vary depending, for example, on the particular antigen-binding protein administered, and / or the particular subject, and / or the type or severity or level of the condition, and / or predisposition (genetic or otherwise) to the condition. Thus, this term should not be construed to limit the invention to a particular amount, e.g., weight or amount of antigen-binding protein; rather, the invention encompasses any amount of antigen-binding protein sufficient to achieve a predetermined result in a subject.

[0215] kit

[0202] The present invention further includes kits comprising one or more of the following: (i) an antigen-binding protein of the invention, or a nucleic acid or expression construct encoding same; (ii) a cell of the invention; or (iii) The pharmaceutical composition of the present invention.

[0216] In the case of a kit for detecting CCR8, the kit may further comprise a detection means, for example linked to the antigen binding protein of the invention.

[0217]

[0204] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharmaceutically acceptable carrier.

[0218]

[0205] Optionally, the kits of the invention are packaged with instructions for use in any of the example methods described herein. [Example]

[0219] Example Example 1 - Generation of anti-human CCR8 antibodies by immunizing mice with human CCR8.

[0179] 10 7mAbs reactive with human CCR8 were generated by inoculating L1.2 human CCR8-transfected cells intraperitoneally (i.p.) five to six times at two-week intervals. The final immunization was intravenously (i.v.). Four days later, the spleen was removed, and the cells were fused with the SP2 / 0 cell line.

[0220] From six different fusions, we isolated six clones specific for hCCR8, none of which showed cross-reactivity to mouse or cynomolgus monkey CCR8.

[0221] Example 2 - Generation of anti-human CCR8 antibodies by immunizing mice with murine CCR8.

[0181] 10 7 mAbs reactive with mouse CCR8 were generated by inoculating L1.2 mouse CCR8-transfected cells intraperitoneally (i.p.) five to six times at two-week intervals. The final immunization was intravenously (i.v.). Four days later, the spleen was removed, and the cells were fused with the SP2 / 0 cell line.

[0222] Example 3 - Isolation of anti-CCR8 2H8 antibody and cross-reactivity with human, mouse and cynomolgus monkey CCR8 After several inoculations, we confirmed that the sera of immunized mice contained antibodies capable of binding to both mouse and human CCR8 (Figure 1). We performed a fusion with one of the mice and screened the hybridomas by flow cytometry.

[0223] Although the present inventors isolated surrogate antibodies specific to mouse CCR8, they unexpectedly found one clone (2H8; isotype IgG2b) that showed reactivity with both the human and mouse receptors (FIG. 1). This is in contrast to the antibody obtained in Example 1 above, which showed reactivity only with human CCR8 (FIG. 1A; anti-hCCR8#1 is shown as a representative example). Furthermore, the present inventors compared this with an existing anti-hCCR8 antibody (clone #L263G8) from Biolegend, which also showed reactivity only with human CCR8 (FIG. 1B).

[0224] Next, we tested the reactivity of the 2H8 antibody with cynomolgus monkey (cyno) CCR8. Surprisingly, 2H8 also showed high reactivity with cynoCCR8 compared to the isotype control (Figure 2), whereas prior art antibodies and some generated using the method of Example 1 showed no reactivity.

[0225] Example 4 - 2H8 does not cross-react with other CCR chemokine receptors

[0185] To confirm the specificity of 2H8 for CCR8, the inventors tested the reactivity of 2H8 with other human CCR chemokine receptors and found no cross-reactivity with any of the CCR chemokine receptors tested, except for human and mouse CCR8 (Figure 3).

[0226] Example 5 - 2H8 binds to human tumor-infiltrating Tregs (TiTregs)

[0186] To determine whether 2H8 can bind to TiTreg, the inventors isolated single cell suspensions from fresh colon tumor patient biopsies and stained the cells with anti-human CD3, anti-human CD4, and anti-human FoxP3 (TiTreg) and 2H8 or an isotype control.

[0227] 2H8 was found to bind to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gate), whereas the isotype control did not (FIG. 4).

[0228] Example 6 - Chimeric 2H8 exhibits similar binding affinity compared to existing anti-hCCR8 antibodies

[0188] The present inventors have generated a chimeric version of the 2H8 antibody comprising the heavy and light chain constant regions of human IgG1 as shown in SEQ ID NOs:58 and 59.

[0229] We used flow cytometry to compare the binding of chimeric 2H8, Shionogi's 19D7, and BMS's 4A19 anti-hCCR8 antibodies to hCCR8-L1.2 transfected cells. All anti-hCCR8 antibodies showed similar binding to hCCR8 (Figure 5).

[0230] Example 7 - Humanized 2H8 anti-CCR8 antibody

[0190] Next, the present inventors prepared a humanized 2H8 antibody. The antibody was humanized by CDR grafting.

[0231] Briefly, using the IMGT / V-QUEST and IMGT / Junction analysis tools, human germline genes whose sequences from both the heavy and light chain variable regions closely aligned with the sequence of the murine antibody 2H8 were identified. The framework sequences of these selected human germline genes were used as acceptor sequences for the 2H8 CDRs. However, murine residues were retained in the critical "Vernier" zone. Humanized VH and VL genes were synthesized by Genscript.

[0232] The binding activity of the humanized 2H8 antibody was compared to that of chimeric 2H8 and Shionogi's 19D7 anti-CCR8 antibody using flow cytometry. The results are shown in Figure 6 and Table 2 below.

[0233] [Table 7]

[0234] Example 8 - 2H8 binds to human tumor-infiltrating Tregs (TiTregs)

[0193] To determine whether 2H8 can bind to TiTreg, the inventors isolated single cell suspensions from fresh colon tumor patient biopsies and stained the cells with anti-human CD3, anti-human CD4, and anti-human FoxP3 (TiTreg) and 2H8 or an isotype control.

[0235] 2H8 was found to bind to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gate), whereas the isotype control did not (FIG. 7).

[0236] Example 9 - Chimeric 2H8 does not inhibit CCL1-induced chemotaxis. Next, we performed a chemotaxis assay to determine whether chimeric 2H8 blocks CCL1-induced chemotaxis. CCL1 is known to induce chemotaxis by binding to the N-terminus of CCR8.

[0237] Briefly, L1.2 cells stably transfected with human CCR8 were maintained in suspension in RPMI 1640 containing 10% heat-inactivated FCS, 2% l-glutamine, and 100 U ml penicillin. For the assay, cells were washed once with PBS and resuspended at 10°C in test buffer (RPMI 1640, 1% endotoxin-free BSA, 100 U / ml penicillin, 100 μg / ml streptomycin). 6 Antibodies were added to 100 μl of cells (1 × 10 5 The cells were preincubated with 5.8 nM human CCL1 (30 min, 37°C) for 30 min and then placed in the upper chamber of a 5 μm pore transwell plate (Corning Life Sciences). The lower chamber contained 5.8 nM human CCL1 (30 min, 37°C), and the assay was incubated at 37°C (5% CO2) for 4 h. Viable cells that migrated to the lower chamber were counted using an LSRII flow cytometer.

[0238] Chimeric 2H8 did not inhibit CCL1-induced chemotaxis, whereas chimeric 19D7 had a significant and dose-dependent inhibitory effect on CCL1-induced chemotaxis (Figure 8). Advantageously, we believe that in environments with high CCL1 concentrations, such as the tumor microenvironment, the binding of chimeric 2H8 to CCR8 is not inhibited by competition with CCL1 because, unlike chimeric 19D7, they do not share the same binding site.

[0239] Example 10 - Epitope mapping of 2H8 To further investigate the epitope binding of 2H8, the present inventors performed epitope mapping. Specifically, the present inventors replaced the N-terminus, extracellular loop 1, extracellular loop 2, and extracellular loop 3 of human CCR8 with the N-terminus, extracellular loop 1, extracellular loop 2, and extracellular loop 3 of human CCR5, respectively. These modified hCCR8s are shown as 5888, 8588, 8858, and 8885 in Table 3, respectively (the amino acid sequence derived from CCR5 is underlined). The results of epitope mapping are shown in Table 3 below.

[0240] [Table 8]

[0241] Interestingly, a mutation to the N-terminus of human CCR8 (5888 in Table 3) prevented 19D7 from binding to CCR8, but 2H8 was still able to bind to CCR8, whereas a mutation to extracellular loop 2 (8858 in Table 3) prevented 2H8 from binding to CCR8, but 19D7 was still able to bind to CCR8.

[0242]

[0200] These results suggest that 2H8 and 19D7 bind to different epitopes of CCR8.

[0243] Example 11 - Chimeric 2H8 demonstrates ADCC potential with efficacy and potency comparable to 19D7.

[0201] To further investigate the functional properties of chimeric 2H8, we performed a dose-dependent antibody-dependent cellular cytotoxicity (ADCC) assay.

[0244] The ability of antibodies to induce ADCC was assessed by flow cytometry. Briefly, hCCR8 L1.2-expressing cells were labeled with a membrane dye, PKH26, to allow for their identification when incubated with effector cells and antibodies. Labeled target cells were washed three times with medium and then cultured at 1 x 10 6 The labeled target cells were resuspended in culture medium at a concentration of 1 × 10 cells / ml. The labeled target cells were dispensed into round-bottom 96-well plates (1 × 10 cells / well in 100 μl). 5 The cells were preincubated with 5 μg / ml, 1 μg / ml, 0.5 μg / ml, or 0.1 μg / ml of antibody for 30 minutes at 37°C. PBMCs were prepared from heparinized blood (obtained from healthy individuals) by centrifugation through a Ficoll gradient. Human natural killer cells were then isolated from the PBMCs using MACS CD56 microbeads and a MACS positive selection column (Miltenyi Biotec) according to the manufacturer's protocol. 2 × 10 cells were then cultured. 5 Purified human NK cells (effector cells) were incubated with the target cell / antibody mixture (E:T ratio = 2:1) for 3 hours at 37°C in the presence of 10% heat-inactivated serum. Treated cells were washed three times and resuspended in 200 μl of PBS. Immediately before analysis on the LSRII flow cytometer, TO-PRO 3 iodide was added to detect cell death. 20 μl / well of CountBright absolute counting beads (Invitrogen) were added as a counting standard to determine the cell concentration of cell subsets. Samples were acquired on the LSRII flow cytometer.

[0245]

[0203] Chimeric 2H8 and Shionogi's chimeric 19D7 anti-CCR8 antibodies induced similar levels of cytotoxicity, as shown in Figure 9 and Table 4 below.

[0246] [Table 9]

[0247] Example 12 - In vivo activity of 2H8 in a mouse model of colon cancer To test the activity of 2H8 in vivo, we inoculated 5×10 5 MC38 colon cells were implanted subcutaneously. Animals were treated with 2H8 mIgG2a (n=9), anti-mPD1 (n=10), and isotype control (n=6). After the first injection of antibody, tumor volume (mm) was measured using digital calipers. 3 ) were measured daily and compared between groups. Treatment was at a dose of 5 mg / kg twice weekly for up to 3 weeks.

[0248] Treatment with 2H8 substantially inhibited tumor growth compared to both the isotype control and anti-mPD1 treated groups (FIG. 10).

[0249] At the endpoint, tumor and spleen tissues were processed, stained, and analyzed by flow cytometry. + CD4 in cells (parent population) + FOXP3 + CD25 + The percentages of cells were collected and compared among the three groups. Treatment with 2H8 depleted Tregs in the tumor but not in the spleen (Figure 11). Furthermore, Treg depletion was significantly greater in the 2H8-treated group compared with anti-PD1 or isotype control.

[0250] We next analyzed CD8+ and other CD4+ T cell populations in tumor samples. We found that treatment with 2H8 increased tumor-specific CD8+ T cells and decreased CD4+ T cells, which was statistically significant compared to anti-PD1 or isotype control (Figure 12).

[0251]

[0208] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which various combinations constitute various alternative aspects of the invention.

Claims

1. An antigen binding protein that binds to or specifically binds to extracellular loop 2 of CCR8.

2. 2. The antigen binding protein of claim 1, wherein the antigen binding protein binds to residues 172-202 of CCR8, preferably residues 172-190 of CCR8, wherein the amino acid sequence of CCR8 is as set forth in SEQ ID NO:

55.

3. 3. The antigen-binding protein of claim 1 or 2, wherein the antigen-binding protein binds to residues 176-179 of CCR8, wherein the amino acid sequence of CCR8 is as set forth in SEQ ID NO:

55.

4. 4. The antigen-binding protein of any one of claims 1 to 3, wherein the antigen-binding protein does not block the binding of CCR8 to CCL1.

5. An antigen binding protein comprising CDRH1, CDRH2 and / or CDRH3 of an antibody having a variable heavy chain as defined in SEQ ID NO:1 and CDRL1, CDRL2 and / or CDRL3 of an antibody having a variable light chain as defined in SEQ ID NO:

2.

6. 1. An antigen binding protein that binds to or specifically binds to CCR8, comprising: a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 2; or VH comprising the sequence set forth in SEQ ID NO: 83 and VL comprising the sequence set forth in SEQ ID NO: 84 An antigen-binding protein that competitively inhibits the binding of an antibody comprising:

7. The antigen-binding protein comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to CCR8, and the antigen-binding domain comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:6, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:7; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 83; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:8, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:9, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 2 or 84; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; (vi) a VH comprising the sequence set forth in SEQ ID NO: 1 or 83; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 2 or 84; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 5, a CDR2 comprising the sequence set forth in SEQ ID NO: 6, and a CDR3 comprising the sequence set forth in SEQ ID NO: 7; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 8, a CDR2 comprising the sequence set forth in SEQ ID NO: 9, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (x) VH comprising the sequence set forth in SEQ ID NO: 1 and VL comprising the sequence set forth in SEQ ID NO: 2; (xi) VH comprising the sequence set forth in SEQ ID NO: 83 and VL comprising the sequence set forth in SEQ ID NO: 84 The antigen-binding protein of any one of claims 1 to 6, comprising at least one of:

8. (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 11; and a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:

12.

2. A VH comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 13, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 14; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 15; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:17, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:18; (iii) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 11, FR2 comprising the sequence set forth in SEQ ID NO: 12, FR3 comprising the sequence set forth in SEQ ID NO: 13, and FR4 comprising the sequence set forth in SEQ ID NO: 14; (iv) a VL comprising an FR1 comprising the sequence set forth in SEQ ID NO: 15, an FR2 comprising the sequence set forth in SEQ ID NO: 16, an FR3 comprising the sequence set forth in SEQ ID NO: 17, and an FR4 comprising the sequence set forth in SEQ ID NO: 18; or (v) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 11, FR2 comprising the sequence set forth in SEQ ID NO: 12, FR3 comprising the sequence set forth in SEQ ID NO: 13, and FR4 comprising the sequence set forth in SEQ ID NO: 14; and VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 15, FR2 comprising the sequence set forth in SEQ ID NO: 16, FR3 comprising the sequence set forth in SEQ ID NO: 17, and FR4 comprising the sequence set forth in SEQ ID NO:

18. The antigen-binding domain of claim 7, further comprising at least one of:

9. (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 60; a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 61; 2. A VH comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:62, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:63; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 64; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 65; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:66, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:67; (iii) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 60, FR2 comprising the sequence set forth in SEQ ID NO: 61, FR3 comprising the sequence set forth in SEQ ID NO: 62, and FR4 comprising the sequence set forth in SEQ ID NO: 63; (iv) a VL comprising an FR1 comprising the sequence set forth in SEQ ID NO: 64, an FR2 comprising the sequence set forth in SEQ ID NO: 65, an FR3 comprising the sequence set forth in SEQ ID NO: 66, and an FR4 comprising the sequence set forth in SEQ ID NO: 67; or (v) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 60, FR2 comprising the sequence set forth in SEQ ID NO: 61, FR3 comprising the sequence set forth in SEQ ID NO: 62, and FR4 comprising the sequence set forth in SEQ ID NO: 63; and VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 64, FR2 comprising the sequence set forth in SEQ ID NO: 65, FR3 comprising the sequence set forth in SEQ ID NO: 66, and FR4 comprising the sequence set forth in SEQ ID NO:

67. The antigen-binding domain of claim 7, further comprising at least one of:

10. The antigen binding protein comprises an antigen binding domain of an antibody, wherein the antigen binding domain binds to or specifically binds to CCR8, and the antigen binding protein comprises: (i) a VH comprising a complementarity determining region (CDR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 21; (ii) a VH comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 1 or 83; (iii) a VL comprising a CDR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:22, a CDR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:23, and a CDR3 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:10; (iv) a VL comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 2 or 84; (v) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; (vi) a VH comprising the sequence set forth in SEQ ID NO: 1 or 83; (vii) a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (viii) a VL comprising the sequence set forth in SEQ ID NO: 2 or 84; (ix) a VH comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 19, a CDR2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the sequence set forth in SEQ ID NO: 21; and a VL comprising a CDR1 comprising the sequence set forth in SEQ ID NO: 22, a CDR2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR3 comprising the sequence set forth in SEQ ID NO: 10; (x) a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 2; or (xi) VH comprising the sequence set forth in SEQ ID NO: 83 and VL comprising the sequence set forth in SEQ ID NO: 84 7. The antigen-binding protein of claim 5 or 6, comprising at least one of:

11. (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 24; and a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:

25.

2. A VH comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:26, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:14; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 27; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 28; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:29, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:18; (iii) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 24, FR2 comprising the sequence set forth in SEQ ID NO: 25, FR3 comprising the sequence set forth in SEQ ID NO: 26, and FR4 comprising the sequence set forth in SEQ ID NO: 14; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 27, FR2 comprising the sequence set forth in SEQ ID NO: 28, FR3 comprising the sequence set forth in SEQ ID NO: 29, and FR4 comprising the sequence set forth in SEQ ID NO: 18; (v) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 24, FR2 comprising the sequence set forth in SEQ ID NO: 25, FR3 comprising the sequence set forth in SEQ ID NO: 26, and FR4 comprising the sequence set forth in SEQ ID NO: 14; and VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 27, FR2 comprising the sequence set forth in SEQ ID NO: 28, FR3 comprising the sequence set forth in SEQ ID NO: 29, and FR4 comprising the sequence set forth in SEQ ID NO:

18. The antigen-binding domain of claim 10, further comprising at least one of:

12. (i) a framework region (FR) 1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 68; a FR comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 69; 2. A VH comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:70, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:63; (ii) FR1 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 71; FR2 comprising a sequence at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 72; a VL comprising an FR3 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:73, and an FR4 comprising a sequence that is at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO:67; (iii) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 68, FR2 comprising the sequence set forth in SEQ ID NO: 69, FR3 comprising the sequence set forth in SEQ ID NO: 70, and FR4 comprising the sequence set forth in SEQ ID NO: 63; (iv) VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 71, FR2 comprising the sequence set forth in SEQ ID NO: 72, FR3 comprising the sequence set forth in SEQ ID NO: 73, and FR4 comprising the sequence set forth in SEQ ID NO: 67; (v) VH comprising FR1 comprising the sequence set forth in SEQ ID NO: 68, FR2 comprising the sequence set forth in SEQ ID NO: 69, FR3 comprising the sequence set forth in SEQ ID NO: 70, and FR4 comprising the sequence set forth in SEQ ID NO: 63; and VL comprising FR1 comprising the sequence set forth in SEQ ID NO: 71, FR2 comprising the sequence set forth in SEQ ID NO: 72, FR3 comprising the sequence set forth in SEQ ID NO: 73, and FR4 comprising the sequence set forth in SEQ ID NO:

67. The antigen-binding domain of claim 10, further comprising at least one of:

13. The antigen-binding protein (i) single chain Fv fragment (scFv); (ii) a dimeric scFv (di-scFv); or (iii) one of (i) or (ii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of an antibody; 13. The antigen-binding protein of any one of claims 1 to 12, in the form:

14. The antigen-binding protein (i) diabodies; (ii) triabodies; (iii) tetrabodies; (iv) Fab; (v)F(ab')2; (vi) Fv; (vii) bispecific antibodies or other forms of multispecific antibodies; or (viii) one of (i)-(vii) linked to the constant region, Fc, or heavy chain constant domain (CH)2 and / or CH3 of an antibody; 13. The antigen-binding protein of any one of claims 1 to 12, in the form:

15. An anti-CCR8 antibody or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region CDR H1 according to SEQ ID NO: 5, CDR H2 according to SEQ ID NO: 6, and CDR H3 according to SEQ ID NO: 7 Including; The light chain variable region CDR L1 according to SEQ ID NO: 8, CDR L2 according to SEQ ID NO: 9, and CDR H3 according to SEQ ID NO: 10 Including, An anti-CCR8 antibody or an antigen-binding fragment thereof.

16. The anti-CCR8 antibody or antigen-binding fragment thereof comprises: FR L1 set forth in SEQ ID NO: 15, FR L2 set forth in SEQ ID NO: 16, FR L3 set forth in SEQ ID NO: 17, and FR L4 set forth in SEQ ID NO: 18; or FR L1 set forth in SEQ ID NO: 64, FR L2 set forth in SEQ ID NO: 65, FR L3 set forth in SEQ ID NO: 66, and FR L4 set forth in SEQ ID NO: 67 The anti-CCR8 antibody or antigen-binding fragment thereof of claim 15, comprising a light chain variable region comprising:

17. The anti-CCR8 antibody or antigen-binding fragment thereof comprises: FR H1 set forth in SEQ ID NO: 11, FR H2 set forth in SEQ ID NO: 12, FR H3 set forth in SEQ ID NO: 13, and FR H4 set forth in SEQ ID NO: 14; or FR H1 set forth in SEQ ID NO: 60, FR H2 set forth in SEQ ID NO: 61, FR H3 set forth in SEQ ID NO: 62, and FR H4 set forth in SEQ ID NO: 63 The anti-CCR8 antibody or antigen-binding fragment thereof according to claim 15 or 16, comprising a heavy chain variable region comprising:

18. An anti-CCR8 antibody or antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region CDR H1 according to SEQ ID NO: 19, CDR H2 according to SEQ ID NO: 20, and CDR H3 according to SEQ ID NO: 21 Including; The light chain variable region CDR L1 according to SEQ ID NO: 22, CDR L2 according to SEQ ID NO: 23, and CDR L3 according to SEQ ID NO: 10 Contains An anti-CCR8 antibody or an antigen-binding fragment thereof.

19. The anti-CCR8 antibody or antigen-binding fragment thereof comprises: FR L1 set forth in SEQ ID NO: 27, FR L2 set forth in SEQ ID NO: 28, FR L3 set forth in SEQ ID NO: 29, and FR L4 set forth in SEQ ID NO: 18; or FR L1 set forth in SEQ ID NO: 71, FR L2 set forth in SEQ ID NO: 72, FR L3 set forth in SEQ ID NO: 73, and FR L4 set forth in SEQ ID NO: 67 19. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 18, comprising a light chain variable region comprising:

20. The anti-CCR8 antibody or antigen-binding fragment thereof comprises: FR H1 set forth in SEQ ID NO: 24, FR H2 set forth in SEQ ID NO: 25, FR H3 set forth in SEQ ID NO: 26, and FR H4 set forth in SEQ ID NO: 14; or FR H1 set forth in SEQ ID NO: 68, FR H2 set forth in SEQ ID NO: 69, FR H3 set forth in SEQ ID NO: 70, and FR H4 set forth in SEQ ID NO: 63 20. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 18 or 19, comprising a heavy chain variable region comprising:

21. The anti-CCR8 receptor antibody or antigen-binding fragment thereof according to any one of claims 15 to 20, wherein the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:

2.

22. The anti-CCR8 receptor antibody or antigen-binding fragment thereof according to any one of claims 15 to 21, wherein the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:

1.

23. The anti-CCR8 receptor antibody or antigen-binding fragment thereof according to any one of claims 15 to 20, wherein the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the sequence of SEQ ID NO:

84.

24. The anti-CCR8 receptor antibody or antigen-binding fragment thereof according to any one of claims 15 to 20 and 23, wherein the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO:

83.

25. 25. The antigen binding protein, anti-CCR8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 24, wherein said antigen binding protein or anti-CCR8 receptor antibody or antigen-binding fragment thereof further comprises up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the amino acid sequence of the indicated SEQ ID NO.

26. 26. The antigen-binding protein, anti-CCR8 antibody or antigen-binding fragment thereof of claim 25, wherein said amino acid insertion, deletion, substitution or combination thereof is not present in said CDR.

27. 27. The antigen-binding protein of any one of claims 1 to 26, comprising a human constant region.

28. 28. The antigen binding protein of claim 27, wherein the human constant region comprises the amino acid sequence set forth in SEQ ID NO: 58 and / or 59.

29. A nucleic acid encoding the antigen-binding protein, antibody or antigen-binding fragment thereof of any one of claims 1 to 28.

30. A vector comprising the nucleic acid of claim 29.

31. 31. A cell comprising the vector of claim 30 or the nucleic acid of claim 29.

32. 24. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1 to 8 or 17 to 20, the antibody or antigen-binding fragment thereof of any one of claims 9 to 20, the nucleic acid of claim 21, the vector of claim 22, or the cell of claim 23, and a pharmaceutically acceptable carrier, diluent, or excipient.

33. 32. A kit or article of manufacture comprising the antigen-binding protein of any one of claims 1 to 14 or 25 to 28, the antibody or antigen-binding fragment thereof of any one of claims 15 to 28, the nucleic acid of claim 29, the vector of claim 30, the cell of claim 31, or the pharmaceutical composition of claim 32.

34. 30. A method for producing the antigen-binding protein of any one of claims 1 to 14 or 25 to 28, or the antibody or antigen-binding fragment of any one of claims 15 to 28, comprising expressing the nucleic acid of claim 29 in a cell or a non-human animal.

35. 31. A method for preventing or treating a condition or disease associated with expression of CCR8 in an individual, the method comprising the step of providing an antigen-binding protein according to any one of claims 1 to 14 or 25 to 28, an antibody or antigen-binding fragment thereof according to any one of claims 15 to 28, a nucleic acid according to claim 29, a vector according to claim 30, a cell according to claim 31, or a pharmaceutical composition according to claim 32 to an individual in need of treatment for said condition or disease.

36. 36. The method of claim 35, wherein the disease or condition is associated with expression of CCR8.

37. 37. The method of claim 35 or 36, wherein the disease or condition is cancer.

38. 32. Use of the antigen-binding protein of any one of claims 1 to 14 or 25 to 28, the antibody or antigen-binding fragment of any one of claims 15 to 28, the nucleic acid of claim 29, the vector of claim 30, the cell of claim 31, or the pharmaceutical composition of claim 32 in the manufacture of a medicament for treating or preventing cancer in a subject.

39. 33. The antigen-binding protein of any one of claims 1 to 14 or 25 to 28, the antibody or antigen-binding fragment of any one of claims 15 to 28, the nucleic acid of claim 29, the vector of claim 30, the cell of claim 31, or the pharmaceutical composition of claim 32 for use in treating or preventing cancer in a subject.

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