Anti-CCR8 Antibodies and Uses Thereof

Abstract

Anti-CCR8 antibodies are provided, including mouse antibodies, humanized antibodies, and those with further optimized CDR sequences. The antibodies have shown high antagonist and ADCC activity, and can be used to treat cancer and inflammatory conditions. According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided that has specificity for human CC motif chemokine receptor 8 (CCR8) protein, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

Description

[Background technology]

[0001] background CC motif chemokine receptor 8 (CCR8) is a seven-transmembrane G protein-coupled receptor (GPCR). CCR8 is preferentially expressed in the thymus. CCR8 and its ligands play an important role in controlling monocyte chemotaxis and thymocyte apoptosis. More specifically, CCR8 may contribute to the proper positioning of activated T cells within the site of antigen challenge and within specific areas of lymphoid tissues.

[0002] CCR8 is selectively expressed on type 2 helper T (Th2) cells, but not on type 1 helper T (Th1) cells. Th2 cells play an important role in the allergic inflammatory response that occurs at the site of allergen exposure. The ligand for CCR8 is the CC chemokine CCL1, which is a chemoattractant for Th2 cells. Thus, the CCR8 / CCL1 receptor / ligand pair may play a role in the development of allergic inflammatory conditions such as asthma, atopic dermatitis and allergic rhinitis. This role includes the recruitment of Th2 cells to sites of allergic inflammation, the production of Th2 cytokines at those sites, and the subsequent migration of eosinophils and basophils.

[0003] CCR8 knockout mice showed impaired Th2 immune responses in allergic inflammation models. In ovalbumin-induced and cockroach antigen-induced allergic lung inflammation, the levels of Th2 cytokines (IL-4, IL-5, and IL-13) were significantly reduced in the lungs of CCR8 knockout mice, and the number of recruited eosinophils was significantly reduced. Therefore, inhibition of CCR8 may be useful in improving the symptoms of allergic inflammatory conditions such as asthma, atopic dermatitis, and allergic rhinitis.

[0004] CCR8 has also been identified as a marker for tumor-infiltrating Tregs. Because CCR8 expression on these Tregs is selectively upregulated in multiple cancers, including breast, colorectal and lung cancers. These CCR8+Tregs represent a highly activated and suppressive subpopulation of Tregs, and high abundance of CCR8+Tregs in these tumor types is associated with poor prognosis. Therefore, CCR8 is a promising therapeutic target for depleting tumor-resident Tregs to enhance antitumor immunity.

[0005] However, chemokine receptors have traditionally been very difficult antigens to raise antibodies against: they are poorly expressed on the cell surface, making them very difficult to access for antibody binding, and antibodies generated against peptides corresponding to the extracellular domains of chemokine receptors often fail to recognize the intact receptors on cells, presumably due to differences in secondary structure.

[0006] CCR8 is an extremely difficult GPCR to raise antibodies against, and there has been limited success in the past in generating species cross-reactive antibodies (cynomolgus monkey and human). Furthermore, finding functional antibodies against GPCRs, including CCR8, is even more difficult. Summary of the Invention [Means for solving the problem]

[0007] overview The present disclosure provides, in various embodiments, antibodies and antigen-binding fragments specific to human CCR8 protein. Experimental tests have shown that these newly identified antibodies can strongly and specifically bind to human CCR8 protein, and most can cross-react with cynomolgus monkey CCR8 protein, which is different from most of the benchmark antibodies, making it feasible to demonstrate preclinical safety in non-human primates. Furthermore, in vitro studies have demonstrated that most of these antibodies can specifically block CCR8 signaling induced by its ligand CCL1. Furthermore, when used with constant regions with intact or enhanced Fc function, these antibodies can induce antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells expressing CCR8. In vivo experiments have shown that these antibodies effectively inhibited tumor growth, especially when used with constant regions with enhanced Fc function.

[0008] According to one embodiment of the present disclosure, there is provided an antibody or antigen-binding fragment thereof having specificity for human CC motif chemokine receptor 8 (CCR8) protein, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0009] In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 61; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 62; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 63 or 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 64; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0010] In another embodiment, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 58; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 59; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50 or 51; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 60.

[0011] In another embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, 65 or 66; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 34; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 43; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 48; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 53.

[0012] In some embodiments, the antibody or fragment is humanized. In some embodiments, the antibody or fragment comprises an Fc fragment with enhanced antibody-dependent cellular cytotoxicity (ADCC). One example of an Fc fragment is a human IgG1 fragment with one or more substitutions selected from the group consisting of L234Y, L235Q, G236W, S239D / M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L / M, I332E, K334A / E, P396L according to EU numbering.

[0013] Also provided are multispecific antibodies comprising an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments that have binding specificity for a target antigen that is not CCR8.

[0014] In another embodiment, a chimeric antigen receptor (CAR) is also provided that comprises an antigen-binding fragment of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

[0015] Polynucleotides encoding the antibodies or antigen-binding fragments thereof or CARs of the present disclosure are also provided. In some embodiments, the polynucleotide is an mRNA, optionally chemically modified.

[0016] Methods and uses for treating cancer and inflammatory conditions using the antibodies or antigen-binding fragments thereof of the present disclosure are also provided. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the binding affinity of the identified antibodies to human CCR8, cyno CCR8 and human CCR4.

[0018] [Figure 2-1] FIG. 2 shows the blocking activity of the chimeric antibodies tested. [Figure 2-2] Same as above.

[0019] [Diagram 3] FIG. 3 shows the ADCC signaling activity of the chimeric antibodies tested.

[0020] [Figure 4] FIG. 4 shows the natural killer cell-mediated ADCC killing effect induced by the antibodies tested.

[0021] [Figure 5-1] FIG. 5 shows the in vivo tumor inhibitory activity of the tested antibodies. [Figure 5-2] Same as above.

[0022] [Figure 6-1] FIG. 6 shows binding of humanized antibodies to human CCR8, cyno CCR8. [Figure 6-2] Same as above.

[0023] [Figure 7] FIG. 7 shows the ADCC activity of the humanized CCR8 antibodies tested.

[0024] [Figure 8] FIG. 8 shows the blocking activity of the humanized CCR8 antibodies tested.

[0025] [Figure 9] FIG. 9 shows the binding and functional properties of anti-CCR8 chimeric antibodies after removal of potential PTM sites.

[0026] [Figure 10] FIG. 10 shows the binding and functional properties of humanized CCR8 antibodies after removal of potential PTM sites.

[0027] [Figure 11] FIG. 11 shows the in vivo tumor inhibitory activity of humanized CCR8 antibodies after removal of potential PTM sites.

[0028] [Figure 12] FIG. 12 shows an in vitro comparison of Hu200C9B9-7 NG-NA antibody with benchmark antibodies.

[0029] [Figure 13-1] FIG. 13 shows an in vivo and ex vivo comparison of Hu200C9B9-7 NG-NA antibody with benchmark antibodies. [Figure 13-2] Same as above.

[0030] [Figure 14] FIG. 14 shows the ADCC activity of different Fc-engineered Hu200C9B9-7 NG-NA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0032] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0033] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegeleisen, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0034] The term antibody encompasses a wide variety of classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE, respectively.

[0035] Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and are therefore within the scope of this disclosure. All immunoglobulin classes are expressly within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides of approximately 23,000 daltons molecular weight and two identical heavy chain polypeptides of 53,000-70,000 daltons molecular weight. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains on either side, beginning at the mouth of the "Y" and continuing through the variable region.

[0036] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure 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 of immunoglobulin molecule.

[0037] As used herein, the term "chimeric antibody" should be taken to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial, or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0038] The antibody disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibody is human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody.In some embodiments, the variable region can be of chondrichthyan origin (e.g., from shark).

[0039] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which may be made by combining polynucleotides that do not normally occur together.

[0040] Hybridoma techniques can be carried out under different "stringency" conditions. Generally, low stringency hybridization reactions are carried out at about 40°C in about 10xSSC or equivalent ionic strength / temperature solutions. Medium stringency hybridization is typically carried out at about 50°C in about 6xSSC, and high stringency hybridization reactions are generally carried out at about 60°C in about 1xSSC. Hybridization reactions can also be carried out under "physiological conditions", which are well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH and Mg2+ concentration normally found in cells. Anti-CCR8 antibody

[0041] As demonstrated in the attached experimental examples, the present inventors were able to generate anti-CCR8 antibodies 84D1-2H3, 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4, 115C5E3B8, 163H9D5, 187B5F10, 195H8D10 and 200C9B9 (Table 1), all of which have high binding affinity to human CCR8 protein. The binding is specific, as they did not bind to CCR4. Except for 163H9D5, all of these antibodies can also cross-react with cyno CCR8 protein, thus facilitating the preclinical development of these antibodies.

[0042] Furthermore, all of these antibodies, except for 84D1-2H3, showed strong antagonist activity, but regardless of their antagonist activity, all of the antibodies were able to induce ADCC in animal models and also inhibit tumor growth.

[0043] Based on the sequence, these antibodies can be classified into three groups. As shown in Table 1A-B, group B includes 163H9D5, 187B5F10, 195H8D10 and 200C9B9, group A includes 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4 and 115C5E3B8, and group C includes 84D1-2H3. Each group of antibodies shares high homology between each CDR, and therefore they are interchangeable.

[0044] According to one embodiment of the present disclosure, an antibody or its antigen-binding fragment is provided.In some embodiments, the antibody or its antigen-binding fragment has binding properties to human CCR8 protein.In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0045] In some embodiments, for antibody Group B, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 61, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 62, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 63 or 39, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 64, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0046] As shown in Table 1B, SEQ ID NO:61 has a sequence of TYXMN, where X is A or V. SEQ ID NO:62 has a sequence of RIRTKSNNYATX1YX2X3X4VKD, where X1 is F, H or Y, X2 is A or V, and X3X4 is DS, DA or ES. SEQ ID NO:63 has a sequence of GTITRLGX1GX2DY, where X1 is A or G, and X2 is L or M. SEQ ID NO:64 has a sequence of RSSKX1LLHSX2X3NTYLY, where X1 is R or S, X2 is N or Q, and X3 is G or A.

[0047] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 61; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 62; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 63; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 64; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0048] In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 61; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 62; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 64; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0049] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or 24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 31, 32, 33, 100, 101, 102 or 103; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 46 or 47; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0050] Sequence analysis revealed that the VL CDR1 sequences (SEQ ID NOs: 46 and 47) contain residues that can potentially be post-translationally modified. To avoid post-translational modification (PTM) risks and thus simplify production, the present disclosure has designed and tested certain de-risked versions of VL CDR1, including SEQ ID NOs: 72, 73, 74 and 75. Thus, in some embodiments, antibodies and fragments are provided having CDR sequences, where VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23 or 24; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 31, 32, 33, 100, 101, 102 and 103; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 46, 47, 72, 73, 74 or 75; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0051] In some embodiments, for an antibody in Group A, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 58; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37 or 38; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 59; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50 or 51; and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 60.

[0052] As shown in Table 1B, SEQ ID NO:58 has a sequence of XIISX2DX3X4NX5YNPSLKX6, in which X1 is F or Y, X2 is F or Y, X3 is G or A, X4 is S, N or Y, X5 is D or N, and X6 is N or T. SEQ ID NO:59 has a sequence of KASDHINNXLA, in which X is R or W. SEQ ID NO:60 has a sequence of QQYWX1X2X3YT, in which X1 is G or S, X2 is T or Y, and X3 is P or S.

[0053] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26, 27, 28, 29 or 30; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44 or 45; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50 or 51; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 54, 55 or 56.

[0054] In some embodiments, VH CDR2, such as VH CDR2 provided in SEQ ID NO: 67-71, may be PTM risk avoided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 67, 68, 69, 70 or 71; VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37 or 38; VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44 or 45; VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50 or 51; VL CDR3 comprises the amino acid sequence of SEQ ID NO: 54, 55 or 56.

[0055] In some embodiments, an antibody or antigen-binding fragment derived from the antibody 84D1-2H3 is provided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:21; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:25; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:34; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:43; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:48; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:53.

[0056] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 21; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, 65 or 66; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 34; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 43; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 48; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 53.

[0057] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1 and further retaining the VH CDR of SEQ ID NO: 1 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 2 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2 and further retaining the VL CDR of SEQ ID NO: 2 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO: 2.

[0058] Thus, in some embodiments, antibodies and antigen-binding fragments are provided that bind to the same epitope on CCR8 as 84D1-2H3. Thus, in some embodiments, antibodies and antigen-binding fragments are provided that compete with 84D1-2H3 for binding to CCR8.

[0059] In some embodiments, an antibody or antigen-binding fragment derived from antibody 86D4E12A5 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; VH CDR2 comprises the amino acid sequence of SEQ ID NO:26; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:35; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:49; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:54.

[0060] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 26 or 67; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 35; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 54.

[0061] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 3 and further retaining the VH CDR of SEQ ID NO: 3 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 4 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4 and further retaining the VL CDR of SEQ ID NO: 4 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 3 and the VL comprises the amino acid sequence of SEQ ID NO: 4.

[0062] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 86D4E12A5. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 86D4E12A5 for binding to CCR8.

[0063] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from the antibody 96G3-1F10. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:27; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:36; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:45; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:50; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:55.

[0064] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 27 or 68; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 36; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 45; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 55.

[0065] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:5 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:5 and further retaining the VH CDR of SEQ ID NO:5 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:6 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6 and further retaining the VL CDR of SEQ ID NO:6 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:5 and the VL comprises the amino acid sequence of SEQ ID NO:6.

[0066] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 96G3-1F10. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 96G3-1F10 for binding to CCR8.

[0067] In some embodiments, an antibody or antigen-binding fragment derived from the antibody 99D1-1E11 is provided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:28; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:37; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:49; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:54.

[0068] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 28 or 69; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 37; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 54.

[0069] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:7 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:7 and further retaining the VH CDR of SEQ ID NO:7 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:8 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:8 and further retaining the VL CDR of SEQ ID NO:8 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid sequence of SEQ ID NO:8.

[0070] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 99D1-1E11. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 99D1-1E11 for binding to CCR8.

[0071] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from the antibody 101D5G10G4. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:29; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:36; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:45; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:50; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:55.

[0072] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 29 or 70; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 36; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 45; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 50; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 55.

[0073] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:9 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:9 and further retaining the VH CDR of SEQ ID NO:9 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO:10 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:10 and further retaining the VL CDR of SEQ ID NO:10 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:9 and the VL comprises the amino acid sequence of SEQ ID NO:10.

[0074] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 101D5G10G4. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 101D5G10G4 for binding to CCR8.

[0075] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from the antibody 115C5E3B8. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; VH CDR2 comprises the amino acid sequence of SEQ ID NO:30; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:38; VL CDR1 comprises the amino acid sequence of SEQ ID NO:44; VL CDR2 comprises the amino acid sequence of SEQ ID NO:51; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:56.

[0076] In some embodiments, the VH CDR2 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 30 or 71; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 38; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 51; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 56.

[0077] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 11 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 11 and further retaining the VH CDR of SEQ ID NO: 11 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 12 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 12 and further retaining the VL CDR of SEQ ID NO: 12 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 12.

[0078] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 115C5E3B8. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 115C5E3B8 for binding to CCR8.

[0079] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from antibody 163H9D5. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:23; VH CDR2 comprises the amino acid sequence of SEQ ID NO:31; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:39; VL CDR1 comprises the amino acid sequence of SEQ ID NO:46; VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0080] In some embodiments, the VL CDR1 is PTM risk averted. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 23; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 31; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 46, 72 or 73; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0081] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 13 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 13 and further retaining the VH CDR of SEQ ID NO: 13 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 14 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 14 and further retaining the VL CDR of SEQ ID NO: 14 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 13 and the VL comprises the amino acid sequence of SEQ ID NO: 14.

[0082] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 163H9D5. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 163H9D5 for binding to CCR8.

[0083] In some embodiments, an antibody or antigen-binding fragment is provided that is derived from the antibody 187B5F10. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:32; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:40; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:47; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0084] In some embodiments, the VL CDR1 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 32, 100 or 101; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 40; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 74 or 75; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0085] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 15 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 15 and further retaining the VH CDR of SEQ ID NO: 15 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 16 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 16 and further retaining the VL CDR of SEQ ID NO: 16 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 15 and the VL comprises the amino acid sequence of SEQ ID NO: 16.

[0086] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 187B5F10. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 187B5F10 for binding to CCR8.

[0087] In some embodiments, an antibody or antigen-binding fragment derived from antibody 195H8D10 is provided. In some embodiments, VH CDR1 comprises the amino acid sequence of SEQ ID NO:24; VH CDR2 comprises the amino acid sequence of SEQ ID NO:32; VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:41; VL CDR1 comprises the amino acid sequence of SEQ ID NO:47; VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0088] In some embodiments, the VL CDR1 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 32, 100 or 101; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 41; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, 74 or 75; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0089] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 17 and further retaining the VH CDR of SEQ ID NO: 17 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 18 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 18 and further retaining the VL CDR of SEQ ID NO: 18 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 18.

[0090] Humanized versions of 195H8D10 are also provided. Examples of humanized VH sequences include SEQ ID NOs: 76-79, and examples of humanized VL sequences include SEQ ID NOs: 80-83. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 76, 77, 78, or 79, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 76, 77, 78, or 79, and further retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 80, 81, 82, or 83, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 80, 81, 82, or 83, and further retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 76, 77, 78 or 79, and the VL comprises the amino acid sequence of SEQ ID NO: 80, 81, 82 or 83.

[0091] PTM re-risked humanized versions of 195H8D10 are also provided. Examples of humanized VH sequences include SEQ ID NOs: 76-79, and examples of humanized VL sequences include SEQ ID NOs: 92-95. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 76, 77, 78, or 79, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 76, 77, 78, or 79, and further retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 92, 93, 94, or 95, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 92, 93, 94, or 95, and further retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 76, 77, 78, or 79, and the VL comprises the amino acid sequence of SEQ ID NO: 92, 93, 94, or 95. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 76, and the VL comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 77, and the VL comprises the amino acid sequence of SEQ ID NO: 94.

[0092] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 195H8D10. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 195H8D10 for binding to CCR8.

[0093] In some embodiments, an antibody or antigen-binding fragment derived from the antibody 200C9B9 is provided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:33; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:42; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:46; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0094] In some embodiments, the VL CDR1 is PTM risk avoided. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 33, 102 or 103; the VH CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 42; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 46, 72 or 73; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 52; the VL CDR3 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0095] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 19 and further retaining the VH CDR of SEQ ID NO: 19 or a PTM re-risked version thereof. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 20 or comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 20 and further retaining the VL CDR of SEQ ID NO: 20 or a PTM re-risked version thereof. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 19 and the VL comprises the amino acid sequence of SEQ ID NO: 20.

[0096] Humanized versions of 200C9B9 are also provided. Examples of humanized VH sequences include SEQ ID NOs: 84-87, and examples of humanized VL sequences include SEQ ID NOs: 88-91. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 84, 85, 86, or 87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 84, 85, 86, or 87, and further retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 88, 89, 90, or 91, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 88, 89, 90, or 91, and further retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 84, 85, 86 or 87, and the VL comprises the amino acid sequence of SEQ ID NO: 88, 89, 90 or 91.

[0097] PTM re-risked humanized versions of 200C9B9 are also provided. Examples of humanized VH sequences include SEQ ID NOs: 84-87, and examples of humanized VL sequences include SEQ ID NOs: 96-99. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 84, 85, 86, or 87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 84, 85, 86, or 87, and further retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 96, 96, 98, or 99, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 96, 96, 98, or 99, and further retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 84, 85, 86, or 87, and the VL comprises the amino acid sequence of SEQ ID NO: 96, 96, 98, or 99. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 85, and the VL comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 84, and the VL comprises the amino acid sequence of SEQ ID NO: 98.

[0098] Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that bind to the same epitope on CCR8 as 200C9B9. Thus, in some embodiments, antibodies and antigen-binding fragments are also provided that compete with 200C9B9 for binding to CCR8.

[0099] In some embodiments, antibodies and antigen-binding fragments are also provided that comprise CDR sequences derived from the CDR sequences of the disclosure with one, two or three amino acid substitutions, deletions and / or additions.

[0100] In some embodiments, the anti-CCR8 antibody is a modified mAb that comprises a modified heavy chain constant region, such as an afucosylated heavy chain, that binds with high affinity to activating Fcγ receptors, mediating enhanced ADCC compared to the unmodified mAb. In some embodiments, the anti-CCR8 antibody comprises a heavy chain that is a human IgG1 variant that includes only one or a combination of L234Y, L235Q, G236W, S239D / M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L / M, I332E, K334A / E, P396L (all EU numbering), which enhances ADCC function.

[0101] In some embodiments, the human IgG1 Fc is Fc-DLE (S239D / A330L / I332E EU numbering), which is preferably symmetric. In some embodiments, the human IgG1 Fc is Fc-DE (S239D / I332E EU numbering), which is preferably symmetric. In some embodiments, the human IgG1 is afucosylated. In some embodiments, the human IgG1 Fc is asymmetric. For example, one of the Fc chains comprises one or more (or all) of the following substitutions: L234Y / L235Q / G236W / S239M / H268D / D270E / S298A, and the opposite Fc chain comprises one or more (or all) of the following substitutions: D270E / K326D / A330M / K334E (EU numbering). In certain preferred embodiments, the IgG1 Fc is afucosylated or asymmetric Fc-DLE. multifunctional molecules

[0102] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for CCR8, such as those disclosed herein, and one or more antibodies or antigen-binding fragments having specificity for a second antigen.

[0103] In some embodiments, the second antigen is a protein expressed on immune cells, such as T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells.

[0104] In some embodiments, the second antigen is against CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0105] Different formats of bispecific antibodies are also provided. In some embodiments, each of the anti-CCR8 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0106] Bifunctional molecules are also provided that do not simply comprise antibodies or antigen-binding fragments.As tumor antigen targeting molecules, CCR8-specific antibodies or antigen-binding fragments, such as those described herein, can be combined with immunocytokines or ligands, optionally via peptide linkers.Linked immunocytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL.Such bifunctional molecules can combine immune checkpoint blocking effects with tumor site local immune regulation. Chimeric Antigen Receptor

[0107] In one embodiment, a chimeric antigen receptor (CAR) is also provided, comprising the antibody or fragment thereof of the present disclosure as a targeting unit. In some embodiments, the CAR comprises the antibody or fragment thereof of the present disclosure, a transmembrane domain, a costimulatory domain, and a CD3ε intracellular domain.

[0108] The transmembrane domain can be designed to be fused to the extracellular domain, including antibody or fragment, optionally via hinge domain.It can also be fused to an intracellular domain, such as a costimulatory domain.In some embodiments, the transmembrane domain can include the natural transmembrane region of the costimulatory domain (e.g., the TM region of CD28 or 4-IBB used as costimulatory domain) or the natural transmembrane domain of the hinge region (e.g., the TM region of CD8alpha or CD28 used as hinge domain).

[0109] In some embodiments, a transmembrane domain may comprise a sequence that spans a cell membrane, but extends into the cytoplasm of the cell and / or into the extracellular space. For example, a transmembrane may comprise a membrane-spanning sequence that itself may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm of the cell and / or into the extracellular space. Thus, a transmembrane domain may comprise a region that spans the membrane, and may further comprise amino acids that extend beyond the inner or outer surface of the membrane itself, and still be considered to be a "transmembrane domain".

[0110] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Optionally, a short peptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. A glycine-serine doublet (GS), a glycine-serine-glycine triplet (GSG), or an alanine-alanine-alanine triplet (AAA) would be a suitable linker.

[0111] In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain is located between the transmembrane domain and the activation domain. Examples of costimulatory domains include CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8a, CD8, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (T FRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A(B cell antigen receptor complex-associated alpha chain), CD79B(B cell antigen receptor complex-associated beta chain), CD84(SLAMF5), CD96(Tactile), CD100(SEMA4D), CD103 (ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KI R2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), C D158F2(KIR2DL5B), CD158K(KTR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD 229(SLAMF3), CD244(SLAMF4), CD247(CD3-zeta), CD258(LIGHT), CD268(BAFFR), CD270(T FSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(KG2D), CD319(SLAMF7), CD335(K-p46 ), CD336(K-p44), CD337(K-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF 18), inducible T cell costimulatory factor (ICOS), LFA-1 (CD 1 1a / CD 18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activating NK cell receptor, Toll ligand receptor, and fragments or combinations thereof.

[0112] In some embodiments, the cytoplasmic portion of the CAR also comprises a signaling / activation domain. In one embodiment, the signaling / activation domain is a CD3 epsilon domain or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the CD3 epsilon domain. Methods for expressing or preparing polynucleotides, mRNA, and antibodies

[0113] The present disclosure also provides a polynucleotide or nucleic acid molecule that encodes the antibody, its variant or derivative, or CAR of the present disclosure.The polynucleotide of the present disclosure can encode the entire heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.In addition, the polynucleotide of the present disclosure can encode the part of the heavy and light chain variable region of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0114] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell for expression of the antibody or fragment thereof.

[0115] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically carried out using linear or circular template DNA, containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. Stringent conditions vary depending on specific applications.

[0116] In some embodiments, for the preparation of mRNA encoding an antibody, the template DNA is transcribed in vitro. A suitable template DNA typically has a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the mRNA encoding the desired antibody (e.g., encoding a heavy or light chain), and a termination signal.

[0117] Using standard methods, the mRNA sequence encoding the desired antibody (e.g., encoding a heavy or light chain) can be determined and incorporated into the template DNA. For example, starting from the desired amino acid sequence (e.g., the desired heavy or light chain sequence), a virtual back-translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select the appropriate codons. Typically, the G / C content can be optimized on the one hand to achieve as high a G / C content as possible, and on the other hand to take into account as much as possible the frequency of tRNA according to the codon usage frequency. The optimized RNA sequence can be established and displayed, for example with the aid of a suitable display device, and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or, respectively, regions of the RNA.

[0118] mRNA can be synthesized as unmodified or modified mRNA.Typically, mRNA is modified to improve stability.Modification of mRNA can include, for example, modification of nucleotide of RNA.Thus, modified mRNA can include, for example, backbone modification, sugar modification or base modification.In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil)), as well as modified nucleotides such as purines and pyrimidine analogs. Analogs or derivatives, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, These include, but are not limited to, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0119] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modification. Typically, backbone modification is a modification in which the backbone phosphate of the nucleotide contained in RNA is chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, positively charged guanidinium, etc., which means that phosphodiester bond is replaced by other anionic, cationic or neutral groups.

[0120] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain sugar modifications. Exemplary sugar modifications are chemical modifications of the sugars of the nucleotides they contain, such as 2'-deoxy-2'-fluoro-oligoribonucleotides (2'-fluoro-2'-deoxycytidine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotides (2'-amino-2'-deoxycytidine 5'-triphosphate, 2'-amino-2'-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotides, 2'-deoxy-2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate, 2'-amino ... and 2'-C-alkyl oligoribonucleotides, and their isomers (2'-aracytidine 5'-triphosphate, 2'-aruridine 5'-triphosphate), or azido triphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0121] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) may contain modifications of the base of a nucleotide (base modification). Modified nucleotides containing base modifications are also referred to as base-modified nucleotides. Examples of such base-modified nucleotides include 2-amino-6-chloropurine riboside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallyluridine 5'-triphosphate, 5-bromocytidine 5'-triphosphate, 5-bromouridine 5'-triphosphate, 5-iodocytidine 5'-triphosphate, 5-iodouridine 5'-triphosphate, 5-methylcytidine 5'-triphosphate, 5-methyluridine 5'-triphosphate, and 6-azacytidine 5'-triphosphate. , 6-azauridine 5'-triphosphate, 6-chloropurine riboside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole riboside 5'-triphosphate, N1-methyladenosine 5'-triphosphate, N1-methylguanosine 5'-triphosphate, N6-methyladenosine 5'-triphosphate, O6-methylguanosine 5'-triphosphate, pseudouridine 5'-triphosphate, puromycin 5'-triphosphate or xanthosine 5'-triphosphate.

[0122] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminal (5') end and a "tail" to the C-terminal (3') end. The presence of the cap is important in making the mRNA resistant to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the mRNA from exonuclease degradation.

[0123] Thus, in some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) includes a 5' cap structure. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase, resulting in a 5'5'5 triphosphate linkage; then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5)A and G(5)ppp(5')G.

[0124] In some embodiments, the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(A) tail structure. The polyA tail at the 3' end of the mRNA typically comprises about 10-300 adenosine nucleotides (e.g., about 10-200 adenosine nucleotides, about 10-175 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-125 adenosine nucleotides, 10-100 adenosine nucleotides, about 10-75 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the mRNA encoding the antibody (e.g., mRNA encoding the heavy and light chains) comprises a 3' poly(C) tail structure. A suitable poly-C tail at the 3' end of an mRNA typically comprises about 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly-C tail can be added to or replace the poly-A tail.

[0125] In some embodiments, the mRNA (e.g., mRNAs encoding the heavy and light chains) comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, e.g., an iron response element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length (e.g., between about 50 and 400 nucleotides in length, between about 50 and 300 nucleotides in length, between about 50 and 200 nucleotides in length, or between about 50 and 100 nucleotides in length).

[0126] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) comprises a sequence encoding a signal peptide, such as those described herein. In certain embodiments, a signal peptide derived from human growth hormone (hGH) is incorporated into the 5' region. Typically, the sequence encoding the signal peptide is linked directly or indirectly to the sequence encoding the heavy or light chain at the N-terminus.

[0127] This technology can be used to deliver any antibody known in the art and that can be raised against a desired antigen using standard methods. The invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0128] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Treatment and Use

[0129] As described herein, the antibodies, variants, derivatives, or antibody-drug conjugates of the disclosure can be used in certain treatment and diagnostic methods.

[0130] The present disclosure further relates to antibody-based therapy, including administering the disclosed antibodies, fragments, or antibody-drug conjugates to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the disclosed antibodies (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding the disclosed antibodies (including variants and derivatives thereof as described herein).

[0131] The antibody of the present disclosure can also be used to treat or inhibit cancer.As provided above, CCR8 can be overexpressed in tumor cells, particularly in liver, stomach, pancreas, esophagus, ovary and lung tumors.The inhibition of CCR8 has been shown to be useful in tumor treatment.

[0132] Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. The method, in one embodiment, entails administering to the patient an effective amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses CCR8.

[0133] Cell therapy, for example, chimeric antigen receptor (CAR) T cell therapy, is also provided in the present disclosure. Suitable cells can be used that are transduced with a vector that encodes or contacts a CAR that includes (or alternatively is engineered to express) the anti-CCR8 antibody of the present disclosure. Thus, by such contact or manipulation, the cells can be introduced into a cancer patient that needs treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0134] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0135] Non-limiting examples of cancer include bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer, and cutaneous T-cell lymphoma.

[0136] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and Carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, These include, but are not limited to, alveolar carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0137] CCR8 is expressed on monocytes and Th2 lymphocytes, as well as in the brain, spleen and thymus. It is the receptor for the chemokine CCL1, which is chemotactic for Th2 cells. CCL1 has also been shown to be involved in eosinophil recruitment. The antibodies disclosed herein can be used to inhibit (therapeutically or prophylactically) CCR8 activity; inhibit CCL1 activity; and to inhibit or treat conditions mediated by CCR8 and / or CCL1, including but not limited to inflammatory disorders and allergic conditions. Advantageously, the antibodies disclosed herein can also be used to inhibit conditions mediated by eosinophils, as well as monocytes, T lymphocytes and other immune system cells that express CCR8, including inflammatory disorders and allergic conditions mediated by these cells (e.g., asthma, atopic dermatitis and allergic rhinitis), as well as T cell leukemia and lymphoma.

[0138] In addition, for some HIV-1 strains, CCR8 acts as a coreceptor for HIV infection together with CD4 when transfected into cells that do not express either of the major coreceptors CCR5 or CXCR4. Therefore, in one embodiment of the present invention, a CCR8 monoclonal antibody is used to block the interaction of CCR8 with HIV surface proteins, thereby preventing HIV infection of CCR8-expressing cells.

[0139] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the number of administrations, excretion rate, drug combinations, and the severity of the particular disease being treated.The judgment of such factors by medical practitioners is within the ordinary skill of the art.The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.The amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0140] Methods of administration of antibodies, fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or transdermal patches), bucally, or as an oral spray or transdermal patch. It can be administered as a nasal spray.

[0141] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0142] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0143] It may be desirable to administer an antigen-binding polypeptide or composition of the disclosure locally to the area in need of treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, nonporous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the proteins do not absorb.

[0144] The amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure that will be effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be used as necessary to help identify optimal dosage ranges. The exact dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. An effective dose can be determined by in vitro assays. Dose-response curves derived from in vitro or animal model test systems can be extrapolated.

[0145] As a general proposition, the dosage of the antibody, fragment, or antibody-drug conjugate of the present disclosure administered to a patient is typically between 0.001 mg and 100 mg per kg of the patient's body weight, between 0.01 mg and 20 mg per kg of the patient's body weight, or between 0.5 mg and 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other animal species due to the immune response to the foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are often possible. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue penetration (e.g., into the brain) by modifications such as lipidation.

[0146] In additional embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0147] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

[0148] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody, fragment, or antibody-drug conjugate and acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., immune checkpoint inhibitor).

[0149] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0150] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0151] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0152] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0153] Example 1 Generation and testing of mouse anti-human CCR8 antibodies This example describes the generation of mouse anti-human CCR8 monoclonal antibodies using hybridoma technology.

[0154] Antigen: plasmid DNA encoding human CCR8 or CHO-K1 cell line expressing human CCR8 (CHO-K1 hCCR8 cells). Plasmid DNA immunization was performed by hydrodynamic tail vein injection. Cell immunization was performed by intraperitoneal (ip) injection of CHO-K1 hCCR8 cell line.

[0155] Immunization: To generate monoclonal antibodies against human CCR8, Balb / c, C57BL / 6 and SJL mice were immunized with full-length CCR8 DNA or the CHO-K1 hCCR8 cell line. To monitor the immune response, titrated sera from the mice were typically screened by flow cytometry after 4-6 weeks of immunization as described below. Sera were screened for antibody binding to multiple CCR8 cell lines, as well as corresponding negative control cell lines that do not express CCR8. CCR8-specific and non-specific Ab responses were measured in each animal, and animals with sufficient titers of anti-hCCR8 Ig were selected for a final boost with the HEK293 hCCR8 cell line 3-6 days prior to hybridoma fusion.

[0156] Cell fusion and hybridoma screening: Spleens were isolated from mice with the final boost as described above. Hybridomas were generated by cell fusion with immortalized mouse myeloma cells by electric field-based electrofusion. Fused cells were plated in 96 flat-bottom microtiter plates for hybridoma selection. Supernatants were screened by FACS using HEK293 hCCR8 cell line, CHO-K1 cyno CCR8 cell line and negative control cell line.

[0157] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parent cell. The subclones were screened using the same approach as the primary clones, and in addition, culture supernatants of positive clones were subjected to a second confirmatory screen by signaling assay using the β-arrestin hCCR8 CHO-K1 cell line to identify blocking antibodies.

[0158] Hybridoma clones 84D1-2H3, 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4, 115C5E3B8, 163H9D5, 187B5F10, 195H8D10 and 200C9B9 were selected for further analysis. The amino acid sequences of the variable regions are provided in Table 1 below and the CDR sequences are summarized in Table 1A. Table 1. Antibody variable region sequences [Table 1-1] [Table 1-2] Table 1A. CDR sequences (with optional mutations to avoid PTMs, Kabat numbering) [Table 1A-1] [Table 1A-1]

[0159] Review of the sequences revealed that antibodies 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4 and 115C5E3B8 are homologous to each other, as well as 163H9D5, 187B5F10, 195H8D10 and 200C9B9, which are designated Groups A and B, respectively, and which share the following consensus sequences for their CDRs: Table 1B. Consensus CDR sequences of antibody groups [Table 1B-1] [Table 1B-2] [Table 1B-3]

[0160] Example 2 Binding activity of anti-hCCR8 chimeric antibodies To evaluate the binding activity of hybridoma clones 84D1-2H3, 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4, 115C5E3B8, 163H9D5, 187B5F10, 195H8D10 and 200C9B9, chimeric mAbs from these clones were subjected to FACS testing using HEK293 hCCR8, CHO-K1 cyno CCR8 and CHO-K1 hCCR4 cell lines.

[0161] Briefly, HEK293 hCCR8, CHO-K1 cyno CCR8 or CHO-K1 hCCR4 cell lines were first incubated with 5-fold serial dilutions and 8 doses of the 10 chimeric Abs starting at 100 nM and hIgG1 control for 30 min at 4°C. After washing with FACS buffer, PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4°C. Samples were washed twice with FACS buffer. PE mean fluorescence intensity (MFI) was evaluated by MACSQuant Analyzer 16.

[0162] As shown in Figure 1A, all anti-hCCR8 antibodies bound specifically to human CCR8 in a dose-dependent manner. As shown in Figure 1B, all anti-hCCR8 antibodies, except for 163H9D5, also bound to cyno CCR8 to different extents. As shown in Figure 1C, none of these antibodies bound to the most closely related family member CCR4, further suggesting that these antibodies are specific to CCR8.

[0163] Example 3 Blockade of hCCL1-induced signaling by anti-CCR8 chimeric antibodies It has been reported that the CCR8 ligand CCL1 is overexpressed by tumor-associated macrophages, cancer-associated fibroblasts or other cell types and secreted into the cancer microenvironment. By activating its receptor CCR8, it induces proliferation, apoptosis resistance, and migration of immunosuppressive regulatory T cells, thus further inhibiting antitumor immunity. Therefore, anti-hCCR8 antibody in chimeric format was tested in a cell-based assay for its ability to inhibit CCL1-mediated downstream signaling.

[0164] In the β-arrestin assay, CCR8 is fused in-frame with the transcription factor Gal4 linked by a protease site. Upon CCL1 binding, the β-arrestin fused with the protease is recruited to the intracellular termini, where the tagged protease then cleaves the transcription factor, which translocates to the nucleus and activates its expression, which is Gal4-dependent luciferase expression. Briefly, β-arrestin hCCR8 CHO-K1 cells were seeded into each well of a 96-well assay plate at a density of 20000 per well and cultured at 37°C and 5% CO2. After 24 hours of incubation, each chimeric antibody was serially diluted and added to the designated columns of the assay plate. Human CCL1 (R&D system) at a final concentration of 10 ng / ml was then added and incubated for another 12-16 hours. Working detection solution (ONE-Glo™ Luciferase Assay System from Promega) was added to all wells of the assay plate and the assay plate was incubated for 3 minutes at room temperature in the dark. Luminescence was read on a multimode plate reader (Envision® 2105).

[0165] Data were plotted using GraphPad Prism software to determine half-maximal inhibitory concentrations (IC 50 ) values ​​were obtained and summarized in Figure 2 A. As shown in Figure 2 A, all antibodies except 84D1-2H3 showed various levels of antagonism in hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells.

[0166] In addition, hCCL1 binding of CCR8 on Chem1 cells induced calcium flux, suggesting that calcium (Ca 2+ Blockade of the hCCL1-mediated pathway by anti-hCCR8 mAbs was tested by performing a flux assay. Chem-1 hCCR8 cells were seeded at a density of 5000 per well in 384-well assay plates and cultured for at least 18 hours at 37°C and 5% CO2. Dye working buffer (Screen Quest™ Fluo-8 No wash calcium assay kit, AAT Bioquest) and different anti-hCCR8 mAbs were added to the cells and incubated for 1 hour at 37°C and 5% CO2 before the addition of 19 nM hCCL1 (R&D system). Ca 2+ Flux fluorescence signals were measured by Max-Min using FLIPR™ TETRA (Molecular Device). IC was calculated using Graphpad Prism. 50 was calculated.

[0167] As shown in Figure 2B, all antibodies except 84D1-2H3 inhibited hCCL1-induced Ca binding to hCCR8 in Chem1 cells. 2+ The two compounds showed varying levels of antagonism in flux, which is consistent with the β-arrestin assay.

[0168] Furthermore, chemotaxis assays were performed to evaluate the inhibitory activity of 86D4E12A5, 96G3-1F10, 187B5F10, 195H8D10 and 200C9B9 for their ability to block CCL1-induced migration of CCR8-expressing cells. BaF3 hCCR8 cell suspensions and diluted antibody samples were added to a 96-well plate and incubated for 1 h at 37°C and 5% CO2. A solution of hCCL1 (80 μl) at a concentration of 40 ng / ml was transferred to the lower compartment of the chemotaxis chamber and the corresponding well of the assemble chamber using a chemotaxis filter with a pore size of 5 μm. The cell / antibody mixture (100 μl) was then transferred to the upper compartment of the chamber and incubated for 3 h at 37°C. Finally, the contents from the lower compartment of the chemotaxis chamber were transferred to a new 96-well plate and 10 μl of resazurin was added to the plate. After 24 hours of incubation at 37° C., plates were read in a fluorescent plate reader at 544 / 590 nM.

[0169] FIG. 2C showed that 86D4E12A5, 96G3-1F10, 187B5F10, 195H8D10 and 200C9B9 all inhibited hCCL1-induced migration of BaF3 hCCR8.

[0170] Example 4 Antibodies induce ADCC signaling in an ADCC reporter bioassay This example compared ADCC signaling of CCR8 antibodies in vitro.

[0171] In this experiment, HEK293 hCCR8 cells were used as target cells, Jurkat cells stably expressing high affinity FcγRIIIa [CD16a(176V)] and a luciferase reporter driven by an NFAT response element were used as effector cells, and the antibodies tested were anti-hCCR8 antibodies with WT hIgG1 Fc or hIgG1 S239D / I332E Fc. Upon simultaneous binding to both target and effector cells, the effector cells transduce an intracellular signal resulting in NFAT-mediated luciferase, which can be quantified by a luminescence reader. In this experiment, target and effector cells were mixed at a 1:1 E:T ratio, and increasing concentrations of antibodies were added and incubated for 6 hours at 37°C, 5% CO2 in a 96-well assay plate. ADCC activity was determined by bioluminescence readout.

[0172] The results (FIG. 3A) showed that chimeric anti-hCCR8 antibodies with wild-type hIgG1 induced ADCC signaling in a dose-dependent manner.

[0173] As shown in FIG. 3B, a chimeric anti-hCCR8 antibody with an enhanced Fc backbone (hIgG1 S239D / I332E) induced stronger ADCC signaling in the same experimental setting.

[0174] Example 5 The antibody induces ADCC in HEK293 hCCR8 cells using NK92 CD16a as effector cells Anti-hCCR8 antibodies with enhanced Fc (hIgG1 S239D / I332E), which showed stronger ADCC reporter activity in the reporter assay, were tested for their ability to mediate killing of target cells (HEK293 hCCR8 cell line) by the natural killer cytotoxic cell line NK-92 overexpressing CD16a(176V).

[0175] In this experiment, ADCC was measured using the DELFIA® EuTDA Cytotoxicity kit (PerkinElmer). Target cells (HEK293 hCCR8 cells) were diluted to a density of 1,000,000 cells / ml in culture medium and labeled with fluorescence-enhancing ligand for 25 min at 37°C. After 3-5 extensive washes, the labeled target cells mixed with NK92 CD16a(176V) cells at a ratio of 1:10 and anti-hCCR8 antibodies at a series of concentrations were added to a microplate for co-culture. After 2 h of incubation, 20 μL of the supernatant was transferred to a new plate, and 200 μL of europium solution was added and incubated at room temperature for 15 min using a DELFIA Plateshake. The signal correlates with the number of lysed cells.

[0176] As shown in FIG. 4, a chimeric anti-hCCR8 antibody with enhanced Fc function (hIgG1 S239D / I332E) was able to induce ADCC effects against CCR8-positive cells by a natural killer cell line.

[0177] Example 6 Efficacy in the MC38 tumor model This example used a syngeneic mouse model to test the in vivo anti-tumor efficacy of functional molecules. The Fc of an anti-CCR8 antibody was engineered into mIgG2a to mimic the stronger ADCC effect of human IgG1.

[0178] 5 × 10 MC38 cells resuspended in PBS in a volume of 0.1 ml 5 The cells were administered subcutaneously (sc) into the right flank of B-hCCR8-humanized C57BL / 6 mice at a concentration of 100x the mean tumor volume was approximately 82mm. 3Once tumor volume reached 100 mg / kg, animals were randomly assigned according to tumor volume into experimental groups with 7 animals in each group. Anti-CCR8 antibodies were tested antibodies including 84D1-2H3-mIgG2a (6 mg / kg and 3 mg / kg), 195H8D10-mIgG2a (6 mg / kg and 3 mg / kg) and 200C9B9-mIgG2a (6 mg / kg and 3 mg / kg). Tested antibodies were administered twice weekly by intraperitoneal injection. Mouse body weight and tumor volume were measured twice weekly.

[0179] As shown in Figure 5, all anti-human CCR8 antibodies with enhanced ADCC activity showed significant tumor growth inhibition at both the 3 mg / kg and 6 mg / kg dose levels. Among these antibodies, 195H8D10 and 200C9B9 showed stronger anti-tumor activity.

[0180] Example 7 Humanization of hCCR8 antibody The variable region genes of antibodies 195H8D10 and 200C9B9 were used to generate a humanized mAb. The amino acid sequences of the VH and VK of 195H8D10 / 200C9B9 were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig sequence.

[0181] For the light chain of 195H8D10, IGKV2-28 * 01 was the best matched germline, and for the heavy chain of 195H8D10, IGHV3-72 * 01 was selected as the humanization backbone. Then, a humanized 195H8D10 CDR-grafted antibody was designed, and CDR-L1, L2 and L3 were ligated to IGKV2-28 * 01 framework sequence, and CDR-H1, H2 and H3 were grafted onto IGHV3-72 *The grafted antibody was grafted onto the framework sequence of 01. A 3D model was then generated to determine the amino acids in the original murine framework regions that are essential for antibody binding and conformation. Based on the analysis, back mutations were performed on the grafted antibody, resulting in the generation of four additional humanized heavy chains and four additional light chains.

[0182] For the light chain of 200C9B9, IGKV2-28 * 01 was the best matched germline, and for the heavy chain of 200C9B9, IGHV3-72 * 01 was selected as the humanized backbone. A humanized 200C9B9 CDR-grafted antibody was then designed, with CDRL1, L2 and L3 substituted for IGKV2-28. * 01 framework sequence, and CDRH1, H2 and H3 were grafted into IGHV3-72 * The 200C9B9 CDR-grafted antibody was grafted onto the framework sequence of 01. A 3D model was then generated to determine the amino acids in the original murine FR region sequence that are essential for antibody binding and conformation. Based on the analysis, back mutations were performed on the 200C9B9 CDR-grafted antibody, resulting in the generation of four additional humanized heavy chains and four additional light chains.

[0183] The resulting humanized sequences are listed in Tables 2-3. Table 2. Humanization of 195H8D10 [Table 2] Table 2A. Humanized antibodies from 195H8D10 [Table 2A-1] [Table 2A-2] Table 3. Humanization of 200C9B9 [Table 3] Table 3A. Humanized antibodies from 200C9B9 [Table 3A]

[0184] Example 8 Binding activity of humanized antibodies Flow cytometry was used to confirm the binding activity of the humanized antibodies using CHO-K1 hCCR8 and CHO-K1 cyno CCR8 cell lines.

[0185] Briefly, first, CHO-K1 hCCR8 cells and CHO-K1 cyno CCR8 cells were incubated with different concentrations of humanized 195H8D10 and 200C9B9 antibodies for 30 min at 4°C. PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4°C. Samples were washed twice with FACS buffer. PE mean fluorescence intensity (MFI) was evaluated by MACSQuant Analyzer 16.

[0186] As shown in Figures 6A and 6B, the humanized 195H8D10 / 200C9B9 antibody was comparable to the corresponding chimeric antibody with respect to human CCR8 binding.

[0187] Furthermore, Figures 6C and 6D showed that the humanized 195H8D10 / 200C9B9 antibodies were comparable to the corresponding chimeric antibodies with respect to cyno CCR8 binding, with the exception of Hu195H8D10-2, Hu195H8D10-4, Hu200C9B9-2, and Hu200C9B9-4.

[0188] Example 9 Humanized antibodies induce ADCC signaling in an ADCC reporter bioassay This example compared ADCC signaling of humanized CCR8 antibodies in vitro.

[0189] In this experiment, CHO-K1 hCCR8 cells were used as target cells, Jurkat CD16a(176V)NFAT luciferase reporter cells were used as effector cells, and the antibodies tested were humanized 195H8D10 and 200C9B9 antibodies, as described in the previous example. The experiment was performed under co-incubation of target and effector cells and test antibodies in 96-well assay plates at 37°C, 5% CO2 for 6 hours according to the manufacturer's instructions. ADCC signaling was determined by bioluminescence readout.

[0190] As shown in Figures 7A and 7B, the humanized 195H8D10 / 200C9B9 antibody induced comparable ADCC signaling as the corresponding chimeric antibody in an ADCC reporter assay.

[0191] Example 10 Inhibition of hCCL1-induced β-arrestin recruitment by humanized antibodies The humanized 195H8D10 / 200C9B9 antibody was again tested to confirm antagonism of CCL1-mediated downstream signaling in CHO-K1 hCCR8 cells.

[0192] The experiment was performed as described in the previous example. Briefly, β-arrestin hCCR8 CHO-K1 cells were plated and mixed with diluted test antibodies. Human CCL1 (R&D system) was then added to the cultures and incubated for 12-16 hours. Detection buffer was added to all wells of the assay plate and luminescence was read on a microplate reader.

[0193] As shown in Figures 8A and 8B, all humanized 195H8D10 / 200C9B9 antibodies demonstrated comparable levels of antagonism in the hCCL1-induced β-arrestin assay as the corresponding chimeric antibodies.

[0194] Example 11 Confirmation of PTM removal of 195H8D10 and 200C9B9 It has been observed that 195H8D10 and 200C9B9 VL CDR1 contain NG residues (Kabat numbering) that are at risk for post-translational modification (PTM) and pose a difficult challenge for future manufacturing. Therefore, this example mutated NG on VL to QG or NA to prevent PTM. The sequences of potential PTM removal sites are listed in Table 4. Table 4. PTM removal of 195H8D10 and 200C9B9 [Table 4]

[0195] As shown in FIG. 9A, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 were comparable to the corresponding parent antibodies with respect to human CCR8 binding.

[0196] As shown in FIG. 9B, 195H8D10-C2 and 200C9B9-C2 maintain cyno-binding capacity compared to the corresponding antibodies.

[0197] As shown in FIG. 9C, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 induced comparable ADCC signaling as the corresponding parental antibodies in an ADCC reporter assay.

[0198] As shown in FIG. 9D, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 exhibited comparable levels of antagonism to the corresponding parental antibodies in hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells.

[0199] Therefore, this example identifies the sequences of humanized 195H8D10 and 200C9B9 (VL CDR1 NG-NA) with potential PTM sites removed, and the sequences are listed in Table 5. Table 5. Humanization of 195H8D10 with removal of potential PTM sites [Table 5-1] [Table 5-2] Table 5A. 195H8D10 humanized antibody with potential PTM sites removed [Table 5A] Table 6. Humanization of 200C9B9 with removal of potential PTM sites. [Table 6-1] [Table 6-2] Table 6A. Humanized antibodies from 200C9B9 with PTMs removed [Table 6A]

[0200] Example 12 Confirmation of PTM removal of humanized 195H8D10 and 200C9B9 To further confirm the performance of the humanized 195H8D10 / 200C9B9 antibody after PTM removal, we tested the binding, ADCC activity, functional blocking activity, and tumor growth inhibition efficacy of Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies.

[0201] As shown in FIG. 10A, the Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies were comparable to the corresponding parental antibodies with respect to human CCR8 binding.

[0202] As shown in FIG. 10B, Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies induced comparable ADCC signaling as the corresponding parental antibodies in an ADCC reporter assay.

[0203] As shown in FIG. 10C, Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies demonstrated comparable levels of antagonism to the corresponding parental antibodies in hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells.

[0204] As shown in Figure 10D, Hu195H8D10-7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies inhibited hCCL1-induced Ca binding to hCCR8 in Chem1 cells. 2+ They showed comparable levels of antagonism in flux, which is consistent with the β-arrestin assay.

[0205] Furthermore, we tested the in vivo antitumor efficacy of Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies using a syngeneic mouse model. The same mouse Fc was used for ADCC-mediated Treg killing in B-hCCR8 mice. Briefly, 5×10 MC38 cells resuspended in PBS were cultured in a volume of 0.1 ml. 5 The cells were administered subcutaneously (sc) into the right flank of B-hCCR8-humanized C57BL / 6 mice at a concentration of 1000x1000 cells / mL. The mean tumor volume was approximately 85 mm 3 Once tumor volume reached 100 mg / kg, animals were randomly assigned to experimental groups with 7 animals in each group according to tumor volume. Anti-CCR8 antibodies (6 mg / kg) were the tested antibodies, and they were administered by intraperitoneal injection twice weekly. Mouse body weights and tumor volumes were measured twice weekly.

[0206] As shown in Figure 11, Hu195H8D10-1 / 7 NG-NA and Hu200C9B9-3 / 7 NG-NA antibodies with enhanced ADCC activity showed significant tumor growth inhibition (TGI). Among these antibodies, Hu200C9B9-7 NG-NA single treatment showed stronger tumor growth inhibition, with an inhibition rate of approximately 70% at the end of the study.

[0207] Example 13 Comparison of Hu200C9B9-7 NG-NA with benchmark antibodies Finally, we compared the Hu200C9B9-7 NG-NA antibody with benchmark antibodies from Gilead Sciences (1-K17.044) and Bristol Myers Squibb (4A19) in vitro and in vivo. The anti-CCR8 antibodies developed by Gilead and BMS are currently in phase I and phase I / II clinical trials, respectively.

[0208] All CCR8 antibodies were fused with the same Fc (human IgG1 kappa). Binding activity, ADCC activity, and functional blocking activity were compared in vitro. As shown in Figure 12A, Hu200C9B9-7 NG-NA was a strong binder of hCCR8, with binding capacity comparable to that of its benchmark as judged by FACS. To determine specificity, we performed a binding assay based on the parental HEK293 without a target. The results (Figure 12B) showed that Hu200C9B9-7 NG-NA did not bind to the parental HEK293 at all, while the benchmark antibodies developed from Gilead and BMS had some degree of non-specific binding to the parental HEK293 cells.

[0209] As mentioned in the previous example, we also compared the ADCC signaling of Hu200C9B9-7 NG-NA and benchmarks. Figure 12C shows that all anti-hCCR8 antibodies with wild-type hIgG1 induced ADCC signaling in a dose-dependent manner in HEK293 hCCR8 and Jurkat CD16a(176V)NFAT luciferase cell co-culture assays.

[0210] In addition, blockade of hCCL1-mediated pathways by Hu200C9B9-7 NG-NA and benchmark antibodies was confirmed by β-arrestin recruitment assay and calcium (Ca 2+) flux assay. Hu200C9B9-7 NG-NA showed comparable levels of antagonism to the benchmark in hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells (Figure S12D). Furthermore, Hu200C9B9-7 NG-NA inhibited hCCL1-mediated Ca2+ upregulation in Chem-1 hCCR8 cells. 2+ It was able to inhibit flux better than both benchmark antibodies (Figure 12E).

[0211] The preclinical efficacy of Hu200C9B9-7 NG-NA and Benchmark was evaluated in a syngeneic mouse model in which the mouse CCR8 gene was replaced with its human counterpart and inoculated with MC38, as described in the previous example. Using the same mouse Fc, Hu200C9B9-7 NG-NA, 1-K17.044 and 4A19 single treatment all showed significant inhibition of tumor growth, with inhibition rates of 61.2%, 53.2% and 59.1%, respectively (Figure 13A). Ex vivo analysis suggested that all of these antibodies efficiently depleted CCR8+Tregs and increased mCD45+ lymphocytes at the tumor site (Figure 13B), but spared peripheral Treg subsets and mCD45+ lymphocytes (Figure 13C).

[0212] Example 14 Fc selection of Hu200C9B9-7 NG-NA Fc engineering is a promising approach to enhance the ADCC effect and improve the antitumor efficacy of monoclonal antibodies (mAbs).

[0213] In order to find the best ADCC enhancement approach for therapeutic antibodies, we produced antibodies with Fc-DLE (introducing the triple mutation S239D / A330L / I332E in both Fc chains), Fc-DE (introducing the double mutation S239D / I332E in both Fc chains), Fc-afucosylated in which the N-glycan residues in the IgG Fc region lack the core fucose sugar unit, Fc-asymmetric (i.e., introducing different substitutions in each Fc region of the heavy chain: one heavy chain: L234Y / L235Q / G236W / S239M / H268D / D270E / S298A and the opposite heavy chain: D270E / K326D / A330M / K334E) and Fc-WT hIgG1. We assessed the binding affinity of these Fc variants or glycomutant Fcs to the FcγRIIIa allotype and the inhibitory FcγRIIb and calculated the ratio of activating to inhibitory FcγR binding (A / I).

[0214] Binding of antibodies with different Fc to recombinant hCD16a-176V, hCD16a-176F and hCD32b proteins was tested in Biacore using the capture method. Proteins were captured using CM5-anti-His chips. Serial dilutions of antibodies were injected onto the captured proteins. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 7. The Fc variant with the DLE mutation was found to have high affinity for both CD16a allotypes and the lowest affinity for CD32b. Thus, the Fc variant with the DLE mutation reached the highest A / I ratio. Table 7. Affinities and A / I ratios measured by Biacore [Table 7-1] [Table 7-2]

[0215] To further evaluate the ability of CCR8 antibody in ADCC signaling, we compared anti-CCR8 antibody with Fc-DLE, Fc-DE, Fc-afucosylated, Fc-asymmetric and Fc-WT (using the same Fab part). ADCC activity was determined by Jurkat hCD16a NFAT or Jurkat hCD16a hCD32b NFAT effector cells incubated with CHO-K1 hCCR8 cells. The results (Figure 14A) showed that anti-hCCR8 antibody with Fc-DLE, Fc-DE, Fc-afucosylated, Fc-asymmetric induced strong ADCC activity compared with WT hIgG1. Furthermore, in the Jurkat hCD16a hCD32b cell culture system expressing inhibitory receptors, anti-hCCR8 antibodies with Fc-DLE, Fc-afucosylated and Fc-asymmetric antibodies showed stronger ADCC activity than Fc-DE and WT hIgG1, which was consistent with the A / I ratio (Figure 14B).

[0216] In addition, antibody-mediated killing of CCR8-expressing HEK293 cells was tested. PBMCs from healthy donors co-incubated with HEK293 hCCR8 cells (50:1 ratio of PBMCs to target cells) were treated overnight at 37°C with serial dilutions of anti-CCR8 antibodies with Fc-DLE, Fc-DE, Fc-afucosylated, Fc-WT and hIgG1 DLE control. Cell lysis was determined by released LDH levels in the supernatant after co-culture. The results showed that anti-hCCR8 antibodies with engineered Fc, especially with the DLE mutation, mediated the most potent killing of CCR8-expressing target cells (Figure 14C). * * *

[0217] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0218] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody or antigen-binding fragment thereof having specificity for human C-C motif chemokine receptor 8 (CCR8) protein, comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3; (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO:61; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 63 or 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; the VL CDR3 comprises the amino acid sequence of SEQ ID NO:57; (b) the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:58; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37, or 38; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:59; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50, or 51; the VL CDR3 comprises the amino acid sequence of SEQ ID NO:60; or (c) the VH CDR1 comprises the amino acid sequence of SEQ ID NO:21; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 25, 65, or 66; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:34; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:43; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:48; the VL CDR3 comprises the amino acid sequence of SEQ ID NO:53; An antibody or an antigen-binding fragment thereof.

2. (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO:61; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 63 or 39; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; the VL CDR3 comprises the amino acid sequence of SEQ ID NO:57; The antibody or antigen-binding fragment thereof described in claim 1.

3. the VH CDR1 comprises the amino acid sequence of SEQ ID NO:61; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence of SEQ ID NO:63; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:52; the VL CDR3 comprises the amino acid sequence of SEQ ID NO:57; The antibody or antigen-binding fragment thereof according to claim 2.

4. the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 being respectively SEQ ID NOs: 23, 31, 39, 46, 52 and 57; SEQ ID NOs: 23, 31, 39, 72, 52 and 57; SEQ ID NOs: 23, 31, 39, 73, 52 and 57; SEQ ID NOs: 24, 32, 40, 47, 52 and 57; SEQ ID NOs: 24, 32, 40, 74, 52 and 57; SEQ ID NOs: 24, 32, 40, 75, 52 and 57; SEQ ID NOs: 24, 32, 41, 47, 52 and 57; SEQ ID NOs: 24, 32, 41, 74, 52 and 57; SEQ ID NOs: 24, 32, 41, 75, 52 and 57; SEQ ID NOs: 24, 33, 42, 46, 52 and 57; SEQ ID NOs: 24, 33, 42, 72, 52 and 57; or SEQ ID NOs: 24, 33, 42, 73, 52 and 57 The antibody or antigen-binding fragment thereof according to claim 2, comprising the amino acid sequence:

5. The antibody or antigen-binding fragment thereof of claim 2, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 24, 32, 41, 74, 52, and 57, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76 to 79, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 95.

7. The antibody or antigen-binding fragment thereof of claim 2, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 24, 32, 41, 47, 52, and 57, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 76 to 79, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 80 to 83.

9. The antibody or antigen-binding fragment thereof of claim 2, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 24, 33, 42, 72, 52, and 57, respectively.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 84 to 87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 96 to 99.

11. The antibody or antigen-binding fragment thereof of claim 2, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NOs: 24, 33, 42, 46, 52, and 57, respectively.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 84 to 87, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 88 to 91, preferably the VH comprises the amino acid sequence of SEQ ID NO: 85, and the VL comprises the amino acid sequence of SEQ ID NO:

98.

13. The antibody or antigen-binding fragment thereof of claim 4, wherein the VH comprises the amino acid sequence of SEQ ID NO: 13 and the VL comprises the amino acid sequence of SEQ ID NO:

14.

14. The antibody or antigen-binding fragment thereof of claim 4, wherein the VH comprises the amino acid sequence of SEQ ID NO: 15 and the VL comprises the amino acid sequence of SEQ ID NO:

16.

15. (b) the VH CDR1 comprises the amino acid sequence of SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:58; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 35, 36, 37, or 38; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:59; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 49, 50, or 51; the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 60; The antibody or antigen-binding fragment thereof described in claim 1.

16. the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 being respectively SEQ ID NOs: 22, 26, 35, 44, 49 and 54; SEQ ID NOs: 22, 67, 35, 44, 49 and 54; SEQ ID NOs: 22, 27, 36, 45, 50 and 55; SEQ ID NOs: 22, 68, 36, 45, 50 and 55; SEQ ID NOs: 22, 28, 37, 44, 49 and 54; SEQ ID NOs: 22, 69, 37, 44, 49 and 54; SEQ ID NOs: 22, 29, 36, 45, 50 and 55; SEQ ID NOs: 22, 70, 36, 45, 50 and 55; SEQ ID NOs: 22, 30, 38, 44, 51 and 56; or SEQ ID NOs: 22, 71, 38, 44, 51 and 56 The antibody or antigen-binding fragment thereof of claim 15, comprising the amino acid sequence:

17. The VH and VL are each SEQ ID NOs: 3 and 4; SEQ ID NOs:5 and 6; SEQ ID NOs: 7 and 8; SEQ ID NOs: 9 and 10; or SEQ ID NOs: 11 and 12 17. The antibody or antigen-binding fragment thereof of claim 16, comprising the amino acid sequence:

18. the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 being respectively SEQ ID NOs: 21, 25, 34, 43, 48 and 53; SEQ ID NOs: 21, 65, 34, 43, 48 and 53; or SEQ ID NOs: 21, 66, 34, 43, 48 and 53 The antibody or antigen-binding fragment thereof according to claim 1, comprising the amino acid sequence:

19. The antibody or antigen-binding fragment thereof of claim 18, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1 and the VL comprises the amino acid sequence of SEQ ID NO:

2.

20. 20. The antibody or fragment thereof of any one of claims 1 to 19, which is a bivalent Fab antibody or a fragment selected from the group consisting of F(ab')2, F(ab)2, Fab', Fab, Fv and scFv.

21. 21. The antibody or fragment thereof of any one of claims 1 to 20, which is humanized.

22. 22. The antibody or fragment thereof of claim 1 , comprising an Fc fragment having enhanced antibody-dependent cellular cytotoxicity (ADCC).

23. 23. The antibody or fragment thereof of claim 22, wherein the Fc fragment is a human IgG1 Fc fragment having one or more substitutions selected from the group consisting of: L234Y, L235Q, G236W, S239D / M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330L / M, I332E, K334A / E, P396L according to EU numbering.

24. The IgG1 Fc fragment, (a) contains the substitutions S239D, A330L and I332E; (b) containing the substitutions S239D and I332E; (c) is afucosylated; or (d) comprising one or more of the substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in one Fc chain and one or more of the substitutions D270E, K326D, A330M, and K334E in the second Fc chain; 24. An antibody or fragment thereof according to claim 23.

25. The IgG1 Fc fragment, (a) contains the substitutions S239D, A330L and I332E; (b) is afucosylated; or (c) comprising one or more of the substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A in one Fc chain and one or more of the substitutions D270E, K326D, A330M, and K334E in the second Fc chain; 24. An antibody or fragment thereof according to claim 23.

26. 26. A multispecific antibody comprising an antigen-binding fragment of any one of claims 1 to 25 and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen that is not CCR8.

27. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment of any one of claims 1 to 25, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular domain.

28. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 26 or the CAR of claim 27.

29. 29. The polynucleotide of claim 28 which is one or more mRNAs.

30. 30. The polynucleotide of claim 29, wherein the mRNA is chemically modified.

31. A cell comprising the polynucleotide of claim 28.

32. A composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 26, or a CAR described in claim 27, and a pharma- ceutically acceptable carrier.

33. 28. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1 to 26, or a CAR of claim 27.

34. 28. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 26, or a CAR according to claim 27, for the preparation of a medicament for treating cancer.

35. 27. A method of treating an inflammatory condition in a patient in need thereof, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 26.

36. 27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 26 for the preparation of a medicament for treating an inflammatory condition.

37. 37. The method of claim 35 or the use of claim 36, wherein the inflammatory condition is selected from the group consisting of asthma, atopic dermatitis, and allergic rhinitis.

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