Anti-CCR8 antibodies

Abstract

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

Description

Technical Field

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

[0002] Related Applications

[0003] This application claims the priority of Australian Provisional Application No. 2022902742, the entire content of which is incorporated herein by reference. Background Art

[0004] Immunotherapy is a rapidly evolving and very promising treatment for various forms of cancer, with many recent achievements. However, some patients have a limited or even no response to current immunotherapies, while other patients exhibit relapse after an initial response.

[0005] The human immune system includes checks and balances to prevent an overactive immune system from harming the body. Regulatory T cells ("Tregs") play an important role in maintaining a functional immune system by suppressing immune responses. Specifically, Tregs are the major population of immune cells that play a key role in maintaining self-tolerance and the resolution of immune responses by adopting multifaceted immunomodulatory mechanisms. However, Tregs are prone to infiltrating into the tumor microenvironment (TME) and suppressing anti-tumor immune responses, thus becoming an obstacle to effective cancer immunotherapy. It has been demonstrated that Treg modulation strategies increase anti-tumor immunity and reduce tumor burden in both preclinical and clinical settings.

[0006] C-C chemokine receptor 8 (CCR8) is a chemokine receptor selectively expressed on tumor-infiltrating Treg subsets with the highest levels of inhibitory markers, and its expression is associated with poor prognosis in multiple tumor types. It has been shown that this CCR8-expressing Treg subset (CCR8+ Tregs) is a major driver of immunosuppression and is crucial for Treg function and suppression.

[0007] Accordingly, there remains a need in the art to develop therapeutic agents targeting CCR8, such as anti-CCR8 antibodies, which can be used for therapeutic purposes in the treatment of cancer.

[0008] The citation of any prior art in the specification does not mean an admission or implication that such prior art forms part of the common general knowledge in any jurisdiction, or that such prior art can reasonably be expected to be understood, regarded as relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0009] In one aspect, the present invention provides an antigen-binding protein that binds or specifically binds to extracellular loop 2 of CCR8, preferably wherein the antigen-binding protein does not block the binding of CCR8 to CCL1.

[0010] In any embodiment, the antigen-binding protein binds to residues 172 to 202 of CCR8, wherein the amino acid sequence of CCR8 is as shown in SEQ ID NO:55. In any embodiment, the antigen-binding protein binds to residues 172 to 190 of CCR8, wherein the amino acid sequence of CCR8 is as shown in SEQ ID NO:55. In any embodiment, the antigen-binding protein binds to residues 176 to 179 of CCR8, wherein the amino acid sequence of CCR8 is as shown in SEQ ID NO:55.

[0011] In one aspect, the present 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.

[0012] 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.

[0013] 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 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.

[0014] In another aspect, the present invention provides an antigen-binding protein for binding to CCR8, the antigen-binding protein comprising:

[0015] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and

[0016] FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a,

[0017] wherein:

[0018] FR1, FR2, FR3, and FR4 are each a framework region;

[0019] CDR1, CDR2, and CDR3 are each a complementarity-determining region;

[0020] FR1a, FR2a, FR3a, and FR4a are each a framework region;

[0021] CDR1a, CDR2a, and CDR3a are each a complementarity-determining region;

[0022] wherein the sequence of any one of the framework regions or complementarity-determining regions is as described herein.

[0023] In another aspect, the present invention provides an antigen-binding protein for binding to CCR8, the antigen-binding protein comprising:

[0024] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and

[0025] FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a,

[0026] wherein:

[0027] FR1, FR2, FR3, and FR4 are each a framework region;

[0028] CDR1, CDR2, and CDR3 are each a complementarity-determining region;

[0029] FR1a, FR2a, FR3a, and FR4a are each a framework region;

[0030] CDR1a, CDR2a, and CDR3a are each a complementarity-determining region;

[0031] wherein the sequence of any one of the complementarity-determining regions has the amino acid sequence as described in Table 1 below. Preferably, the framework region has the amino acid sequence as also described in Table 1 below, including amino acid variations at specific residues, which amino acid variations can be determined by aligning the various framework regions derived from each antibody. The present invention also includes the following cases: wherein CDR1, CDR2, and CDR3 are sequences from the variable heavy chain (VH) of an antibody, and CDR1a, CDR2a, and CDR3a are sequences from the variable light chain (VL) of an antibody; or wherein CDR1, CDR2, and CDR3 are sequences from VL, and CDR1a, CDR2a, and CDR3a are sequences from VH.

[0032] In any embodiment, the antigen-binding protein described herein comprises:

[0033] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a.

[0034] As defined herein, the linker can be a chemical substance, one or more amino acids, or a disulfide bond formed between two cysteine residues.

[0035] In certain preferred embodiments, the present invention provides an antigen-binding protein comprising the amino acid sequences of SEQ ID NO:1 and 2 (in N-to-C or C-to-N order), consisting essentially of or consisting of the same.

[0036] Optionally, the antigen-binding protein comprises SEQ ID NO:2 (VL) – linker – SEQ ID NO:1 (VH), or comprises SEQ ID NO:1 (VH) – linker – SEQ ID NO:2 (VL).

[0037] In certain preferred embodiments, the present invention provides an antigen-binding protein comprising the amino acid sequences of SEQ ID NO:83 and 84 (in N-to-C or C-to-N order), consisting essentially of or consisting of the same.

[0038] Optionally, the antigen-binding protein comprises SEQ ID NO:84 (VL) – linker – SEQ ID NO:83 (VH), or comprises SEQ ID NO:83 (VH) – linker – SEQ ID NO:84 (VL).

[0039] In any embodiment, the present invention provides an antigen-binding protein that binds to or specifically binds to CCR8, wherein the antigen-binding protein competitively inhibits the binding of an antibody comprising a VH and a VL, wherein the VH comprises the sequence as shown in SEQ ID NO:1 and the VL comprises the sequence as shown in SEQ ID NO:2.

[0040] In any embodiment, the present invention provides an antigen-binding protein that binds to or specifically binds to CCR8, wherein the antigen-binding protein competitively inhibits the binding of an antibody comprising a VH and a VL, wherein the VH comprises the sequence as shown in SEQ ID NO:83 and the VL comprises the sequence as shown in SEQ ID NO:84.

[0041] In one aspect, the present invention further provides an antigen-binding protein comprising the antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to CCR8, and wherein the antigen-binding domain comprises at least one of the following:

[0042] (i) A VH that comprises complementarity determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises 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 to the sequence set forth in SEQ ID NO:5, CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:6, and CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:7;

[0043] (ii) A VH that comprises 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 to the sequence set forth in SEQ ID NO:1;

[0044] (iii) A VL that comprises CDRs 1, 2, and 3, wherein CDR1 comprises 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 to the sequence set forth in SEQ ID NO:8, CDR2 comprises 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 to the sequence set forth in SEQ ID NO:9, and CDR3 comprises 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 to the sequence set forth in SEQ ID NO:10;

[0045] (iv) A VL that comprises 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 to the sequence set forth in SEQ ID NO:2;

[0046] (v) A VH that comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO:5, the CDR2 comprises the sequence shown in SEQ ID NO:6, and the CDR3 comprises the sequence shown in SEQ ID NO:7;

[0047] (vi) A VH that comprises the sequence shown in SEQ ID NO:1;

[0048] (vii) A VL that comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO:8, the CDR2 comprises the sequence shown in SEQ ID NO:9, and the CDR3 comprises the sequence shown in SEQ ID NO:10;

[0049] (viii) A VL that comprises the sequence shown in SEQ ID NO:2;

[0050] (ix) A VH that comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO:5, the CDR2 comprises the sequence shown in SEQ ID NO:6, and the CDR3 comprises the sequence shown in SEQ ID NO:7; and a VL that comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO:8, the CDR2 comprises the sequence shown in SEQ ID NO:9, and the CDR3 comprises the sequence shown in SEQ ID NO:10; or

[0051] (x) A VH that comprises the sequence shown in SEQ ID NO:1; and a VL that comprises the sequence shown in SEQ ID NO:2.

[0052] In one aspect, the present invention further provides an antigen-binding protein comprising the antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to CCR8, and wherein the antigen-binding domain comprises at least one of the following:

[0053] (i) A VH that includes complementarity determining regions (CDRs) 1, 2, and 3, where CDR1 includes 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 to the sequence shown in SEQ ID NO:5, CDR2 includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO:6, and CDR3 includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO:7;

[0054] (ii) A VH that includes 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 to the sequence shown in SEQ ID NO:83;

[0055] (iii) A VL that includes CDRs 1, 2, and 3, where CDR1 includes 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 to the sequence shown in SEQ ID NO:8, CDR2 includes 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 to the sequence shown in SEQ ID NO:9, and CDR3 includes 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 to the sequence shown in SEQ ID NO:10;

[0056] (iv) A VL that includes 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 to the sequence shown in SEQ ID NO:84;

[0057] (v) A VH that comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:5, CDR2 comprises the sequence set forth in SEQ ID NO:6, and CDR3 comprises the sequence set forth in SEQ ID NO:7;

[0058] (vi) A VH that comprises the sequence set forth in SEQ ID NO:83;

[0059] (vii) A VL that comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:8, CDR2 comprises the sequence set forth in SEQ ID NO:9, and CDR3 comprises the sequence set forth in SEQ ID NO:10;

[0060] (viii) A VL that comprises the sequence set forth in SEQ ID NO:84;

[0061] (ix) A VH that comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:5, CDR2 comprises the sequence set forth in SEQ ID NO:6, and CDR3 comprises the sequence set forth in SEQ ID NO:7; and a VL that comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:8, CDR2 comprises the sequence set forth in SEQ ID NO:9, and CDR3 comprises the sequence set forth in SEQ ID NO:10; or

[0062] (x) A VH that comprises the sequence set forth in SEQ ID NO:83; and a VL that comprises the sequence set forth in SEQ ID NO:84.

[0063] In any aspect of the invention, the antigen-binding domain further comprises at least one of the following:

[0064] (i) A VH that includes framework regions (FR) 1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO: 11; FR2 includes 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 to the sequence shown in SEQ ID NO: 12; FR3 includes 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 to the sequence shown in SEQ ID NO: 13; and FR4 includes 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 to the sequence shown in SEQ ID NO: 14;

[0065] (ii) A VL that includes FR1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO: 15; FR2 includes 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 to the sequence shown in SEQ ID NO: 16; FR3 includes 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 to the sequence shown in SEQ ID NO: 17; and FR4 includes 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 to the sequence shown in SEQ ID NO: 18;

[0066] (iii) A VH which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:11, FR2 comprises the sequence shown in SEQ ID NO:12, FR3 comprises the sequence shown in SEQ ID NO:13, and FR4 comprises the sequence shown in SEQ ID NO:14;

[0067] (iv) A VL which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:15, FR2 comprises the sequence shown in SEQ ID NO:16, FR3 comprises the sequence shown in SEQ ID NO:17, and FR4 comprises the sequence shown in SEQ ID NO:18; or

[0068] (v) A VH which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:11, FR2 comprises the sequence shown in SEQ ID NO:12, FR3 comprises the sequence shown in SEQ ID NO:13, and FR4 comprises the sequence shown in SEQ ID NO:14; and a VL which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:15, FR2 comprises the sequence shown in SEQ ID NO:16, FR3 comprises the sequence shown in SEQ ID NO:17, and FR4 comprises the sequence shown in SEQ ID NO:18.

[0069] In any aspect of the present invention, the antigen-binding domain further comprises at least one of the following:

[0070] (i) A VH that includes framework regions (FR) 1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO: 60; FR2 includes 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 to the sequence shown in SEQ ID NO: 61; FR3 includes 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 to the sequence shown in SEQ ID NO: 62; and FR4 includes 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 to the sequence shown in SEQ ID NO: 63;

[0071] (ii) A VL that includes FR1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO: 64; FR2 includes 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 to the sequence shown in SEQ ID NO: 65; FR3 includes 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 to the sequence shown in SEQ ID NO: 66; and FR4 includes 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 to the sequence shown in SEQ ID NO: 67;

[0072] (iii) A VH that comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:60, FR2 comprises the sequence shown in SEQ ID NO:61, FR3 comprises the sequence shown in SEQ ID NO:62, and FR4 comprises the sequence shown in SEQ ID NO:63;

[0073] (iv) A VL that comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:64, FR2 comprises the sequence shown in SEQ ID NO:65, FR3 comprises the sequence shown in SEQ ID NO:66, and FR4 comprises the sequence shown in SEQ ID NO:67; or

[0074] (v) A VH that comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:60, FR2 comprises the sequence shown in SEQ ID NO:61, FR3 comprises the sequence shown in SEQ ID NO:62, and FR4 comprises the sequence shown in SEQ ID NO:63; and a VL that comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO:64, FR2 comprises the sequence shown in SEQ ID NO:65, FR3 comprises the sequence shown in SEQ ID NO:66, and FR4 comprises the sequence shown in SEQ ID NO:67.

[0075] 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 wherein the antigen-binding domain comprises at least one of the following:

[0076] (i) A VH that comprises complementarity-determining regions (CDRs) 1, 2 and 3, wherein CDR1 comprises 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 to the sequence shown in SEQ ID NO:19, CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO:20, and CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO:21;

[0077] (ii) VH that comprises 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 to the sequence set forth in SEQ ID NO:1;

[0078] (iii) VL that comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises 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 to the sequence set forth in SEQ ID NO:22, CDR2 comprises 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 to the sequence set forth in SEQ ID NO:23, and CDR3 comprises 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 to the sequence set forth in SEQ ID NO:10;

[0079] (iv) VL that comprises 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 to the sequence set forth in SEQ ID NO:2;

[0080] (v) VH that comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:19, CDR2 comprises the sequence set forth in SEQ ID NO:20, and CDR3 comprises the sequence set forth in SEQ ID NO:21;

[0081] (vi) VH that comprises the sequence set forth in SEQ ID NO:1;

[0082] (vii) VL that comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:22, CDR2 comprises the sequence set forth in SEQ ID NO:23, and CDR3 comprises the sequence set forth in SEQ ID NO:10;

[0083] (viii) VL that comprises the sequence set forth in SEQ ID NO:2;

[0084] (ix) A VH that includes CDR1, CDR2, and CDR3, where the CDR1 includes the sequence set forth in SEQ ID NO:19, the CDR2 includes the sequence set forth in SEQ ID NO:20, and the CDR3 includes the sequence set forth in SEQ ID NO:21; and a VL that includes CDR1, CDR2, and CDR3, where the CDR1 includes the sequence set forth in SEQ ID NO:22, the CDR2 includes the sequence set forth in SEQ ID NO:23, and the CDR3 includes the sequence set forth in SEQ ID NO:10; or

[0085] (x) A VH that includes the sequence set forth in SEQ ID NO:1; and a VL that includes the sequence set forth in SEQ ID NO:2.

[0086] 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 wherein the antigen-binding domain comprises at least one of the following:

[0087] (i) A VH that includes complementarity-determining regions (CDRs) 1, 2, and 3, where the CDR1 includes 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 to the sequence set forth in SEQ ID NO:19, the CDR2 includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:20, and the CDR3 includes a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence set forth in SEQ ID NO:21;

[0088] (ii) A VH that includes 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 to the sequence set forth in SEQ ID NO:83;

[0089] (iii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises 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 to the sequence shown in SEQ ID NO: 22, the CDR2 comprises 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 to the sequence shown in SEQ ID NO: 23, and the CDR3 comprises 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 to the sequence shown in SEQ ID NO: 10;

[0090] (iv) VL, wherein the VL comprises 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 to the sequence shown in SEQ ID NO: 84;

[0091] (v) VH, wherein the VH comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 19, the CDR2 comprises the sequence shown in SEQ ID NO: 20, and the CDR3 comprises the sequence shown in SEQ ID NO: 21;

[0092] (vi) VH, wherein the VH comprises the sequence shown in SEQ ID NO: 83;

[0093] (vii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 22, the CDR2 comprises the sequence shown in SEQ ID NO: 23, and the CDR3 comprises the sequence shown in SEQ ID NO: 10;

[0094] (viii) VL, wherein the VL comprises the sequence shown in SEQ ID NO: 84;

[0095] (ix) A VH that includes CDR1, CDR2, and CDR3, where the CDR1 includes the sequence shown in SEQ ID NO:19, the CDR2 includes the sequence shown in SEQ ID NO:20, and the CDR3 includes the sequence shown in SEQ ID NO:21; and a VL that includes CDR1, CDR2, and CDR3, where the CDR1 includes the sequence shown in SEQ ID NO:22, the CDR2 includes the sequence shown in SEQ ID NO:23, and the CDR3 includes the sequence shown in SEQ ID NO:10;

[0096] (x) A VH that includes the sequence shown in SEQ ID NO:83; and a VL that includes the sequence shown in SEQ ID NO:84.

[0097] In any aspect of the present invention, the antigen-binding domain further includes at least one of the following:

[0098] (i) A VH that includes framework regions (FR) 1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO:24, FR2 includes 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 to the sequence shown in SEQ ID NO:25, FR3 includes 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 to the sequence shown in SEQ ID NO:26, and FR4 includes 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 to the sequence shown in SEQ ID NO:14;

[0099] (ii) VL, where the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO:27, the FR2 comprises 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 to the sequence shown in SEQ ID NO:28, the FR3 comprises 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 to the sequence shown in SEQ ID NO:29, and the FR4 comprises 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 to the sequence shown in SEQ ID NO:18;

[0100] (iii) VH, where the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:24, the FR2 comprises the sequence shown in SEQ ID NO:25, the FR3 comprises the sequence shown in SEQ ID NO:26, and the FR4 comprises the sequence shown in SEQ ID NO:14;

[0101] (iv) VL, where the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:27, the FR2 comprises the sequence shown in SEQ ID NO:28, the FR3 comprises the sequence shown in SEQ ID NO:29, and the FR4 comprises the sequence shown in SEQID NO:18; or

[0102] (v) A VH that includes FR1, FR2, FR3, and FR4, where FR1 includes the sequence shown in SEQ ID NO:24, FR2 includes the sequence shown in SEQ ID NO:25, FR3 includes the sequence shown in SEQ ID NO:26, and FR4 includes the sequence shown in SEQ ID NO:14; and a VL that includes FR1, FR2, FR3, and FR4, where FR1 includes the sequence shown in SEQ ID NO:27, FR2 includes the sequence shown in SEQ ID NO:28, FR3 includes the sequence shown in SEQ ID NO:29, and FR4 includes the sequence shown in SEQ ID NO:18.

[0103] In any aspect of the present invention, the antigen-binding domain further comprises at least one of the following:

[0104] (i) A VH that includes framework regions (FR) 1, FR2, FR3, and FR4, where FR1 includes 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 to the sequence shown in SEQ ID NO:68, FR2 includes 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 to the sequence shown in SEQ ID NO:69, FR3 includes 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 to the sequence shown in SEQ ID NO:70, and FR4 includes 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 to the sequence shown in SEQ ID NO:63;

[0105] (ii) VL, where the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO:71, the FR2 comprises 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 to the sequence shown in SEQ ID NO:72, the FR3 comprises 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 to the sequence shown in SEQ ID NO:73, and the FR4 comprises 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 to the sequence shown in SEQ ID NO:67;

[0106] (iii) VH, where the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:68, the FR2 comprises the sequence shown in SEQ ID NO:69, the FR3 comprises the sequence shown in SEQ ID NO:70, and the FR4 comprises the sequence shown in SEQ ID NO:63;

[0107] (iv) VL, where the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:71, the FR2 comprises the sequence shown in SEQ ID NO:72, the FR3 comprises the sequence shown in SEQ ID NO:73, and the FR4 comprises the sequence shown in SEQID NO:67; or

[0108] (v) VH, which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 68, FR2 comprises the sequence shown in SEQ ID NO: 69, FR3 comprises the sequence shown in SEQ ID NO: 70, and FR4 comprises the sequence shown in SEQ ID NO: 63; and VL, which comprises FR1, FR2, FR3 and FR4, wherein FR1 comprises the sequence shown in SEQ ID NO: 71, FR2 comprises the sequence shown in SEQ ID NO: 72, FR3 comprises the sequence shown in SEQ ID NO: 73, and FR4 comprises the sequence shown in SEQ ID NO: 67.

[0109] As described herein, the antigen-binding protein may be in the following forms:

[0110] (i) Single-chain Fv fragment (scFv);

[0111] (ii) Dimeric scFv (di-scFv); or

[0112] (iii) One of (i) or (ii), which is linked to an antibody constant region, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0113] In addition, as described herein, the antigen-binding protein may be in the following forms:

[0114] (i) Diabody;

[0115] (ii) Triabody;

[0116] (iii) Tetrabody;

[0117] (iv) Fab;

[0118] (v) F(ab')2;

[0119] (vi) Fv;

[0120] (vii) Bispecific antibody or other forms of multispecific antibodies; or

[0121] (viii) One of (i) to (vii), which is linked to an antibody constant region, Fc or heavy chain constant domain (CH)2 and / or CH3.

[0122] The aforementioned antigen-binding protein may also be referred to as the antigen-binding domain of an antibody.

[0123] In certain embodiments, the complementarity-determining region sequences (CDRs) of the antigen-binding protein of the present invention may be defined according to the IMGT numbering system, Kabat or Chothia system.

[0124] In any aspect or embodiment, the antigen-binding protein can comprise a human constant region.

[0125] The reference herein to a protein or antibody that "binds" CCR8 provides literal support for a protein or antibody that "specifically binds" CCR8.

[0126] Preferably, the antigen-binding protein as described herein is an antibody or an antigen-binding fragment thereof. Generally, the antigen-binding protein is an antibody, such as a monoclonal antibody. The antigen-binding protein can be in the form of a recombinant or modified antibody (e.g., chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, primatized antibody, deimmunized antibody, synthetic humanized antibody, half antibody, bispecific antibody, trispecific antibody, or multispecific antibody). The antibody can also comprise chemical modifications, such as conjugation to an active agent or a radiolabel or a reagent for improving solubility, or other modifications as described herein.

[0127] 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.

[0128] In another aspect, the invention also provides an anti-CCR8 antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region,

[0129] wherein the heavy chain variable region comprises:

[0130] - CDR H1 as shown in SEQ ID NO:5, CDR H2 as shown in SEQ ID NO:6, and CDR H3 as shown in SEQ ID NO:7; and

[0131] wherein the light chain variable region comprises:

[0132] - CDR L1 as shown in SEQ ID NO:8, CDR L2 as shown in SEQ ID NO:9, and CDR L3 as shown in SEQ ID NO:10.

[0133] In any embodiment, the anti-CCR8 antibody or an antigen-binding fragment thereof comprises a light chain variable region that comprises FR L1 as shown in SEQ ID NO:15, FR L2 as shown in SEQ ID NO:16, FR L3 as shown in SEQ ID NO:17, and FR L4 as shown in SEQ ID NO:18.

[0134] In any embodiment of the present invention, the anti-CCR8 antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region that comprises FR H1 as set forth in SEQ ID NO:11, FR H2 as set forth in SEQ ID NO:12, FR H3 as set forth in SEQ ID NO:13, and FR H4 as set forth in SEQ ID NO:14.

[0135] In any embodiment, the anti-CCR8 antibody or an antigen-binding fragment thereof comprises a light-chain variable region that comprises FR L1 as set forth in SEQ ID NO:64, FR L2 as set forth in SEQ ID NO:65, FR L3 as set forth in SEQ ID NO:66, and FR L4 as set forth in SEQ ID NO:67.

[0136] In any embodiment of the present invention, the anti-CCR8 antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region that comprises FR H1 as set forth in SEQ ID NO:60, FR H2 as set forth in SEQ ID NO:61, FR H3 as set forth in SEQ ID NO:62, and FR H4 as set forth in SEQ ID NO:63.

[0137] In another aspect, the present invention also provides an anti-CCR8 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment comprises a light-chain variable region and a heavy-chain variable region,

[0138] wherein the heavy-chain variable region comprises:

[0139] - 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:

[0140] 21; and

[0141] wherein the light-chain variable region comprises:

[0142] - 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:

[0143] 10.

[0144] In any embodiment, the anti-CCR8 antibody or an antigen-binding fragment thereof comprises a light-chain variable region that comprises FR L1 as set forth in SEQ ID NO:27, FR L2 as set forth in SEQ ID NO:28, FR L3 as set forth in SEQ ID NO:29, and FR L4 as set forth in SEQ ID NO:18.

[0145] In any embodiment of the invention, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising FR H1 as set forth in SEQ ID NO:24, FR H2 as set forth in SEQ ID NO:25, FR H3 as set forth in SEQ ID NO:26, and FR H4 as set forth in SEQ ID NO:14.

[0146] In any embodiment, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising FR L1 as set forth in SEQ ID NO:71, FR L2 as set forth in SEQ ID NO:72, FR L3 as set forth in SEQ ID NO:73, and FR L4 as set forth in SEQ ID NO:67.

[0147] In any embodiment of the invention, the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising FR H1 as set forth in SEQ ID NO:68, FR H2 as set forth in SEQ ID NO:69, FR H3 as set forth in SEQ ID NO:70, and FR H4 as set forth in SEQ ID NO:63.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence of SEQ ID NO:2, and the heavy chain variable region comprising the sequence of SEQ ID NO:1.

[0153] In any embodiment, the anti-CCR8 receptor antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising the sequence of SEQ ID NO:84, and the heavy chain variable region comprising the sequence of SEQ ID NO:83.

[0154] In any aspect or embodiment, the antigen-binding protein or anti-CCR8 receptor antibody or antigen-binding fragment thereof may also comprise up to 15, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably has 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the amino acid sequence of the indicated SEQ ID NO. Preferably, these amino acid insertions, deletions, substitutions or combinations thereof are not in the CDRs.

[0155] In any aspect of the present invention and in any antigen-binding protein described herein, an Fc region is also included, which is engineered to have a reduced or increased ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the reduced or increased ability to induce ADCC is conferred by mutations, deletions or modifications of amino acids in the Fc region that interact with Fc receptors.

[0156] In another aspect, the present invention provides an anti-CCR8 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, which comprises an antigen-binding protein having a sequence as described herein or comprising CDR and / or FR sequences as described herein.

[0157] In any aspect, the antigen-binding protein is an antibody of the IgG2b isotype.

[0158] The antigen-binding protein as described herein may comprise a human constant region, such as an IgG constant region, such as an IgG1, IgG2, IgG3 or IgG4 constant region or a mixture thereof. Where the antibody or protein comprises V H and V L , V H may be linked to the heavy chain constant region, and V L may be linked to the light chain constant region.

[0159] In one instance, an antigen-binding protein as described herein comprises a constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one instance, 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)).

[0160] In one instance, the antigen-binding protein comprises a V as disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as described above). H and the V L is linked or fused to a κ light chain constant region.

[0161] In any aspect of the invention, the antibody is a naked antibody. Specifically, the antibody is in a non-conjugated form and is not adapted to form a conjugate.

[0162] In any aspect of the invention, the antibody exhibits antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, the antibody exhibits the same or a similar level of ADCC as 19D7 (the Shionogi anti-CCR8 antibody). ADCC can be measured by any method known in the art or as described herein (including Example 11).

[0163] In any aspect of the invention, the antigen-binding protein depletes or reduces tumor-infiltrating T regulatory (Treg) cells in a tumor; optionally wherein the antigen-binding protein does not deplete or reduce Treg in the spleen.

[0164] In any aspect of the invention, the antigen-binding protein increases tumor-specific CD8+ T cells and / or reduces CD4+ T cells or tumor-specific CD4+ T cells.

[0165] In another aspect, the 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 conjugated to a label or cytotoxic agent.

[0166] In aspects of the invention related to multiple polypeptide chains that form an antigen-binding protein, the expression construct comprises a nucleic acid encoding a polypeptide comprising, for example, VH operably linked to a promoter and a nucleic acid encoding a polypeptide comprising, for example, VL operably linked to a promoter.

[0167] In another example, the expression construct is a bicistronic expression construct that, for example, in a 5' to 3' order, comprises the following operably linked components:

[0168] (i) A promoter

[0169] (ii) A nucleic acid encoding a first polypeptide;

[0170] (iii) An internal ribosome entry site; and

[0171] (iv) A nucleic acid encoding a second polypeptide,

[0172] wherein the first polypeptide comprises VH and the second polypeptide comprises VL, or vice versa.

[0173] In another aspect, the invention also contemplates separate expression constructs, where one expression construct encodes a first polypeptide comprising VH and another expression construct encodes a second polypeptide comprising VL. For example, the invention also provides a composition comprising:

[0174] (i) A first expression construct that comprises a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter; and

[0175] (ii) A second expression construct that comprises a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter.

[0176] 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 the expression construct of the invention or:

[0177] (i) A first expression construct that comprises a nucleic acid encoding a polypeptide comprising VH operably linked to a promoter; and

[0178] (ii) A second expression construct that comprises a nucleic acid encoding a polypeptide comprising VL operably linked to a promoter,

[0179] wherein the first polypeptide and the second polypeptide bind to form the antigen-binding protein of the invention.

[0180] Examples of the cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0181] In another aspect, the present invention provides a nucleic acid encoding an antigen-binding protein, an immunoglobulin variable domain, an antibody or an antigen-binding fragment thereof, a scFv, a Fab, a Fab', an F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate as described herein.

[0182] In another aspect, the present invention provides a vector comprising the nucleic acid as described herein.

[0183] In another aspect, the present invention provides a cell comprising the vector or nucleic acid as described herein.

[0184] In any embodiment, the nucleic acid may comprise a nucleotide sequence as shown in any one of SEQ ID NO:3, 4 or 30 to 54, preferably SEQ ID NO: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.

[0185] In another aspect, the present invention provides a pharmaceutical composition comprising an antigen-binding protein as described herein (or comprising CDR and / or FR sequences as described herein) or an immunoglobulin variable domain, an antibody or an antigen-binding fragment thereof, a scFv, a Fab, a Fab', an F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate, and a pharmaceutically acceptable carrier, diluent or excipient.

[0186] In another aspect, the present invention provides a kit or article comprising an antigen-binding protein as described herein (or comprising CDR and / or FR sequences as described herein) or an immunoglobulin variable domain, an antibody or an antigen-binding fragment thereof, a scFv, a Fab, a Fab', an F(ab')2, an Fv fragment, a diabody, a triabody, a linear antibody, a single-chain antibody molecule or a multispecific antibody, a fusion protein or a conjugate.

[0187] In another aspect, the present invention provides the use of a sequence according to one or more of CDR1, CDR2, CDR3, FR1, FR2, FR3 and FR4 as described herein for producing an antigen-binding protein for binding to CCR8.

[0188] In another aspect, the present invention provides a method for producing an antigen-binding protein, an antibody or an antigen-binding fragment for binding to CCR8 as described herein, the method comprising expressing the nucleic acid as described herein in a cell or animal as described herein.

[0189] The functional characteristics of the antigen-binding proteins of the present invention, with necessary modifications, will be applicable to the antibodies or antigen-binding fragments of the present invention.

[0190] The antigen-binding proteins as described herein can be purified, substantially purified, isolated, and / or recombinant.

[0191] The antigen-binding proteins of the present invention can be a part of the supernatant taken from the culture medium in which hybridomas expressing the antigen-binding proteins of the present invention have been cultured.

[0192] In another aspect, the present invention provides a method for preventing or treating a disorder or disease associated with the expression of CCR8 in an individual, the method comprising the step of providing to an individual in need of treatment for said disorder 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 disorder associated with the expression of CCR8 is preferably cancer.

[0193] In another aspect, the present invention further provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject an antigen-binding protein, antibody or antigen-binding fragment or pharmaceutical composition of the present invention, thereby treating or preventing cancer in the subject. As used herein, a method for treating cancer includes methods for inhibiting, preventing or minimizing the spread or progression of cancer, including inhibiting or preventing the metastasis of cancer.

[0194] In another aspect, the present invention further provides a method for reducing or depleting regulatory T cells in a tumor, the method comprising administering to the subject an antigen-binding protein, antibody or antigen-binding fragment or pharmaceutical composition of the present invention, thereby reducing or depleting regulatory T cells in the tumor.

[0195] In another aspect, the present invention further provides a method for increasing tumor-specific CD8+ T cells and / or reducing CD4+ T cells in a tumor, the method comprising administering to the 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 reducing CD4+ T cells in the tumor.

[0196] In another aspect, the present invention further provides the use of the antigen-binding protein, antibody or antigen-binding fragment or pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing cancer in a subject.

[0197] In another aspect, the present invention further provides the use of the antigen-binding protein, antibody or antigen-binding fragment or pharmaceutical composition of the present invention in the manufacture of a medicament for:

[0198] · Reduce or deplete regulatory T cells in a tumor; or

[0199] · Increase tumor - specific CD8+ T cells in a tumor and / or reduce CD4+ T cells.

[0200] In another aspect, the present invention provides an antigen - binding protein, antibody or antigen - binding fragment or pharmaceutical composition of the present invention for treating or preventing cancer in a subject.

[0201] In another aspect, the present invention provides an antigen - binding protein, antibody or antigen - binding fragment or pharmaceutical composition of the present invention for:

[0202] · Reduce or deplete regulatory T cells in a tumor; or

[0203] · Increase tumor - specific CD8+ T cells in a tumor and / or reduce CD4+ T cells.

[0204] As used herein, unless the context otherwise requires, the term "comprising" or its variants, such as "including" and "containing", are not intended to exclude additional additives, components, integers or steps.

[0205] Other aspects of the present invention and other embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] Figure 1 . Reactivity of the 2H8 antibody with two transfected CHO - Flpn cell lines of human and mouse. Reactivity of 2H8 with mouse CCR8 (blue) or human CCR8 (orange) CHO - Flpn cells and untransfected CHO Flpn (red) compared to (A) anti - hCCR8 #1 antibody or (B) anti - hCCR8 (clone #L263G8 Biolegend).

[0207] Figure 2 . Reactivity of the 2H8 antibody and other anti - hCCR8 antibodies with Cyno CCR8. HEK293T cells were transfected with a plasmid encoding cynomolgus monkey CCR8 and stained with various anti - hCCR8 antibodies (blue) or isotype controls (red).

[0208] Figure 3. Reactivity of the 2H8 antibody against other CCR chemokine receptors in transfected L1.2 cells. 2H8-B4 does not cross-react with: untransfected cells (purple); human CCR3 (pink); human CCR6 (dark green); human CCR7 (orange-green); or human CCR10 (blue) - in the order from top to bottom in the non-binding histograms. 2H8-B4 binds to both human CCR8 (orange)- and mouse CCR8 (red)-transfected L1.2 cells - in the order from top to bottom in the binding histograms.

[0209] Figure 4 . 2H8 binds to human tumor-infiltrating Tregs (TiTregs). Fresh patient colorectal biopsies were used to isolate single-cell suspensions. Cells were stained with anti-human CD3; anti-human CD4; anti-human FoxP3; and 2H8-B4 or isotype control. The histograms show that 2H8-B4 (red) binds to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gate), while the isotype control (gray) does not bind to them.

[0210] Figure 5 . The hCCR8 antibody binds to hCCR8-L1.2 transfected cells. By flow cytometry, the chimeric 2H8-B4 antibody was compared with chimeric 19D7 (Shionogi antibody) and chimeric 4A19 (BMS antibody) on hCCR8-L1.2 transfected cells using increasing concentrations of purified mAb.

[0211] Figure 6 . Humanized 2H8 binds to hCCR8-L1.2 transfected cells. By flow cytometry, humanized 2H8-B4 was compared with chimeric 2H8 B4 (parent) and 19D7 (Shionogi antibody) on hCCR8-L1.2 transfected cells using increasing concentrations of purified mAb.

[0212] Figure 7 . Fresh patient colorectal biopsies were used to isolate single-cell suspensions. Cells were stained with anti-human CD3; anti-human CD4; anti-human FoxP3; and 2H8-B4 (right panel) or isotype control (left panel). 2H8-B4 (right panel) binds to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gate), while the isotype control (left panel) does not bind to them.

[0213] Figure 8. Chimeric 2H8 does not inhibit CCL1-induced chemotaxis. 200,000 hCCR8 L1.2 cells / well were pre-incubated with different concentrations of the antibody at 37 °C for 15 minutes. Then the cells were transferred to a chemotaxis plate and incubated overnight (4 hours) to achieve chemotaxis at the EC90 of the antibody at the same concentration gradient (hCCL1 - 5.84 nM). Then 100 μL was collected from the bottom well, centrifuged and resuspended in 50 μL of FACS buffer. Then 15 μL was counted on an LSRIIb machine.

[0214] Figure 9 . Chimeric 2H8 exhibits ADCC potential, and its efficacy and potency are comparable to those of 19D7. A dose-response curve of the ADCC assay was constructed, and the potential to induce cytotoxicity of both chimeric 2H8 and chimeric 19D7 was analyzed by using L1.2-hCCR8 cells incubated with different concentrations of the antibody. The highest antibody concentration was 13.33 nM, and it was diluted fourfold to generate a dose-response curve. The antibody concentrations were transformed to a logarithmic scale and placed on the x-axis. Statistical analysis was performed using a best-fit sigmoidal standard curve to calculate the IC50 and R2 of each antibody.

[0215] Figure 10 . 2H8 inhibits tumor growth in the MC38 model of colorectal cancer. In C57BL / 6 mice, the individual volumes of MC38 tumors implanted subcutaneously at 5×10 5 cells per injection are shown in the graph. Animals were treated with 2H8 mIgG2a (n = 9), anti-mPD1 (n = 10), and isotype control (n = 6). After the first injection of the antibody, the tumor volume (mm 3 ) was measured daily with digital calipers and compared between groups. Treatments were performed twice a week at a dose of 5 mg / kg for a maximum of 3 weeks.

[0216] Figure 11 . 2H8 causes depletion of Tregs in tumors but not in the spleen. At the endpoint, tumor tissues and spleen tissues were processed, stained and analyzed by flow cytometry. Cells were collected, and the percentages of CD4 + FOXP3 + CD25 + cells in TCR-β + cells (parental population) were compared between the three groups. All graphs present the mean and individual values, shown as histograms, scatter plots, and error bars representing SEM. (A) Representative flow cytometry scatter plots of the three groups, showing CD4 + FOXP3 + CD25 +Percentage of the mean ± SEM of cells. (B) Percentage of T-regs in tumors of C57BL / 6 wild-type mice. (C) Percentage of T-regs 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 the 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.

[0217] Figure 12 . Treatment with 2H8 increases tumor-specific CD8+ T cells and decreases CD4+ T cells. At the end point, tumor tissues and spleen tissues of MC38 tumors in C57BL / 6 mice were treated, these tissues were stained and analyzed by flow cytometry. Cells were collected, and the percentages of CD4+ cells and CD8+ cells in TCR-β+ cells (parent population) were compared among the three groups. All curves show the mean and individual values, presented as histograms, scatter plots, and error bars representing SEM. (A) Representative flow cytometry scatter plots of tumors of the 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 on tumors of the three different groups. (F) Percentage of CD62LloCD44hi CD8+ T cells in tumors. (G) Percentage of CD62LhiCD44hi CD8+ T cells in tumors. (H) Percentage of CD62LloCD44lo CD8+ T cells in tumors. (I) Percentage of CD62LhiCD44lo CD8+ T cells in tumors. 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

[0218] Sequence Listing

[0219] Table 1. Sequences of the present invention

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] Detailed embodiments

[0227] Other aspects of the present invention and other embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description given by way of example and with reference to the accompanying drawings.

[0228] Certain embodiments of the present invention will now be described in detail. While the present invention will be described in connection with the embodiments, it should be understood that the intention is not to limit the present invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims.

[0229] Various antigen-binding proteins that bind to CCR8 are known, but lack one or more properties that make them suitable for preclinical or clinical development. The CCR8-binding antigen-binding proteins described herein exhibit favorable binding properties not only to human CCR8 but also to mouse CCR8 and cynomolgus monkey (cyno) CCR8. This provides an opportunity to study the properties of the antigen-binding proteins in two preclinical models, mice and cynomolgus monkeys. Additionally, the antigen-binding proteins also exhibit specificity for CCR8 as they do not bind to many closely related receptors (as shown herein). Surprisingly, by immunizing mice with mouse CCR8, CCR8 antigen-binding proteins that bind to human, mouse, and cynomolgus monkey CCR8 are produced as described in the examples.

[0230] Additionally, the resulting CCR8 antibodies are then humanized and, surprisingly, retain the binding affinity for human CCR8 as well as the ability to bind to live cells.

[0231] General definitions

[0232] Throughout the specification, unless specifically stated otherwise or the context requires, when referring to a single step, a composition of matter, a group of steps, or a group of compositions of matter, one and more than one (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter shall be covered. 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 a single as well as two or more; reference to "an" includes a single species as well as two or more species; reference to "the" includes a single entity as well as two or more entities, and so on.

[0233] Those skilled in the art will understand that the present invention is susceptible to variations and modifications in addition to those specifically described. It should be understood that the present invention encompasses all such variations and modifications. The present invention also includes all steps, features, compositions, and compounds individually or jointly mentioned or pointed out in this specification, as well as any and all combinations of said steps or features, or any two or more of them.

[0234] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0235] All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

[0236] The scope of the present invention is not limited by the specific examples described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention.

[0237] For the purpose of interpreting this specification, the following definitions will apply, and terms used in the singular shall also include the plural form, and vice versa, where appropriate. If any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0238] Unless otherwise specifically defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0239] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures well known to those of skill in the art. Such techniques are described and explained throughout the literature in sources such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0240] The description and definition of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein can be further elucidated by the discussion in the following: 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.

[0241] The term “and / or”, e.g., “X and / or Y” shall be understood to mean “X and Y” or “X or Y” and shall be considered to provide explicit support for both meanings or either meaning.

[0242] As used herein, the term “derived from” shall be understood to indicate that the specified entity can be obtained from a particular source, although not necessarily directly from that source.

[0243] As used herein, the term “CCR8” or “C-C 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 the expression of CCR8, FOXp3, CD39, granzyme B, and IL-10 in a STAT3-dependent manner. See, e.g., Barsheshet et al., PNAS 114(23):6086-91 (June 6, 2017). CCR8 is expressed primarily on Treg cells and, to a lesser extent, on a subset of TH2 cells, monocytes, NK cells, and CD8+ cells. CCR8 is a transmembrane receptor that interacts with G proteins and has seven transmembrane domains, an extracellular N-terminal domain, and an intracellular C-terminal domain. 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.

[0244] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, due to its source or the source from which it is derived, is not associated with the natural associated components that accompany it in its native state; and is substantially free of other proteins from the same source. A protein can be rendered substantially free of natural associated components or be substantially purified by separation using protein purification techniques known in the art. "Substantially purified" means that the protein is substantially free of 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 contaminants.

[0245] The term "recombinant" should be understood to mean the product of artificial genetic recombination. Thus, in the context of a recombinant protein containing an antibody antigen-binding domain, the term does not cover antibodies that occur naturally in a subject, i.e., the products of natural recombination that occur during B cell maturation. However, if such an antibody is isolated, it is considered an isolated protein containing an antibody antigen-binding domain. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant methods, the resulting protein is a recombinant protein containing an antibody antigen-binding domain. Recombinant proteins also cover proteins that are expressed by artificial recombinant methods when they are in, for example, a cell, tissue, or subject in which the protein is expressed.

[0246] The term "protein" should be understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds or a series of polypeptide chains that are covalently or non-covalently linked to each other (i.e., a polypeptide complex). For example, this 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 can contain one or more non-natural amino acids.

[0247] The term "polypeptide" or "polypeptide chain" will be understood from the previous paragraph to mean a series of consecutive amino acids linked by peptide bonds.

[0248] As used herein, the term "antigen-binding domain" should be understood to mean the region of an antibody that is capable of specifically binding an antigen, i.e., V H or V L or an Fv containing both VH and VL. The antigen-binding domain need not be in the context of a full antibody; for example, it can be in another form as described herein, such as an scFv.

[0249] For the purposes of the present disclosure, the term "antibody" includes proteins capable of specifically binding one or several closely related antigens through antigen-binding domains contained within the Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies).

[0250] Antibodies typically contain constant domains, which can be arranged into constant regions or constant fragments or crystallizable fragments (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain two covalently linked heavy chains (about 50 kD to 70 kD) and two light chains (each about 23 kDa). Light chains generally contain a variable region (if present) and a constant domain and are either κ light chains or λ light chains in mammals. Heavy chains generally contain a variable region and one or two constant domains linked to additional constant domains by a hinge region. Heavy chains of mammals are 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, the two heavy chains as well as the heavy and light chains are held together by interchain disulfide bonds and by non-covalent interactions. The number of interchain disulfide bonds can vary between different types of antibodies. Each chain has an N-terminal variable region (V H or V L , each having a length of about 110 amino acids) and one or more constant domains at the C-terminus. The constant domain of the light chain (C L , with a length of about 110 amino acids) aligns with and is bonded by a disulfide bond to the first constant domain of the heavy chain (C H1 , with a length of 330 to 440 amino acids). The variable region of the light chain aligns with the variable region of the heavy chain. The heavy chain of the antibody can contain 2 or more additional C H domains (such as C H2 , C H3 , etc.) and can contain a hinge region between the C H1 and C H2 constant domains. Antibodies can 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 instance, the antibody is a murine (mouse or rat) antibody or a primate (such as human) antibody. In one instance, the heavy chain of the antibody lacks a C-terminal lysine residue. In one instance, the antibody is humanized, synthetic humanized, chimeric, CDR-grafted, or deimmunized.

[0251] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably and refer to an antibody in a substantially intact form as opposed to an antigen-binding fragment of the antibody. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain that contain an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or an amino acid sequence variant thereof.

[0252] As used herein, "variable region" refers to the portion of the light chain and / or heavy chain of an antibody that is capable of specifically binding an antigen as defined herein and contains the complementarity-determining regions (CDRs), namely CDR1, CDR2, and CDR3; and the amino acid sequence of the framework regions (FRs). For example, the variable region contains three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. V H refers to the variable region of the heavy chain. V L refers to the variable region of the light chain.

[0253] As used herein, the term "complementarity-determining region" (synonym CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of the antibody variable region that are the major contributors to specific antigen binding. Each variable domain (V H or V L ) typically has three CDRs, which are designated CDR1, CDR2, and CDR3. The CDRs of V H are also referred to herein as CDR H1, CDRH2, and CDR H3, respectively, where CDR H1 corresponds to CDR 1 of V H , CDR H2 corresponds to CDR 2 of V H , and CDR H3 corresponds to CDR 3 of V H . Similarly, the CDRs of V L are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR1 of V L , CDR L2 corresponds to CDR 2 of V L , and CDR L3 corresponds to CDR 3 of V LCDR 3. In one example, the amino acid positions assigned to CDRs and FRs are defined according to 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 example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The present invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including normalized numbering systems or the following numbering systems: 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; Honnegher and Plükthun J. Mol. Biol. 309:657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25:206-211 1997.

[0254] The "framework regions" (FRs) 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, where FR H1 corresponds to the FR 1 of V H and FR H2 corresponds to the FR 2 of V H and FR H3 corresponds to the FR 3 of V H and FR H4 corresponds to the FR 4 of V H Similarly, the FRs of V L are referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to the FR 1 of V L and FR L2 corresponds to the FR 2 of V L and FR L3 corresponds to the FR 3 of V L and FRL4 corresponds to the FR 4 of V L

[0255] As used herein, the term "Fv" should be understood to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which V L and V​H Associate and form a complex having an antigen-binding domain, i.e., capable of specifically binding an antigen. The V that forms the antigen-binding domain H and V L can be located in a single polypeptide chain or in different polypeptide chains. In addition, the Fv of the present invention (and any protein of the present invention) can have multiple antigen-binding domains that can or cannot bind the same antigen. This term should be understood to cover fragments directly derived from antibodies and proteins corresponding to such fragments produced using recombinant methods. In some instances, V H is not linked to the heavy chain constant domain (C H )1 and / or V L is not linked to the light chain constant domain (C L ). Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetra-bodies or higher-order complexes, or any of the foregoing, such as microantibodies, linked to their constant regions or domains such as CH2 or CH3 domains.

[0256] A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digesting the whole antibody with papain to produce a fragment consisting of the complete light chain and a portion of the heavy chain, or can be produced using recombinant methods. The "Fab' fragment" of an antibody can be obtained by treating the whole antibody with pepsin and then reducing it to produce a molecule consisting of the complete light chain and a portion of the heavy chain containing V H and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be produced using recombinant methods. The "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked together by two disulfide bonds and is obtained by treating the whole antibody molecule with pepsin but without subsequent reduction. A "Fab2" fragment is a recombinant fragment that contains two Fab fragments linked, for example, by a leucine zipper or a CH3 domain. A "single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragments (Fv) of an antibody, wherein the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0257] As used herein, the term "binding" with respect to the interaction of an antigen-binding protein or its antigen-binding domain with an antigen means that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than to a general protein. If an antibody binds to epitope "A", then in a reaction containing labeled "A" and the protein, the presence of molecules containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" that binds to the antibody.

[0258] As used herein, the term "specifically binds" should be understood to mean that the antigen-binding protein of the present invention reacts or associates with a specific antigen or a cell expressing the specific antigen more frequently, more rapidly, for a longer duration, and / or with a higher affinity than it reacts or associates with an alternative antigen or cell. For example, the antigen-binding protein binds to CCR8 with a much higher 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) than it binds 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 an example of the present invention, the antigen-binding protein "specifically binds" to CCR8 with an affinity that is at least 1.5-fold or 2-fold or higher (e.g., 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or 200-fold) higher than it binds 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 means specific binding, and each term should be understood to provide express support for the other term.

[0259] As used herein, the term "undetectably binds" should be understood to mean that an antigen-binding protein (e.g., an antibody) binds to a candidate antigen at a level less than 10%, or 8%, or 6%, or 5% above background. The background can be the level of the binding signal detected in the absence of the 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 binding level is detected using a biosensor assay (e.g., Biacore), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0260] As used herein, the term "insignificant binding" should be 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 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 level of binding detected in the presence of a negative control polypeptide. Binding levels are detected using a biosensor assay (e.g., Biacore or Blitz), in which the antigen-binding protein is immobilized and contacted with the antigen.

[0261] As used herein, the term "epitope" (synonym "antigenic determinant") should be understood to mean the region of CCR8 to which an antigen-binding protein containing the antigen-binding domain of an antibody binds. Unless otherwise defined, the term need not be limited to the specific residues or structures contacted by the antigen-binding protein. For example, the term includes regions spanning the amino acids contacted by the antigen-binding protein and 5-10 (or more) or 2-5 or 1-3 amino acids outside of that region. In some instances, an epitope comprises a series of discontinuous amino acids that are positioned close to each other when the antigen-binding protein is folded, i.e., a "conformational epitope". One of ordinary skill in the art will also appreciate that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes the chemically reactive surface groups of a molecule, such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and in certain instances, can have specific three-dimensional structural features and / or specific charge characteristics.

[0262] As used herein, the term "prevent" includes administering an antigen-binding protein of the invention so as to halt or prevent the development of at least one symptom of a disorder. The term also encompasses treating a subject in remission to prevent or stop a recurrence.

[0263] As used herein, the term "treat" includes administering an antigen-binding protein as described herein so as to reduce or eliminate at least one symptom of a particular disease or disorder.

[0264] As used herein, the term "subject" should be understood to mean any animal, including a human, e.g., a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0265] As used herein, the terms "engineered cell" and "genetically modified cell" may be used interchangeably. These terms mean a cell that contains and / or expresses a foreign gene or nucleic acid sequence that in turn modifies the genotype or phenotype of the cell or its progeny.

[0266] antibody

[0267] In one instance, an antigen-binding protein as described herein according to any instance is an antibody.

[0268] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, CCR8 or a region thereof (e.g., the extracellular region), or an immunogenic fragment or an epitope thereof, or a cell expressing and presenting it (i.e., an immunogen) is administered to a non-human animal (e.g., a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient. The immunogen can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0269] The production of polyclonal antibodies can be monitored by collecting blood samples from the immunized animal at multiple time points after immunization. If necessary to achieve the desired antibody titer, one or more further immunization regimens can be carried out. The process of repeated boosting and titration is carried out until a suitable titer is reached. When the desired immunogenic level is obtained, the immunized animal is bled, the serum is separated and stored, and / or the animal is used to produce monoclonal antibodies (mAbs).

[0270] Monoclonal antibodies are an exemplary form of the antibodies contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous population of antibodies that are capable of binding to the same antigen (e.g., the same epitope within the antigen). The term is not intended to limit the source of the antibody or the manner of its preparation.

[0271] For the production of monoclonal antibodies, any of a number of known techniques can be used, such as those illustrated in US 4,196,265 or in Harlow and Lane (1988) above.

[0272] 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. Mice that have been genetically engineered to express human antibodies (e.g., mice that do not express murine antibodies) can also be used to produce the antibodies of the present invention (e.g., as described in WO 2002 / 066630).

[0273] After immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells), are selected for the mAb production protocol. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes or from peripheral blood samples. The B cells from the immunized animal are then fused with immortalized myeloma cells, which are typically derived from the same species as the animal immunized with the immunogen.

[0274] The hybrids are amplified by culturing in a selective medium that contains an agent that blocks de novo nucleotide synthesis in tissue culture medium. Exemplary agents are aminopterin, methotrexate, and azaserine.

[0275] Functional selection of the amplified hybridomas for antibody specificity and / or titer is performed, such as by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.).

[0276] Alternatively, the ABL-MYC technique (NeoClone, Madison WI, USA 53713) is used to generate cell lines secreting mAbs (e.g., as described in Largaespada et al., J. Immunol. Methods. 197:85-95, 1996).

[0277] Antibodies can also be generated or isolated by screening display libraries (e.g., phage display libraries), such as those described in US6300064 and / or US5885793.

[0278] The antibodies of the invention can be synthetic antibodies. For example, the antibody is a chimeric antibody, a humanized antibody, a synthetic humanized antibody, a primatized antibody, or a deimmunized antibody.

[0279] There are several in vitro methods for determining the potency of an antibody to initiate ADCC. These include the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Generally, a labeled target cell line expressing a certain surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are generally co-incubated with the antibody-labeled target cells. Subsequently, target cell lysis is usually measured by the release of an intracellular marker, e.g., by a scintillation counter or spectrophotometry. Preferred assays are detailed in the Examples.

[0280] A protein containing an antibody-binding domain

[0281] Diabodies, triabodies, tetra-bodies

[0282] In some instances, the proteins of the invention are or comprise diabodies, triabodies, tetra-bodies or higher-order protein complexes such as those described in WO98 / 044001 and / or WO94 / 007921.

[0283] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure V L -X-V H or V H-X-V L , wherein V L is the variable region of an antibody light chain, V H is the variable region of an antibody heavy chain, and X is a linker or the absence of residues that include insufficient residues to permit association of V H and V L (or formation of Fv) in a single polypeptide chain, and wherein the VH of one polypeptide chain binds to the V L of another polypeptide chain to form an antigen-binding domain, i.e., to form an Fv molecule capable of specifically binding one or more antigens. In each polypeptide chain, V L and V H can be the same, or V L and V H in each polypeptide chain can be different to form a bispecific diabody (i.e., containing two Fvs with different specificities).

[0284] Single-chain Fc (scFv)

[0285] One skilled in the art will appreciate that scFv contains V H and V L regions in a single polypeptide chain, and contains a polypeptide linker between V H and V L that enables the scFv to form the desired structure for antigen binding (i.e., to associate the V H and V L of a single polypeptide chain with each other to form an Fv). For example, the linker contains more than 12 amino acid residues, and (Gly4Ser)3 is one of the more preferred linkers for scFv.

[0286] The present invention also contemplates disulfide-stabilized Fv (or diFv or dsFv), wherein a single cysteine residue is introduced into the FR of V H and the FR of V L , and the cysteine residues are linked by a disulfide bond to produce a stabilized Fv.

[0287] Alternatively, or in addition thereto, the present invention encompasses dimeric scFv, i.e., a protein comprising two scFv molecules linked by non-covalent or covalent bonds, such as by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, the two scFvs are linked by a peptide linker that is long enough to permit both scFvs to form and bind antigen, e.g., as described in US20060263367

[0288] Other antibodies and proteins containing their antigen-binding domains

[0289] The present invention also contemplates other antibodies and proteins containing their antigen-binding domains, such as:

[0290] (i) A "keyhole" bispecific protein as described in US5731168;

[0291] (ii) A heteroconjugate protein, e.g., as described in US4676980;

[0292] (iii) A heteroconjugate protein produced using a chemical crosslinker, e.g., as described in US4676980;

[0293] (iv) Fab3 (e.g., as described in EP19930302894).

[0294] In any of the above antibody structures, the binding domain of the protein can be linked by a linker. For example, in the context of scFv, the linker between VH and VL can be a combination of one or more amino acid residues to provide a flexible linker. Those skilled in the art will be familiar with the appropriate linker sequences to utilize. In a typical example, the linker consists of one or more glycine residues and serine residues. In one example, the linker can contain the sequence G4S (i.e., GGGGS), etc. The linker can also contain repeats of glycine residues and serine residues, such as (G4S)3, but it should be understood that any variation thereon is also suitable, such as (G4S)3T.

[0295] Mutations of the protein

[0296] The present invention also provides an antigen-binding protein having at least 80% identity with the sequences disclosed herein or a nucleic acid encoding the same. In one example, the antigen-binding protein or nucleic acid of the present invention comprises a sequence that is at least about 85% or 90% or 95% or 97% or 98% or 99% identical to the sequences disclosed herein.

[0297] Alternatively or additionally, the antigen-binding protein comprises CDRs that are at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the CDRs of V H or V L as described herein according to any example (e.g., three CDRs).

[0298] In another example, the nucleic acid of the present invention comprises a sequence that is at least about 80% or 85% or 90% or 95% or 97% or 98% or 99% identical to the sequence encoding an antigen-binding protein having the function as described herein according to any example. The present invention also encompasses nucleic acids encoding the antigen-binding proteins of the present invention, which are different from the sequences exemplified herein due to the degeneracy of the genetic code.

[0299] 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 = 5 and a gap extension penalty = 0.3. The length of the query sequence is at least 50 residues, and GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the length of the query sequence is at least 100 residues 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 length.

[0300] The present invention also contemplates nucleic acids that hybridize to a nucleic acid encoding an antigen-binding site described herein under stringent hybridization conditions. "Moderate stringency" is defined herein as hybridization and / or washing carried out at a temperature in the range of 45°C to 65°C in 2×SSC buffer, 0.1% (w / v) SDS or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing carried out at a temperature of at least 65°C in 0.1×SSC buffer, 0.1% (w / v) SDS or lower salt concentration or equivalent conditions. The specific stringency levels mentioned herein encompass equivalent conditions using wash / hybridization solutions other than SSC known to those skilled in the art. For example, methods for calculating the temperature at which a double-stranded nucleic acid strand dissociates (also known as the melting temperature or Tm) are known in the art. Temperatures similar (e.g., within 5°C or within 10°C) or equal to the Tm of a nucleic acid are considered 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.

[0301] The present invention also encompasses mutant forms of the antigen-binding proteins of the present invention that contain one or more conservative amino acid substitutions compared to the sequences shown herein. In some instances, the antigen-binding protein contains 10 or fewer, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 conservative amino acid substitutions. "Conservative amino acid substitution" is where an amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydrophobicity and / or hydrophilicity.

[0302] Families of amino acid residues with similar side chains have been defined in the art and include 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). Hydrophobicity indices are described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982 and hydrophilicity indices are described, for example, in US4554101.

[0303] The invention also contemplates non-conservative amino acid changes. For example, of particular interest is the substitution of a charged amino acid with another charged amino acid and with a neutral or positively charged amino acid. In some instances, the antigen-binding protein comprises 10 or fewer, such as 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 non-conservative amino acid substitutions.

[0304] In one instance, the mutation occurs within the FR of the antigen-binding domain of the antigen-binding protein of the invention. In another instance, the mutation occurs within the CDR of the antigen-binding protein of the invention.

[0305] Exemplary methods for generating mutant forms of the antigen-binding protein include:

[0306] · 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

[0307] WO1999 / 058661);

[0308] · introducing a nucleic acid encoding a polypeptide into mutant cells such as XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene);

[0309] · DNA shuffling, for example, as disclosed in Stemmer, Nature 370:389-91, 1994; and

[0310] · Site-directed mutagenesis, e.g., as described in Dieffenbach (ed.) and Dveksler (ed.) (see: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1995).

[0311] Exemplary methods for determining the biological activity of the mutant antigen-binding proteins of the invention will be apparent to those skilled in the art and / or as 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.

[0312] Constant region

[0313] The invention encompasses the antigen-binding proteins and / or antibodies described herein that comprise the constant region of an antibody. This includes antigen-binding fragments of antibodies fused to Fc.

[0314] Sequences that can be used to generate the constant region of the proteins of the invention can be obtained from many different sources. In some instances, the constant region of the protein 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).

[0315] In one instance, the Fc region of the constant region has an increased ability to induce effector functions, e.g., compared to the native or wild-type human IgG1 or IgG3 Fc region. In one instance, the effector functions are 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 functions of Fc-containing proteins are known in the art and / or as described herein.

[0316] Additional modifications

[0317] The invention also contemplates additional modifications to antibodies or antigen-binding proteins that comprise an Fc region or constant region.

[0318] For example, the antibody comprises one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody comprises an Fc region that comprises one or more amino acid substitutions that increase the affinity of the Fc region for the neonatal Fc receptor (FcRn). For example, the Fc region has increased affinity for FcRn at a lower pH, such as about pH 6.0, to facilitate Fc / FcRn binding in endosomes. In one instance, the Fc region has increased affinity for FcRn at about pH 6 compared to the affinity of the Fc region at about pH 7.4, which promotes the release of Fc back into the blood after cellular recycling. These amino acid substitutions can be used to extend the half-life of the protein by reducing clearance from the blood.

[0319] Exemplary amino acid substitutions include T250Q and / or M428L according to the EU numbering system or T252A, T254S, and T266F or M252Y, S254T, and T256E or H433K and N434F. Additional or alternative amino acid substitutions are described, for example, in US20070135620 or US7083784.

[0320] Protein production

[0321] In one instance, the antigen-binding protein described herein according to any instance is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or as known in the art.

[0322] Recombinant expression

[0323] In another instance, the antigen-binding protein described herein according to any instance is recombinant.

[0324] In the case of a recombinant protein, the nucleic acid encoding the recombinant protein can be cloned into an expression construct or vector and then transfected into a host cell such as an Escherichia coli (E. coli) cell, a yeast cell, an insect cell or a mammalian cell (such as a simian COS cell, a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a myeloma cell), which do not otherwise produce the protein. Exemplary cells for expressing proteins are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques for achieving these purposes are known in the art and are described, for example, in Ausubel et al., (eds.), 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 variety of cloning and in vitro amplification methods are applicable to the construction of recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US 4,816,567 or US 5,530,101.

[0325] After isolation, the nucleic acid operably linked to a promoter is inserted into an expression construct or expression vector for further cloning (amplification of DNA) or for expression in a cell-free system or in a cell.

[0326] As used herein, the term "promoter" is to be understood in its broadest sense and includes transcriptional regulatory sequences of a genomic gene (including the TATA box or initiator element required for accurate transcription initiation), with or without additional regulatory elements (e.g., upstream activation sequences, transcription factor binding sites, enhancers and silencers) that respond to developmental and / or external stimuli or alter nucleic acid expression in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or a derivative that confers, activates or enhances the expression of a nucleic acid operably linked to the promoter. An exemplary promoter may contain additional copies of one or more specific regulatory elements to further enhance the expression of the nucleic acid and / or to alter the spatial and / or temporal expression of the nucleic acid.

[0327] As used herein, the term "operably linked" means positioning the promoter relative to the nucleic acid such that the expression of the nucleic acid is controlled by the promoter.

[0328] Many vectors for expression in cells can be obtained. Vector components typically include, but are not limited to, one or more of the following: signal sequences, sequences encoding proteins (e.g., derived from the information provided herein), enhancer elements, promoters, and transcription termination sequences. Those skilled in the art will know the appropriate sequences for expressing proteins. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader sequence, α-factor leader sequence, or acid phosphatase leader sequence), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0329] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), the human elongation factor 1-α promoter (EF1), the 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; hybrid regulatory elements comprising the CMV enhancer / β-actin promoter or immunoglobulin promoter or active fragments thereof. Examples of available mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); the human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture); the baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0330] Typical promoters suitable for expression in yeast cells such as yeast cells selected from Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces 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.

[0331] Means for introducing an isolated nucleic acid or an expression construct comprising the same into cells for expression are known to those skilled in the art. The techniques for a given cell depend on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, liposome-mediated transfection such as by using lipofectamine (Gibco, Maryland, USA) and / or cellfectin (Gibco, Maryland, USA), PEG-mediated DNA uptake, electroporation, and particle bombardment such as by using tungsten or gold particles coated with DNA (Agracetus Inc., Wisconsin, USA), and the like.

[0332] Host cells for producing proteins can be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's Fl0 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0333] Isolation of Proteins

[0334] Methods for isolating proteins are known in the art and / or as described herein.

[0335] When the antigen-binding protein is secreted into the medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (such as PMSF) can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants. Alternatively or additionally, the supernatant can be filtered and / or separated from the cells expressing the protein, for example, using continuous centrifugation.

[0336] Antigen-binding proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (such as protein A affinity chromatography or protein G chromatography), or any combination of the foregoing methods. These methods are known in the art and are described, for example, in WO99 / 57134 or Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0337] Those skilled in the art will also be aware that proteins can be modified to include tags that facilitate purification or detection, such as polyhistidine tags (e.g., hexahistidine tags), or influenza virus hemagglutinin (HA) tags, or simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting proteins are then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexahis tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds to the hexahis tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0338] Determination of binding of the antigen-binding protein

[0339] It will be apparent to those skilled in the art that the antigen-binding proteins of the present invention bind to CCR8. Methods for assessing binding to a protein are known in the art, for example, as described in Scopes (Protein purification: principles and practice, 3rd edition, Springer Verlag, 1994). Such methods generally involve immobilizing the antigen-binding protein and contacting it with a labeled antigen (CCR8). After washing to remove non-specifically bound protein, the amount of label is detected and thus the bound antigen is detected. Of course, the antigen-binding protein can be labeled and the antigen immobilized. A panning assay can also be used. Alternatively or additionally, a surface plasmon resonance assay can be used.

[0340] Optionally, the dissociation constant (Kd), association constant (Ka), and / or affinity constant (KD) of the immobilized antigen-binding protein for CCR8 or its epitope are determined. In one example, the "Kd" or "Ka" or "KD" of a CCR8-binding protein is measured by a radiolabeled or fluorescently labeled CCR8 ligand-binding assay. In the case of "Kd", the assay equilibrates the antigen-binding protein with the lowest 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 is determined, which indicates the Kd of the protein.

[0341] According to another example, K d 、K a or K D is measured by using a surface plasmon resonance assay, such as a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) using immobilized dysfunctional P2X7 receptor or a region thereof or immobilized antigen-binding protein.

[0342] Disease to be treated

[0343] The antigen-binding proteins of the present invention have particular utility in the manufacture of medicaments for the treatment of cancer (e.g., antibodies, antibody-drug conjugates).

[0344] Examples of cancers that can be treated according to the methods of the present invention include pre-neoplastic and neoplastic diseases. Extensive examples include breast tumors, colorectal tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, liver cancer / cholangiocarcinoma, malignant epithelial tumors, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthomas, seminomas, non-seminomas, stromal interstitial 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 tumors.

[0345] Examples of specific cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myelogenous leukemia, adenocystic carcinoma, adrenocortical carcinoma, idiopathic myelofibrosis, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, sclerosing hemangioma, angiomatosis, amine precursor uptake and decarboxylation tumor, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, bowel cancer, brainstem glioma, brain and CNS tumors, breast cancer, branchioma, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancers, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, cutaneous T-cell lymphoma, malignant epithelial tumors (such as Walker carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, Krebs 2, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, squamous cell carcinoma, and transitional cell carcinoma), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes of breast, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine system cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extrahepatic bile duct cancer, eye cancer, eye: melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, genitourinary cancer, germ cell tumors, gestational trophoblastic disease, glioma, gynecologic cancer, giant cell tumor, ganglioneuroma, glioma, glomus tumor, granulosa cell tumor, ovary gynandroblastoma, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal cancer, hamartoma, hemangioendothelioma, hemangioma, hemangiopericytoma, angiosarcoma, angiosarcoma, histiocytic disorders, malignant histiocytosis, histiocytoma, hepatoma, hidradenoma, chondrosarcoma, immunoproliferative small, opoma, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, leiomyosarcoma, leukemia (such as b-cell leukemia, mixed cell leukemia, null cell leukemia, t-cell leukemia, t-cell chronic leukemia, htlv-ii related leukemia, lymphangiosarcoma leukemia,Acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and granulocytic leukemia), leukemic sarcoma, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangiosarcoma, lymphangioleiomyoma, lymphangiosarcoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, oral cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disease, malignant carcinoid syndrome carcinoid heart disease, medulloblastoma, meningioma, melanoma, stromal tumor, mesonephroma, mesothelioma, myoblastoma, myoma, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, Wilms tumor, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small cell lung cancer (nsclc), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, tumors (such as bone tumors, breast tumors, digestive system tumors, colorectal tumors, liver tumors), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, fistula ovarian cancer, pancreatic cancer, nasal cancer, parathyroid cancer, parotid cancer, penile cancer, peripheral neuroectodermal tumor, pituitary cancer, polycythemia, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, nonchromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogene, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (sclc), small intestine cancer, soft tissue sarcoma, spinal cord tumor, squamous cell carcinoma (skin), stomach cancer, synovial sarcoma, sarcoma (such as Ewing's experimental sarcoma, 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), trophoblastic carcinoma, teratoma, follicular cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, urothelial cancer, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0346] Other diseases and disorders include various inflammatory disorders. Examples can include proliferative components. Specific examples include acne, angina, arthritis, aspiration pneumonia, disease, empyema, gastroenteritis, inflammation, intestinal flu, nee, necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis, pid, pleurisy, laryngitis, redness, inflammation, sore throat, stomach flu, and urinary tract infection, chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyneuropathy, or chronic inflammatory demyelinating polyneuropathy.

[0347] Composition

[0348] In some instances, an antigen-binding protein as described herein can be administered orally, parenterally, by inhalation spray, adsorbed, absorbed, topically, rectally, nasally, buccally, vaginally, intraventricularly, via an implantable cartridge in a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, and intracranial injection or infusion techniques.

[0349] Methods for preparing an antigen-binding protein in a form suitable for administration to a subject (e.g., a pharmaceutical composition) are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Co., Easton, Pa., 1984).

[0350] The pharmaceutical compositions of the invention are particularly useful for parenteral administration, such as intravenous administration or administration into the lumen of a body cavity, organ, or joint. Compositions for administration will generally comprise a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline, and the like. These compositions can contain pharmaceutically acceptable adjunct substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the antigen-binding protein of the invention in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, etc., according to the particular mode 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 a blend of oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle can contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffering agents and preservatives.

[0351] The antigen-binding proteins of the invention can be formulated for topical or local administration, such as for topical application to the skin or tissue in need of treatment. Formulations for topical administration generally comprise a topical vehicle in combination with the active agent, with or without additional optional components. The pharmaceutical compositions of the invention can be in the form of a spray, cream, gel, emulsion, etc., for topical administration.

[0352] Suitable topical vehicles and additional components are well known in the art, and clearly the choice of vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include: organic solvents such as alcohols (e.g., ethanol, isopropanol or glycerol), diols such as butanediol, isopentanediol or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohols and glycerol, lipid-based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, protein-based materials such as collagen and gelatin, silicone-based materials (non-volatile and volatile), and hydrocarbon-based materials such as microsponges and polymeric matrices.

[0353] The composition may also contain one or more components suitable for improving the stability or effectiveness of the administered formulation, such as stabilizers, suspending agents, emulsifying agents, viscosity modifiers, gelling agents, preservatives, antioxidants, skin penetration enhancers, humectants and sustained release materials. Examples of such components are described in Martindale – The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. The formulation may include microcapsules such as hydroxy methyl cellulose or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

[0354] Topical formulations can be prepared in a variety of physical forms, which include for example solids, pastes, creams, foams, emulsions, gels, powders, aqueous liquids, lotions, sprays and skin patches. The physical appearance and viscosity of these forms can be controlled by the presence and amount of emulsifying agents and viscosity modifiers present in the formulation. Solids are generally hard and non-pourable and are usually formulated as sticks or bars, or in particulate form. Solids can be opaque or transparent and optionally may contain solvents, emulsifying agents, humectants, emollients, fragrances, dyes / colorants, preservatives and other active ingredients that enhance or augment the efficacy of the final product. Creams and emulsions are generally similar to each other, the main difference being their viscosity. Both emulsions and creams can be opaque, translucent or transparent and usually contain emulsifying agents, solvents and viscosity modifiers, as well as humectants, emollients, fragrances, dyes / colorants, preservatives and other active ingredients that enhance or augment the efficacy of the final product.

[0355] Gels can be formulated to have a range from thick or highly viscous to thin or low viscosity. Formulations such as emulsions and creams can also contain solvents, emulsifiers, humectants, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or augment the efficacy of the final product. Liquids are thinner than creams, emulsions, or gels and generally do not contain emulsifiers. Liquid topical products typically contain solvents, emulsifiers, humectants, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that enhance or augment the efficacy of the final product.

[0356] Emulsifiers for topical formulations include, but are not limited to, ionic emulsifiers, cetearyl alcohol, non-ionic emulsifiers such as polyoxyethylene oleyl ether, PEG-40 stearate, cetearyl alcohol ethoxylate-12, cetearyl alcohol ethoxylate-20, cetearyl alcohol ethoxylate-30, cetearyl alcohol ethoxylate alcohol, PEG-100 stearate, and glyceryl stearate. Suitable viscosity modifiers include, but are not limited to, protective colloids or non-ionic gums such as hydroxyethyl cellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin wax, and cetyl palmitate. Gel compositions can be formed by adding gelling agents such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaternium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carbomer, or glycyrrhizic acid ammoniate. Suitable surfactants include, but are not limited to, non-ionic surfactants, amphoteric surfactants, ionic surfactants, and anionic surfactants. For example, one or more of polydimethylsiloxane 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 lauryl ether sulfate can be used in topical formulations.

[0357] Preservatives include, but are not limited to, antimicrobial agents such as methyl paraben, propyl paraben, 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 humectants include, but are not limited to, lactic acid and other hydroxy acids and 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 pigments include, but are not limited to, 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 formers, conditioners, propellants, sunscreens, pH regulators, and protectants.

[0358] Typical delivery modes of topical compositions include application using a finger, using a physical application device (such as a cloth, tissue, swab, rod, or brush), spraying (including mist, aerosol, or foam spray), application with a dropper, sprinkling, soaking, and rinsing. A controlled release vehicle may also be used, and the composition may be formulated for transdermal application (e.g., as a transdermal patch).

[0359] After formulation, the antigen-binding proteins of the present invention will be administered in a manner compatible with the dosage formulation and in a therapeutically / prophylactically effective amount. The formulations are readily administered in a variety of dosage forms, such as the injectable solution types described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules, or other solids for oral administration, suppositories, vaginal suppositories, nasal solutions or sprays, aerosols, inhalants, liposomal forms, etc. Drug "sustained release" capsules or compositions may 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.

[0360] WO2002 / 080967 describes compositions and methods for administering nebulized compositions containing antibodies for treating, e.g., asthma, and these compositions and methods are also applicable for administering the antigen-binding proteins of the present invention.

[0361] Dosage and timing of administration

[0362] The appropriate dosage of the 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. A skilled physician is capable of determining the appropriate dosage, e.g., by starting with a suboptimal dose and gradually varying the dose to determine the optimal or useful dose. Alternatively, to determine the appropriate dosage for treatment / prophylaxis, data from cell culture assays or animal studies are used, where the appropriate dosage is within the range of circulating concentrations that include the ED 50 of the active compound and has little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. The therapeutically / prophylactically effective dosage can be initially estimated from cell culture assays. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations that include the IC 50 (i.e., the concentration or amount of the compound that achieves half-maximal inhibition of the symptoms). Such information can be used to more accurately determine the useful dosage in humans. The levels in plasma can be measured, e.g., by high performance liquid chromatography.

[0363] In some instances, the methods of the present invention include administering a prophylactically or therapeutically effective amount of the proteins described herein.

[0364] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the prognosis and / or condition of the subject and / or reduces or suppresses one or more symptoms of a clinical disorder described herein to a level below that observed and accepted as a clinical diagnosis or clinical feature of the disorder. The amount to be administered to the subject will depend on the specific characteristics of the disorder to be treated, the type and stage of the disorder to be treated, the mode of administration, and the characteristics of the subject such as general health, other diseases, age, gender, genotype, and weight. Those skilled in the art will be able to determine the appropriate dose based on these and other factors. Thus, the term should not be construed as limiting the invention to a specific amount such as the weight or quantity of a protein, but rather the invention encompasses any amount of the antigen-binding protein sufficient to achieve the desired result in the subject.

[0365] As used herein, the term "prophylactically effective amount" should be understood to mean an amount of a protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical disorder. Those skilled in the art will know that such amount will vary depending on, for example, the specific antigen-binding protein administered and / or the type or severity or level of the specific subject and / or disorder and / or susceptibility (genetic or otherwise) to the disorder. Thus, the term should not be construed as limiting the invention to a specific amount such as the weight or quantity of an antigen-binding protein, but rather the invention encompasses any amount of the antigen-binding protein sufficient to achieve the desired result in the subject.

[0366] Kit

[0367] The invention also includes a kit comprising one or more of the following:

[0368] (i) The antigen-binding protein of the invention or a nucleic acid or expression construct encoding the antigen-binding protein;

[0369] (ii) The cells of the invention; or

[0370] (iii) The pharmaceutical composition of the invention.

[0371] In the case of a kit for detecting CCR8, the kit may further comprise, for example, a detection means linked to the antigen-binding protein of the invention.

[0372] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharmaceutically acceptable carrier.

[0373] Optionally, the kit of the invention is packaged with instructions for use in the methods described herein according to any example.

[0374] Example

[0375] Example 1 - Generation of anti-human CCR8 antibodies by immunizing mice with human CCR8.

[0376] C57BL / 6 mice were immunized intraperitoneally (i.p.) five to six times at two-week intervals with 10 7 L1.2 cells transfected with human CCR8 to generate mAbs reactive with human CCR8. The final immunization was by intravenous injection (i.v.). Four days later, spleens were removed and the cells were fused with the SP2 / 0 cell line.

[0377] The inventors managed to isolate six clones specific for hCCR8 from six different fusions. None of the clones showed cross-reactivity with murine or cynomolgus monkey CCR8.

[0378] Example 2 - Generation of anti-human CCR8 antibodies by immunizing mice with murine CCR8.

[0379] C57BL / 6 mice were immunized intraperitoneally (i.p.) five to six times at two-week intervals with 10 7 L1.2 cells transfected with murine CCR8 to generate mAbs reactive with murine CCR8. The final immunization was by intravenous injection (i.v.). Four days later, spleens were removed and the cells were fused with the SP2 / 0 cell line.

[0380] Example 3 - Isolation of anti-CCR8 2H8 antibody and cross-reactivity with human, murine, and cynomolgus monkey CCR8

[0381] After several immunizations, the inventors identified that the sera of immunized mice contained antibodies capable of binding both murine CCR8 and human CCR8 ( Figure 1 ). The inventors fused one of the mice and screened the hybridomas by flow cytometry.

[0382] The inventors isolated an alternative antibody specific for murine CCR8, but unexpectedly also found that one clone (2H8; isotype IgG2b) showed reactivity with both human and murine receptors ( Figure 1 ). In contrast, the antibodies obtained from Example 1 above showed reactivity only with human CCR8 ( Figure 1 A; anti-hCCR8#1 provided as a representative example). In addition, the inventors compared an existing anti-hCCR8 antibody from Biolegend (clone #L263G8), which similarly showed reactivity only with human CCR8 ( Figure 1 B).

[0383] Next, the inventors tested the reactivity of the 2H8 antibody with cynomolgus monkey (cyno) CCR8. Surprisingly, 2H8 also showed high reactivity with cynomolgus monkey CCR8 compared to the isotype control ( Figure 2), while the prior art antibodies and some antibodies generated using the method of Example 1 did not exhibit high reactivity against cynomolgus monkey CCR8.

[0384] Example 4 - 2H8 does not cross-react with other CCR chemokine receptors

[0385] To confirm the specificity of 2H8 for CCR8, the inventors tested the reactivity of 2H8 against other human CCR chemokine receptors. No cross-reactivity with the tested CCR chemokine receptors was found, except for human and mouse CCR8 ( Figure 3 ).

[0386] Example 5 - 2H8 binds to human tumor-infiltrating Treg (TiTreg)

[0387] To determine whether 2H8 could bind to TiTreg, the inventors isolated single-cell suspensions from fresh colorectal tumor patient biopsies and stained the cells with anti-human CD3, anti-human CD4, anti-human FoxP3 (TiTreg), and 2H8 or isotype control.

[0388] It was found that 2H8 bound to tumor-infiltrating Treg (CD3+; CD4+; FoxP3+ gate), while the isotype control did not bind to it ( Figure 4 ).

[0389] Example 6 - Chimeric 2H8 shows similar binding affinity compared to existing anti-hCCR8 antibodies

[0390] The inventors prepared a chimeric form of the 2H8 antibody, which contains the heavy-chain constant region and light-chain constant region of human IgG1 as shown in SEQ ID NO: 58 and 59.

[0391] The inventors 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 ).

[0392] Example 7 - Humanized 2H8 anti-CCR8 antibody

[0393] The inventors then prepared a humanized 2H8 antibody. Antibody humanization was performed by the CDR grafting method.

[0394] Briefly, human germline genes were identified using the IMGT / V-QUEST and IMGT / Junctions analysis tools, where the sequences from the variable regions of both the heavy and light chains were closely aligned with the sequence of the murine antibody 2H8. 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 key "cursor" regions. The humanized VH and VL genes were synthesized by Genscript.

[0395] The binding activities of the humanized 2H8 antibody were compared with those of the chimeric 2H8 and Shionogi's 19D7 anti-CCR8 antibody using flow cytometry. The results are as Figure 6 shown in Table 2 below.

[0396] Table 2. EC50 values of anti-CCR8 antibodies

[0397]

[0398] Example 8 - Binding of 2H8 to human tumor-infiltrating Tregs (TiTregs)

[0399] To determine whether 2H8 could bind to TiTregs, the inventors isolated single-cell suspensions from fresh colorectal tumor patient biopsies and stained the cells with anti-human CD3, anti-human CD4, anti-human FoxP3 (TiTregs), and 2H8 or an isotype control.

[0400] It was found that 2H8 bound to tumor-infiltrating Tregs (CD3+; CD4+; FoxP3+ gate), while the isotype control did not bind to them ( Figure 7 ).

[0401] Example 9 - Chimeric 2H8 does not inhibit CCL1-induced chemotaxis.

[0402] Next, the inventors performed chemotaxis assays to determine whether chimeric 2H8 would block CCL1-induced chemotaxis. It is known that CCL1 binds to the N-terminus of CCR8 to induce chemotaxis.

[0403] 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-1 penicillin. For this assay, the cells were washed once in PBS and resuspended at 106 cells ml-1 in assay buffer (RPMI 1640, 1% endotoxin-free BSA, 100 U mL-1 penicillin, and 100 μg mL-1 streptomycin). Antibodies were pre-incubated with 100 μL of cells (1 × 105 cells) for 30 minutes and placed in the upper chamber of a Transwell plate (Corning Life Sciences) with 5-μm pores. In the lower chamber, 5.8 nM human CCL1 was placed (30 minutes, 37 °C), and the assay system was incubated at 37 °C for 4 hours (5% CO2). Live cells that migrated to the lower chamber were counted using an LSRII flow cytometer.

[0404] Chimeric 2H8 did not inhibit CCL1-induced chemotaxis, while chimeric 19D7 had a significant and dose-dependent inhibitory effect on CCL1-induced chemotaxis ( Figure 8 ). Advantageously, the inventors believe that in an environment with a high CCL1 concentration such as the tumor microenvironment, the binding of chimeric 2H8 to CCR8 would not be inhibited by competition with CCL1 because they do not share the same binding site, unlike chimeric 19D7.

[0405] Example 10 - Epitope mapping of 2H8

[0406] To further study the epitope binding of 2H8, the inventors performed epitope mapping. Specifically, the 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, where the amino acid sequences from CCR5 are underlined. The results of the epitope mapping are shown in Table 3 below.

[0407] Table 3. Epitope mapping of 2H8 on chimeric transfected cells and hCCR8 2ECL mutants

[0408]

[0409] Interestingly, mutation of the N-terminus of human CCR8 (5888 in Table 3) blocked the binding of 19D7 to CCR8, but 2H8 was still able to bind to CCR8. However, mutation of extracellular loop 2 (8858 in Table 3) blocked the binding of 2H8 to CCR8, but 19D7 was still able to bind to CCR8.

[0410] These results indicate that 2H8 and 19D7 bind to different epitopes on CCR8.

[0411] Example 11 - Chimeric 2H8 exhibits ADCC potential with efficacy and potency comparable to 19D7

[0412] To further investigate the functional properties of chimeric 2H8, the inventors performed dose - dependent antibody - dependent cell - mediated cytotoxicity (ADCC) assays.

[0413] The ability of the antibodies to induce ADCC was evaluated by flow cytometry. Briefly, cells expressing hCCR8L1.2 were labeled with the membrane dye PKH26 for discrimination during incubation with effector cells and antibodies. The labeled target cells were washed three times with medium and resuspended in medium at a concentration of 1×10 6 / mL. The labeled target cells were aliquoted in round - bottom 96 - well plates (1×10 5 at 100 μL / well) and pre - incubated with antibodies at 5 μg / mL, 1 μg / mL, 0.5 μg / mL, or 0.1 μg / mL for 30 minutes at 37°C. PBMCs were prepared from heparinized blood (obtained from healthy individuals) by centrifugation on a Ficoll gradient. Subsequently, human natural killer cells were isolated from PBMCs using MACS CD56 microbeads and a MACS positive selection column (Miltenyi Biotec) according to the manufacturer's protocol. Then, 2×10 5 purified human NK cells (effector cells) were incubated with the target cell / antibody mixture (E:T ratio = 2:1) at 37°C in the presence of 10% heat - inactivated serum for 3 hours. The treated cells were washed three times and resuspended in 200 μL of PBS. TO - PRO 3 iodide was added to detect cell death before analysis on an LSRII flow cytometer. As a counting standard, 20 μL / well of CountBright absolute counting beads (Invitrogen) was added to determine the cell concentration of the cell subsets. Samples were acquired on an LSRII flow cytometer.

[0414] Chimeric 2H8 and Shionogi's chimeric 19D7 anti - CCR8 antibody induced similar levels of cytotoxicity, as Figure 9 shown in Table 4 below.

[0415] Table 4. Cytotoxicity induced by 2H8 in the ADCC assay

[0416]

[0417] Example 12 - In vivo activity of 2H8 in a murine model of colorectal cancer

[0418] To test the in vivo activity of 2H8, the inventors subcutaneously implanted 5×10 5 MC38 colorectal cells into C57BL / 6 mice. 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 (mm3) was measured daily with digital calipers and compared between groups. Treatments were performed twice weekly at a dose of 5 mg / kg for up to 3 weeks.

[0419] Treatment with 2H8 substantially inhibited tumor growth compared to both the isotype control and the anti-mPD1 treatment group ( Figure 10 ).

[0420] At the endpoint, tumor tissue and spleen tissue were processed, stained, and analyzed by flow cytometry. Cells were collected, and the percentage of CD4 + FOXP3 + CD25 + cells in TCR-β + cells (parent population) was compared between the three groups. Treatment with 2H8 caused Treg depletion in tumors but not in spleens ( Figure 11 ). Additionally, Treg depletion was significantly higher in the 2H8 treatment group compared to anti-PD1 or isotype control.

[0421] The inventors next analyzed the CD8+ T cell population and other CD4+ T cell populations in tumor samples. The inventors 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 ).

[0422] It should be understood that the invention disclosed and defined in this specification includes all optional combinations of two or more separate features mentioned in the text or drawings or that are obvious from the text or drawings. All such different combinations constitute various optional aspects of the invention.

Claims

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

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

55.

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

55.

4. The antigen-binding protein according to 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 that comprises 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. An antigen-binding protein that binds or specifically binds to CCR8, and wherein the antigen-binding protein competitively inhibits the binding of an antibody that comprises VH and VL, wherein the VH comprises the sequence as shown in SEQ ID NO: 1 and the VL comprises the sequence as shown in SEQ ID NO: 2; or the VH comprises the sequence as shown in SEQ ID NO: 83 and the VL comprises the sequence as shown in SEQ ID NO:

84.

7. The antigen-binding protein according to any one of claims 1 to 6, wherein the antigen-binding protein comprises an antigen-binding domain of an antibody, wherein the antigen-binding domain binds or specifically binds to CCR8, and wherein the antigen-binding domain comprises at least one of the following: (i) VH, which comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises 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 to the sequence shown in SEQ ID NO: 5, CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 6, and CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 7; (ii) VH, wherein the VH comprises 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 to the sequence shown in SEQ ID NO: 1 or 83; (iii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises 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 to the sequence shown in SEQ ID NO: 8, the CDR2 comprises 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 to the sequence shown in SEQ ID NO: 9, and the CDR3 comprises 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 to the sequence shown in SEQ ID NO: 10; (iv) VL, wherein the VL comprises 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 to the sequence shown in SEQ ID NO: 2 or 84; (v) VH, wherein the VH comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 5, the CDR2 comprises the sequence shown in SEQ ID NO: 6, and the CDR3 comprises the sequence shown in SEQ ID NO: 7; (vi) VH, wherein the VH comprises the sequence shown in SEQ ID NO: 1 or 83; (vii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 8, the CDR2 comprises the sequence shown in SEQ ID NO: 9, and the CDR3 comprises the sequence shown in SEQ ID NO: 10; (viii) VL, wherein the VL comprises the sequence shown in SEQ ID NO: 2 or 84; (ix) A VH, wherein the VH comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO:5, the CDR2 comprises the sequence shown in SEQ ID NO:6, and the CDR3 comprises the sequence shown in SEQ ID NO:7; and a VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO:8, the CDR2 comprises the sequence shown in SEQ ID NO:9, and the CDR3 comprises the sequence shown in SEQ ID NO:10; (x) A VH, wherein the VH comprises the sequence shown in SEQ ID NO:1; and a VL, wherein the VL comprises the sequence shown in SEQ ID NO:2, (xi) A VH, wherein the VH comprises the sequence shown in SEQ ID NO:83; and a VL, wherein the VL comprises the sequence shown in SEQ ID NO:

84.

8. The antigen-binding domain according to claim 7, wherein the antigen-binding domain further comprises at least one of the following: (i) A VH, wherein the VH comprises framework regions (FR) 1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO:11, the FR2 comprises 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 to the sequence shown in SEQ ID NO:12, the FR3 comprises 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 to the sequence shown in SEQ ID NO:13, and the FR4 comprises 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 to the sequence shown in SEQ ID NO:14; (ii) VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO:15, the FR2 comprises 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 to the sequence shown in SEQ ID NO:16, the FR3 comprises 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 to the sequence shown in SEQ ID NO:17, and the FR4 comprises 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 to the sequence shown in SEQ ID NO:18; (iii) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:11, the FR2 comprises the sequence shown in SEQ ID NO:12, the FR3 comprises the sequence shown in SEQ ID NO:13, and the FR4 comprises the sequence shown in SEQ ID NO:14; (iv) VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:15, the FR2 comprises the sequence shown in SEQ ID NO:16, the FR3 comprises the sequence shown in SEQ ID NO:17, and the FR4 comprises the sequence shown in SEQ ID NO:18; or (v) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:11, the FR2 comprises the sequence shown in SEQ ID NO:12, the FR3 comprises the sequence shown in SEQ ID NO:13, and the FR4 comprises the sequence shown in SEQ ID NO:14; and VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:15, the FR2 comprises the sequence shown in SEQ ID NO:16, the FR3 comprises the sequence shown in SEQ ID NO:17, and the FR4 comprises the sequence shown in SEQ ID NO:

18.

9. The antigen-binding domain according to claim 7, wherein the antigen-binding domain further comprises at least one of the following: (i) A VH, wherein the VH comprises framework regions (FR) 1, FR2, FR3, and FR4, and FR1 comprises 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 to the sequence shown in SEQ ID NO: 60, FR2 comprises 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 to the sequence shown in SEQ ID NO: 61, FR3 comprises 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 to the sequence shown in SEQ ID NO: 62, and FR4 comprises 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 to the sequence shown in SEQ ID NO: 63; (ii) A VL, wherein the VL comprises FR1, FR2, FR3, and FR4, and FR1 comprises 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 to the sequence shown in SEQ ID NO: 64, FR2 comprises 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 to the sequence shown in SEQ ID NO: 65, FR3 comprises 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 to the sequence shown in SEQ ID NO: 66, and FR4 comprises 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 to the sequence shown in SEQ ID NO: 67; (iii) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 60, the FR2 comprises the sequence shown in SEQ ID NO: 61, the FR3 comprises the sequence shown in SEQ ID NO: 62, and the FR4 comprises the sequence shown in SEQ ID NO: 63; (iv) VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 64, the FR2 comprises the sequence shown in SEQ ID NO: 65, the FR3 comprises the sequence shown in SEQ ID NO: 66, and the FR4 comprises the sequence shown in SEQ ID NO: 67; or (v) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 60, the FR2 comprises the sequence shown in SEQ ID NO: 61, the FR3 comprises the sequence shown in SEQ ID NO: 62, and the FR4 comprises the sequence shown in SEQ ID NO: 63; and VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 64, the FR2 comprises the sequence shown in SEQ ID NO: 65, the FR3 comprises the sequence shown in SEQ ID NO: 66, and the FR4 comprises the sequence shown in SEQ ID NO:

67.

10. The antigen-binding protein according to claim 5 or 6, wherein the antigen-binding protein comprises the antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to CCR8, and wherein the antigen-binding domain comprises at least one of the following: (i) VH, wherein the VH comprises complementarity-determining regions (CDRs) 1, 2 and 3, the CDR1 comprises 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 to the sequence shown in SEQ ID NO: 19, the CDR2 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 20, and the CDR3 comprises a sequence that is at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99% identical to the sequence shown in SEQ ID NO: 21; (ii) VH, wherein the VH comprises 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 to the sequence shown in SEQ ID NO: 1 or 83; (iii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, and the CDR1 comprises 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 to the sequence shown in SEQ ID NO: 22, the CDR2 comprises 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 to the sequence shown in SEQ ID NO: 23, and the CDR3 comprises 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 to the sequence shown in SEQ ID NO: 10; (iv) VL, wherein the VL comprises 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 to the sequence shown in SEQ ID NO: 2 or 84; (v) VH, wherein the VH comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 19, the CDR2 comprises the sequence shown in SEQ ID NO: 20, and the CDR3 comprises the sequence shown in SEQ ID NO: 21; (vi) VH, wherein the VH comprises the sequence shown in SEQ ID NO: 1 or 83; (vii) VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO: 22, the CDR2 comprises the sequence shown in SEQ ID NO: 23, and the CDR3 comprises the sequence shown in SEQ ID NO: 10; (viii) VL, wherein the VL comprises the sequence shown in SEQ ID NO: 2 or 84; (ix)VH, wherein the VH comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO:19, the CDR2 comprises the sequence shown in SEQ ID NO:20, and the CDR3 comprises the sequence shown in SEQ ID NO:21; and VL, wherein the VL comprises CDR1, CDR2 and CDR3, the CDR1 comprises the sequence shown in SEQ ID NO:22, the CDR2 comprises the sequence shown in SEQ ID NO:23, and the CDR3 comprises the sequence shown in SEQ ID NO:10; (x)VH, wherein the VH comprises the sequence shown in SEQ ID NO:1; and VL, wherein the VL comprises the sequence shown in SEQ ID NO:2; or (xi)VH, wherein the VH comprises the sequence shown in SEQ ID NO:83; and VL, wherein the VL comprises the sequence shown in SEQ ID NO:

84.

11. The antigen-binding domain according to claim 10, wherein the antigen-binding domain further comprises at least one of the following: (i)VH, wherein the VH comprises framework regions (FR) 1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO:24, the FR2 comprises 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 to the sequence shown in SEQ ID NO:25, the FR3 comprises 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 to the sequence shown in SEQ ID NO:26, and the FR4 comprises 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 to the sequence shown in SEQ ID NO:14; (ii) VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises 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 to the sequence shown in SEQ ID NO: 27, the FR2 comprises 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 to the sequence shown in SEQ ID NO: 28, the FR3 comprises 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 to the sequence shown in SEQ ID NO: 29, and the FR4 comprises 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 to the sequence shown in SEQ ID NO: 18; (iii) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 24, the FR2 comprises the sequence shown in SEQ ID NO: 25, the FR3 comprises the sequence shown in SEQ ID NO: 26, and the FR4 comprises the sequence shown in SEQ ID NO: 14; (iv) VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 27, the FR2 comprises the sequence shown in SEQ ID NO: 28, the FR3 comprises the sequence shown in SEQ ID NO: 29, and the FR4 comprises the sequence shown in SEQ ID NO: 18; or (v) VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 24, the FR2 comprises the sequence shown in SEQ ID NO: 25, the FR3 comprises the sequence shown in SEQ ID NO: 26, and the FR4 comprises the sequence shown in SEQ ID NO: 14; and VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO: 27, the FR2 comprises the sequence shown in SEQ ID NO: 28, the FR3 comprises the sequence shown in SEQ ID NO: 29, and the FR4 comprises the sequence shown in SEQ ID NO:

18.

12. The antigen-binding domain according to claim 10, wherein the antigen-binding domain further comprises at least one of the following items: (i) A VH, wherein the VH comprises framework regions (FR) 1, FR2, FR3, and FR4, and the FR1 comprises 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 to the sequence shown in SEQ ID NO: 68, the FR2 comprises 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 to the sequence shown in SEQ ID NO: 69, the FR3 comprises 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 to the sequence shown in SEQ ID NO: 70, and the FR4 comprises 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 to the sequence shown in SEQ ID NO: 63; (ii) A VL, wherein the VL comprises FR1, FR2, FR3, and FR4, and the FR1 comprises 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 to the sequence shown in SEQ ID NO: 71, the FR2 comprises 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 to the sequence shown in SEQ ID NO: 72, the FR3 comprises 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 to the sequence shown in SEQ ID NO: 73, and the FR4 comprises 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 to the sequence shown in SEQ ID NO: 67; (iii)VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:68, the FR2 comprises the sequence shown in SEQ ID NO:69, the FR3 comprises the sequence shown in SEQ ID NO:70, and the FR4 comprises the sequence shown in SEQ ID NO:63; (iv)VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:71, the FR2 comprises the sequence shown in SEQ ID NO:72, the FR3 comprises the sequence shown in SEQ ID NO:73, and the FR4 comprises the sequence shown in SEQ ID NO:67; or (v)VH, wherein the VH comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:68, the FR2 comprises the sequence shown in SEQ ID NO:69, the FR3 comprises the sequence shown in SEQ ID NO:70, and the FR4 comprises the sequence shown in SEQ ID NO:63; and VL, wherein the VL comprises FR1, FR2, FR3 and FR4, the FR1 comprises the sequence shown in SEQ ID NO:71, the FR2 comprises the sequence shown in SEQ ID NO:72, the FR3 comprises the sequence shown in SEQ ID NO:73, and the FR4 comprises the sequence shown in SEQ ID NO:

67.

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

14. The antigen-binding protein according to any one of claims 1 to 12, wherein the antigen-binding protein is in the following form: (i) diabody; (ii) triabody; (iii) tetrabody; (iv) Fab; (v) F(ab')2; (vi) Fv; (vii) bispecific antibody or other form of multispecific antibody; or (viii) one of (i) to (vii), which is linked to an antibody constant region, Fc or heavy chain constant domain (CH)2 and / or CH3.

15. An anti-CCR8 antibody or an antigen-binding fragment thereof, the anti-CCR8 antibody or an antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises: - CDR H1 as shown in SEQ ID NO:5, CDR H2 as shown in SEQ ID NO:6 and CDR H3 as shown in SEQ ID NO:7; and wherein the light chain variable region comprises: -CDR L1 as shown in SEQ ID NO:8, CDR L2 as shown in SEQ ID NO:9, and CDR L3 as shown in SEQ ID NO:

10.

16. The anti-CCR8 antibody or antigen-binding fragment thereof according to claim 15, wherein the anti-CCR8 antibody or antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises: FR L1 as shown in SEQ ID NO:15, FR L2 as shown in SEQ ID NO:16, FR L3 as shown in SEQ ID NO:17, and FR L4 as shown in SEQ ID NO:18; or FR L1 as shown in SEQ ID NO:64, FR L2 as shown in SEQ ID NO:65, FR L3 as shown in SEQ ID NO:66, and FR L4 as shown in SEQ ID NO:

67.

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

63.

18. An anti-CCR8 antibody or antigen-binding fragment thereof, the anti-CCR8 antibody or antigen-binding fragment thereof comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises: -CDR H1 as shown in SEQ ID NO:19, CDR H2 as shown in SEQ ID NO:20, and CDR H3 as shown in SEQ ID NO:21; and wherein the light chain variable region comprises: -CDR L1 as shown in SEQ ID NO:22, CDR L2 as shown in SEQ ID NO:23, and CDR L3 as shown in SEQ ID NO:

10.

19. The anti-CCR8 antibody or antigen-binding fragment thereof according to claim 18, wherein the anti-CCR8 antibody or antigen-binding fragment thereof comprises a light chain variable region, and the light chain variable region comprises: FR L1 as shown in SEQ ID NO:27, FR L2 as shown in SEQ ID NO:28, FR L3 as shown in SEQ ID NO:29, and FR L4 as shown in SEQ ID NO:18; or FR L1 as shown in SEQ ID NO:71, FR L2 as shown in SEQ ID NO:72, FR L3 as shown in SEQ ID NO:73, and FR L4 as shown in SEQ ID NO:

67.

20. The anti-CCR8 antibody or antigen-binding fragment thereof according to claim 18 or 19, wherein the anti-CCR8 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and the heavy chain variable region comprises: FR H1 as shown in SEQ ID NO:24, FR H2 as shown in SEQ ID NO:25, FR H3 as shown in SEQ ID NO:26, and FR H4 as shown in SEQ ID NO:14; or FR H1 as shown in SEQ ID NO:68, FR H2 as shown in SEQ ID NO:69, FR H3 as shown in SEQ ID NO:70, and FR H4 as shown in SEQ ID NO:

63.

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, and the light chain variable region comprises 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, and the heavy chain variable region comprises 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, and the light chain variable region comprises 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, and the heavy chain variable region comprises the sequence of SEQ ID NO:

83.

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

26. The antigen-binding protein, anti-CCR8 antibody or antigen-binding fragment thereof according to claim 25, wherein the amino acid insertions, deletions, substitutions or combinations thereof are not in the CDRs.

27. The antigen-binding protein according to any one of claims 1 to 26, wherein the antigen-binding protein comprises a human constant region.

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

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

30. A vector comprising the nucleic acid according to claim 29.

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

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

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

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

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

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

37. The method according to claim 35 or 36, wherein the disease or disorder is cancer.

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

39. An antigen-binding protein according to any one of claims 1 to 14 or 25 to 28, an antibody or an 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 for treating or preventing cancer in a subject.

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