Anti-CCR8 antibodies and uses thereof

Abstract

The present disclosure provides anti-CCR8 antibodies, including murine antibodies, humanized antibodies, and antibodies having further optimized CDR sequences. The newly disclosed antibodies have high antagonism and ADCC activity, and can be used for treating cancer and inflammation.

Description

[0001] This application is a divisional application of a patent application with an application date of December 23, 2022, application number 202280092377.7, and invention name “Anti-CCR8 antibodies and their applications”. Background Art

[0002] 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 important roles in the regulation of monocyte chemotaxis and thymocyte apoptosis. More specifically, CCR8 may contribute to the correct localization of activated T cells to sites of antigenic attack and specialized areas of lymphoid tissue.

[0003] 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 allergic inflammatory responses that occur at sites of allergen exposure. The ligand for CCR8 is the CC chemokine CCL1, which is a chemotactic attractant for Th2 cells. Therefore, 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 these sites, and the subsequent mobilization of eosinophils and basophils.

[0004] CCR8 knockout mice showed impaired Th2 immune responses in allergic inflammation models. In allergic lung inflammation induced by ovalbumin and cockroach antigen, the levels of Th2 cytokines (IL-4, IL-5, and IL-13) and the number of recruited eosinophils in the lungs of CCR8 knockout mice were significantly reduced. Therefore, inhibiting CCR8 helps to improve the symptoms of allergic inflammation such as asthma, atopic dermatitis, and allergic rhinitis.

[0005] CCR8 has also been identified as a marker for tumor-infiltrating regulatory T cells (Tregs), as CCR8 expression is selectively upregulated in these regulatory T cells in a variety of cancers, including breast, colorectal, and lung cancers. These CCR8+ regulatory T cells represent a highly activated and suppressive subset of regulatory T cells, and their high abundance in these tumor types correlates with poor prognosis. Therefore, CCR8 is a promising therapeutic target to deplete regulatory T cells in tumors to enhance antitumor immunity.

[0006] However, chemokine receptors have traditionally been very difficult antigens to develop antibodies against. They are expressed at low levels on the cell surface and are not very easy to bind to antibodies. In addition, antibodies generated against peptides corresponding to the extracellular domains of chemokine receptors often fail to recognize the intact receptors on cells, likely due to differences in secondary structure.

[0007] CCR8 is a particularly challenging GPCR for antibody development, with limited success in the past in generating cross-species reactive antibodies (cynomolgus monkey and human). In addition, finding functional antibodies for GPCRs including CCR8 is even more challenging. Summary of the invention

[0008] In various embodiments of the present disclosure, antibodies and antigen-binding fragments specific to human CCR8 proteins are provided. Experimental tests show that these newly identified antibodies can bind strongly and specifically to human CCR8 proteins, and most of them can cross-react with cynomolgus monkey CCR8 proteins, which is different from most benchmark antibodies, making it possible to prove preclinical safety in non-human primates. In addition, in vitro studies have shown that most of these antibodies can specifically block CCR8 signaling induced by its ligand CCL1. In addition, when there is a constant region with complete or enhanced Fc function, these antibodies can induce antibody-dependent cell-mediated cytotoxicity (ADCC) of target cells expressing CCR8. In vivo experiments show that these antibodies with a constant region with enhanced Fc function can effectively inhibit tumor growth.

[0009] According to one embodiment of the present disclosure, an antibody or an antigen-binding fragment thereof is provided, which is specific to human CC motif chemokine receptor 8 (CCR8) protein and includes a heavy chain variable region (VH) containing VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region (VL) containing VL CDR1, VL CDR2 and VL CDR3.

[0010] In one embodiment, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:61; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:63 or 39; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

[0011] In another embodiment, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:58; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:59; the VLCDR2 comprises the amino acid sequence shown in SEQ ID NO:49, 50 or 51; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:60.

[0012] In another embodiment, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:21; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:25, 65 or 66; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:34; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:43; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:48; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:53.

[0013] In some embodiments, the antibody or its fragment is humanized. In some embodiments, the antibody or its fragment comprises an Fc fragment with enhanced antibody-dependent cellular toxicity (ADCC). Exemplary Fc fragments are human IgG1 fragments 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.

[0014] Also provided are multispecific antibodies comprising an antigen-binding fragment of the disclosure and one or more antibodies or antigen-binding fragments having binding specificity for a target antigen other than CCR8.

[0015] In another embodiment, a chimeric antigen receptor (CAR) is also provided, which comprises the antigen binding fragment of the present disclosure, a transmembrane domain, a co-stimulatory domain and a CD3 intracellular domain.

[0016] Also provided are polynucleotides encoding antibodies or antigen-binding fragments thereof or CARs of the present disclosure.In some embodiments, the polynucleotide is mRNA, which is optionally chemically modified.

[0017] Also provided are methods and uses of the disclosed antibodies or antigen-binding fragments thereof for treating cancer and inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The binding affinities of the identified antibodies to human CCR8, cynomolgus monkey CCR8 and human CCR4 are shown.

[0019] Figure 2 The blocking activity of the tested chimeric antibodies is shown.

[0020] Figure 3 The ADCC signaling activities of the tested chimeric antibodies are shown.

[0021] Figure 4 The natural killer cell-mediated ADCC killing effect induced by the tested antibodies is shown.

[0022] Figure 5 In vivo tumor suppressor activity of the tested antibodies is shown.

[0023] Figure 6 The binding of the humanized antibody to human CCR8 and cynomolgus monkey CCR8 is shown.

[0024] Figure 7 The ADCC activity of the tested humanized CCR8 antibodies is shown.

[0025] Figure 8 The blocking activity of the tested humanized CCR8 antibodies is shown.

[0026] Fig. 9 Binding and functional properties of anti-CCR8 chimeric antibodies after removal of potential PTM sites are shown.

[0027] Fig.10 Shown are the binding and functional properties of humanized CCR8 antibodies after removal of potential PTM sites.

[0028] Fig.11 The in vivo tumor suppressor activity of humanized CCR8 antibodies after removal of potential PTM sites was shown.

[0029] Fig.12 Comparison of Hu200C9B9-7 NG-NA with benchmark antibodies in vitro is shown.

[0030] Fig.13 Comparison of Hu200C9B9-7 NG-NA with benchmark antibodies in vivo and in vitro is shown.

[0031] Fig.14 ADCC activity of different Fc-engineered Hu200C9B9-7 NG-NAs is shown.

[0032] Detailed description

[0033] definition

[0034] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0035] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody and any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Such embodiments include, but are not limited to, a heavy or light chain complementarity determining region (CDR) 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.

[0036] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment will bind to 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.

[0037] The term antibody encompasses a wide variety of polypeptides that can be distinguished biochemically. It will be appreciated by those skilled in the art that the heavy chain can be classified as γ, μ, α, δ, or ε, with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, whether IgG, IgM, IgA IgG, or IgE.

[0038] Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to have functional specificities. Improved versions of these classes and isotypes can be easily discerned by those skilled in the art in light of the present disclosure, and therefore, they are all within the scope of the present disclosure. All classes of immunoglobulins are obviously within the scope of the present disclosure, and the following discussion is generally directed to IgG class immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are typically connected by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, starting at the mouth of the "Y" and extending to the variable region.

[0039] The antibodies, antigen-binding polypeptides, variants or derivatives of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure may be 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 molecules.

[0040] As used herein, the term "chimeric antibody" refers to any antibody in which an immunologically active region or site is obtained or derived from a first species, while a constant region (which may be complete, partial or modified, depending on the disclosure) is obtained from a second species. In certain embodiments, the binding region or site of interest will be from a non-human source (such as a mouse or primate), while the constant region is human.

[0041] The antibodies disclosed herein can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In some embodiments, the variable region can be of cartilaginous origin (e.g., from shark).

[0042] As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be produced by combining polynucleotides that do not normally occur together.

[0043] Hybridoma technology can be performed under conditions of varying "stringency". Generally, low stringency hybridization reactions are performed at a temperature of about 40°C in about 10×SSC or a solution of equivalent ionic strength / temperature. Moderate stringency hybridizations are typically performed in about 6×SSC at about 50°C, and highly stringent hybridization reactions are typically performed in about 1×SSC at about 60°C. Hybridization reactions can also be performed under "physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg2+ concentrations normally found in cells.

[0044] Anti-CCR8 Antibody

[0045] As shown in the attached experimental examples, the 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. This binding is specific because they do not bind to CCR4. Except for 163H9D5, all of these antibodies can also cross-react with cynomolgus monkey CCR8 protein, thereby facilitating the preclinical development of these antibodies.

[0046] In addition, except for 84D1-2H3, all of these antibodies showed strong antagonistic activity. However, regardless of their antagonistic activity, all antibodies could induce ADCC and were able to inhibit tumor growth in animal models.

[0047] According to the sequence, these antibodies can be divided 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; Group C includes 84D1-2H3. Each CDR of each group of antibodies has high homology, so they are interchangeable.

[0048] According to one embodiment of the present disclosure, an antibody or an antigen binding fragment thereof is provided. In some embodiments, the antibody or its antigen binding fragment has binding specificity to human CCR8 protein. In some embodiments, the antibody or its antigen binding fragment comprises a heavy chain variable region (VH), including VH CDR1, VH CDR2 and VHCDR3, and a light chain variable region (VL), including VL CDR1, VL CDR2 and VL CDR3.

[0049] In some embodiments, with respect to Group B antibodies, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:61; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:63 or 39; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

[0050] As shown in Table 1B, SEQ ID NO:61 has a TYXMN sequence, wherein X is A or V. SEQ ID NO:62 has a RIRTKSNNYATX1YX2X3X4VKD sequence, wherein X1 is F, H or Y, X2 is A or V, and X3X4 is DS, DA or ES. SEQ ID NO:63 has a GTITRLGX1GX2DY sequence, wherein X1 is A or G, and X2 is L or M. SEQ ID NO:64 has a RSSKX1LLHSX2X3NTYLY sequence, wherein X1 is R or S, X2 is N or Q, and X3 is G or A.

[0051] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:61; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:63; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

[0052] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:61; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:62; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:39; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:64; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

[0053] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:23 or 24; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:31, 32, 33, 100, 101, 102 or 103; the VHCDR3 comprises the amino acid sequence shown in SEQ ID NO:39, 40, 41 or 42; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:46 or 47; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:57.

[0054] Sequence analysis shows that the VL CDR1 sequences (SEQ ID NOs: 46 and 47) include residues that may be post-translationally modified. In order to avoid the risk of post-translational modification (PTM) and thus simplify the production process, the present disclosure designed and tested certain risk-free versions of VL CDR1, including SEQ ID NOs: 72, 73, 74 and 75. Thus, in some embodiments, antibodies and fragments having CDR sequences are provided, wherein the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 23 or 24; the VHCDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, 32, 33, 100, 101, 102 or 103; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO: 39, 40, 41 or 42; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO: 46, 47, 72, 73, 74 or 75; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO: 52; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO: 57.

[0055] In some embodiments, with respect to Group A antibodies, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:58; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:59; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:49, 50 or 51; the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:60.

[0056] As shown in Table 1B, SEQ ID NO:58 has the sequence X1ISX2DX3X4NX5YNPSLKX6, wherein 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 the sequence KASDHINNXLA, wherein X is R or W. SEQ ID NO:60 has the sequence QQYWX1X2X3YT, wherein X1 is G or S, X2 is T or Y, and X3 is P or S.

[0057] In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:26, 27, 28, 29 or 30; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:44 or 45; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:49, 50 or 51; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:54, 55 or 56.

[0058] In some embodiments, the VH CDR2 can be PTM de-risked, such as those provided in SEQ ID NOs: 67-71. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NOs: 26, 27, 28, 29, 30, 67, 68, 69, 70, or 71; the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NOs: 35, 36, 37, or 38; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NOs: 44 or 45; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NOs: 49, 50, or 51; and the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NOs: 54, 55, or 56.

[0059] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 84D1-2H3. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:25; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:43; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:48; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:53.

[0060] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:21; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:25, 65 or 66; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO:43; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:48; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:53.

[0061] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 1, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1, while retaining the VH CDRs or PTM-derisked versions thereof of SEQ ID NO: 1. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2, while retaining the VL CDRs or PTM-derisked versions thereof of SEQ ID NO: 2. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 1, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0062] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 84D1-2H3 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 84D1-2H3 for binding to CCR8 are also provided.

[0063] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 86D4E12A5. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:26; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:35; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:49; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:54.

[0064] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26 or 67; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54.

[0065] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:3, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:3, while retaining the VH CDRs or PTM-derisked versions thereof of SEQ ID NO:3. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO:4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:4, while retaining the VL CDRs or PTM-derisked versions thereof of SEQ ID NO:4. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:3, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:4.

[0066] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 86D4E12A5 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 86D4E12A5 for binding to CCR8 are also provided.

[0067] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 96G3-1F10. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:27; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:50; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:55.

[0068] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27 or 68; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 50; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55.

[0069] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:5, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:5, while retaining the VH CDRs or PTM-derisked versions thereof of SEQ ID NO:5. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO:6, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:6, while retaining the VL CDRs or PTM-derisked versions thereof of SEQ ID NO:6. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:5, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:6.

[0070] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 96G3-1F10 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 96G3-1F10 for binding to CCR8 are also provided.

[0071] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 99D1-1E11. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:28; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:49; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:54.

[0072] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28 or 69; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54.

[0073] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:7, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:7, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO:7. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO:8, or a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:8, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO:8. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO:7, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:8.

[0074] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 99D1-1E11 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 99D1-1E11 for binding to CCR8 are also provided.

[0075] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 101D5G10G4. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:29; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:45; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:50; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:55.

[0076] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 29 or 70; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 50; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 55.

[0077] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 9. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 10. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 10.

[0078] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 101D5G10G4 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 101D5G10G4 for binding to CCR8 are also provided.

[0079] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 115C5E3B8. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:30; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:38; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:44; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:51; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:56.

[0080] In some embodiments, the VH CDR2 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30 or 71; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 44; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 51; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 56.

[0081] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 11. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 12, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 12. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 12.

[0082] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 115C5E3B8 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 115C5E3B8 for binding to CCR8 are also provided.

[0083] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 163H9D5. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:23; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:31; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:39; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:46; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0084] In some embodiments, the VL CDR1 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:31; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:39; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:46, 72 or 73; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0085] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 13. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 14. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 14.

[0086] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 163H9D5 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 163H9D5 for binding to CCR8 are also provided.

[0087] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 187B5F10. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:24; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:32; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:47; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0088] In some embodiments, the VL CDR1 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, 100 or 101; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 47, 74 or 75; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0089] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 15, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 15. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 16, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 16. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 15, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 16.

[0090] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 187B5F10 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 187B5F10 for binding to CCR8 are also provided.

[0091] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 195H8D10. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO:24; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:32; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41; the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO:47; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO:52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0092] In some embodiments, the VL CDR1 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 32, 100 or 101; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 41; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 47, 74 or 75; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0093] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 17, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 17. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 18. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 17, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 18.

[0094] Humanized versions of 195H8D10 are also provided. Exemplary humanized VH sequences include SEQ ID NOs: 76-79, and exemplary humanized VL sequences include SEQ ID NOs: 80-83. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NOs: 76, 77, 78, or 79, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 76, 77, 78, or 79, while retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 80, 81, 82, or 83, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 80, 81, 82, or 83, while retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:76, 77, 78 or 79, and the VL comprises the amino acid sequence shown in SEQ ID NO:80, 81, 82 or 83.

[0095] Also provided are PTM-de-risked humanized versions of 195H8D10. Exemplary humanized VH sequences include SEQ ID NOs: 76-79, and exemplary humanized VL sequences include SEQ ID NOs: 92-95. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NOs: 76, 77, 78, or 79, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 76, 77, 78, or 79, while retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 92, 93, 94, or 95, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 92, 93, 94, or 95, while retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 76, 77, 78 or 79, and the VL comprises the amino acid sequence shown in SEQ ID NO: 92, 93, 94 or 95. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 76, and the VL comprises the amino acid sequence shown in SEQ ID NO: 92. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 77, and the VL comprises the amino acid sequence shown in SEQ ID NO: 94.

[0096] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 195H8D10 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 195H8D10 for binding to CCR8 are also provided.

[0097] In some embodiments, the antibodies or antigen-binding fragments provided are derived from antibody 200C9B9. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:24; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:33; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:42; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:46; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57.

[0098] In some embodiments, the VL CDR1 is PTM de-risked. In some embodiments, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, 102 or 103; the VH CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42; the VLCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 46, 72 or 73; the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 52; and the VL CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57.

[0099] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VH CDRs or PTM de-risked versions thereof of SEQ ID NO: 19. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 20, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, while retaining the VL CDRs or PTM de-risked versions thereof of SEQ ID NO: 20. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 19, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 20.

[0100] Humanized versions of 200C9B9 are also provided. Exemplary humanized VH sequences include SEQ ID NOs: 84-87, and exemplary humanized VL sequences include SEQ ID NOs: 88-91. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NOs: 84, 85, 86, or 87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 84, 85, 86, or 87, while retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 88, 89, 90, or 91, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 88, 89, 90, or 91, while retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:84, 85, 86 or 87, and the VL comprises the amino acid sequence shown in SEQ ID NO:88, 89, 90 or 91.

[0101] Also provided are PTM-de-risked humanized versions of 200C9B9. Exemplary humanized VH sequences include SEQ ID NOs: 84-87, and exemplary humanized VL sequences include SEQ ID NOs: 96-99. In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NOs: 84, 85, 86, or 87, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 84, 85, 86, or 87, while retaining the VH CDRs. In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 96, 97, 98, or 99, or a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 96, 97, 98, or 99, while retaining the VL CDRs. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 84, 85, 86 or 87, and the VL comprises the amino acid sequence shown in SEQ ID NO: 96, 97, 98 or 99. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 85, and the VL comprises the amino acid sequence shown in SEQ ID NO: 98. In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 84, and the VL comprises the amino acid sequence shown in SEQ ID NO: 98.

[0102] In some embodiments, antibodies and antigen-binding fragments thereof that bind to the same epitope on CCR8 as 200C9B9 are also provided. In some embodiments, antibodies and antigen-binding fragments thereof that compete with 200C9B9 for binding to CCR8 are also provided.

[0103] In some embodiments, antibodies and antigen-binding fragments are also provided, which include CDR sequences derived from the present disclosure, with one, two or three amino acid substitutions, deletions and / or additions.

[0104] In some embodiments, the anti-CCR8 antibody is a modified mAb comprising a modified heavy chain constant region (e.g., a non-fucosylated heavy chain), which binds to activated Fcγ receptors with higher affinity to mediate enhanced ADCC compared to unmodified mAbs. In some embodiments, the anti-CCR8 antibody comprises a heavy chain belonging to a human IgG1 variant, including a single 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 can enhance ADCC function.

[0105] In some embodiments, human IgG1 Fc is Fc-DLE (S239D / A330L / I332E EU numbering), which is preferably symmetrical. In some embodiments, human IgG1 Fc is Fc-DE (A330L / I332E EU numbering), which is preferably symmetrical. In some embodiments, human IgG1 is non-fucosylated. In some embodiments, human IgG1 Fc is asymmetric. For example, one of the Fc chains includes one or more (or all) of L234Y / L235Q / G236W / S239M / H268D / D270E / S298A replacements, and the relative Fc chain includes one or more (or all) of D270E / K326D / A330M / K334E (EU numbering). In certain preferred embodiments, IgG1 Fc is Fc-DLE, non-fucosylated or asymmetric.

[0106] Multifunctional molecules

[0107] A multifunctional molecule comprising an antibody or antigen-binding fragment specific for CCR8 as disclosed herein, and one or more antibodies or antigen-binding fragments specific for a second antigen.

[0108] 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.

[0109] In some embodiments, the second antigen is 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.

[0110] Different forms of bispecific antibodies are also provided. In some embodiments, the anti-CCR8 fragment and the second fragment are each 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.

[0111] Also provided are bifunctional molecules that not only include antibodies or antigen binding fragments. As tumor antigen targeting molecules, antibodies or antigen binding fragments specific for CCR8 as described herein can be selected to be combined with immune cytokines or ligands through peptide linkers. The immune cytokines or ligands connected 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-, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. This bifunctional molecule can combine the immune checkpoint blocking effect with local immune regulation at the tumor site.

[0112] Chimeric Antigen Receptor

[0113] In one embodiment, a chimeric antigen receptor (CAR) is also provided, which includes an antibody or fragment thereof disclosed herein as a targeting unit. In some embodiments, the CAR includes an antibody or fragment thereof disclosed herein, a transmembrane domain, a costimulatory domain, and a CD3 intracellular domain.

[0114] The transmembrane domain can be designed to be fused to an extracellular domain including an antibody or fragment, optionally fused via a hinge domain. Similarly, it can also be fused to an intracellular domain (such as a costimulatory domain). In some embodiments, the transmembrane domain may include a natural transmembrane domain of a costimulatory domain (e.g., a TM region of CD28 or 4-IBB used as a costimulatory domain) or a natural transmembrane domain of a hinge region (e.g., a TM region of CD8α or CD28 used as a hinge domain).

[0115] In some embodiments, a transmembrane domain may include a sequence that spans the cell membrane but extends into the cytoplasm and / or the extracellular space of the cell. For example, a transmembrane domain may include a transmembrane sequence, which itself may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids that extend into the cytoplasm and / or the extracellular space of the cell. Thus, a transmembrane domain includes a transmembrane region and may further include amino acids that extend beyond the inner or outer surface of the membrane itself; such a sequence may still be considered a "transmembrane domain".

[0116] In some embodiments, the transmembrane domain is fused to the cytoplasmic domain via a short linker. Alternatively, a short peptide or polypeptide linker preferably between 2 and 10 amino acids in length can form a connection between the transmembrane domain and the proximal cytoplasmic signaling domain of the chimeric receptor. Glycine-serine doublets (GS), glycine-serine-glycine triplet (GSG) or alanine-alanine-alanine triplet (AAA) provide suitable linkers.

[0117] In some embodiments, CAR further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain is located between the transmembrane domain and the activation domain. Exemplary co-stimulatory domains include, but are not limited to, CD2, CD3δ, CD3ε, CD3γ, 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 α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (HECT), CD100 (SEMA4D), CD 103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1)), CD158A(KIR2DL1), CD158B1( KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(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 co-stimulator (ICOS), LFA-1 (CD 1la / CD18), KG2C, DAP-10, ICAM-1, Kp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, Toll ligand receptor and fragments or combinations thereof.

[0118] In some embodiments, the cytoplasmic portion of CAR also includes a signal transduction / activation domain. In one embodiment, the signal transduction / activation domain is a CD3 domain, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a CD3 domain.

[0119] Polynucleotides, mRNA and methods of expressing or preparing antibodies

[0120] The present disclosure also provides polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof, or CARs of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode partial heavy chains and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules.

[0121] In some embodiments, the polynucleotide is an mRNA molecule. In some embodiments, the mRNA can be introduced into a target cell to express the antibody or fragment thereof.

[0122] mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). In short, IVT generally uses a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, and an appropriate RNA polymerase (such as T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase and / or RNAse inhibitor. The specific conditions will vary depending on the specific application.

[0123] In some embodiments, to prepare mRNA encoding an antibody, a DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by a desired nucleotide sequence and termination signal encoding the desired antibody (e.g., encoding a heavy chain or light chain) mRNA.

[0124] Standard methods can be used to determine the required coding antibody (for example, encoding heavy chain or light chain) mRNA sequence and merge it into the DNA template. For example, starting from the required amino acid sequence (for example, required heavy chain or light chain sequence), virtual reverse translation is performed based on the degenerate genetic code. Then an optimization algorithm can be used to select suitable codons. Usually, the G / C content can be optimized on the one hand to achieve the highest possible G / C content, and on the other hand, the frequency of tRNA can be considered as best as possible according to codon use. The RNA sequence optimized can be established and displayed, for example, by means of a suitable display device and compared with the original (wild type) sequence. The secondary structure can also be analyzed to calculate the stable and unstable characteristics or regions of RNA respectively.

[0125] mRNA can be synthesized as unmodified or modified mRNA. Generally, mRNA is modified to enhance stability. Modification of mRNA may include, for example, modification of nucleotides of RNA. Therefore, modified mRNA may include, for example, backbone modification, sugar modification, or base modification. In some embodiments, antibodies encoding mRNA (e.g., heavy and light chains encoding mRNA) may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2- Methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thiocytosine, 3-methyl-cytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil ), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-hydroxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic 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. Patent 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, and 5,700,642, the disclosures of which are incorporated herein in their entirety.

[0126] In some embodiments, the mRNA (e.g., mRNA encoding heavy and light chains) may contain RNA backbone modifications. Typically, backbone modifications are modifications in which the phosphates of the backbones of the nucleotides contained in the RNA are chemically modified. Exemplary backbone modifications typically include, but are not limited to, modifications in the group consisting of methylphosphonates, methylphosphoamidates, phosphoamidates, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphoric acid), borophosphates, positively charged guanidine groups, etc., i.e., replacing phosphodiester connections with other anions, cations, or neutral groups.

[0127] In some embodiments, the mRNA (e.g., mRNA encoding heavy and light chains) may contain sugar modifications. Typical sugar modifications are chemical modifications of the sugars of the nucleotides it contains, including but not limited to 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-alkyl The group consisting of oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine 5'-triphosphate, 2'-methyluridine 5'-triphosphate), 2'-C-alkyl oligoribonucleotides and their isomers (2'-arabinocytidine 5'-triphosphate, 2'-arabinocytidine 5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine 5'-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0128] In some embodiments, the mRNA (e.g., mRNA encoding heavy and light chains) may contain modifications of the bases of the nucleotides (base modifications). Modified nucleotides containing base modifications are also referred to as base modified nucleotides. Examples of such base modified nucleotides include, but are not limited to, 2-amino-6-chloropurine nucleoside 5'-triphosphate, 2-aminoadenosine 5'-triphosphate, 2-thiocytidine 5'-triphosphate, 2-thiouridine 5'-triphosphate, 4-thiouridine 5'-triphosphate, 5-aminoallylcytidine 5'-triphosphate, 5-aminoallylcytidine 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 , 6-azacytidine 5'-triphosphate, 6-azauridine 5'-triphosphate, 6-chloropurine nucleoside 5'-triphosphate, 7-deazaadenosine 5'-triphosphate, 7-deazaguanosine 5'-triphosphate, 8-azaadenosine 5'-triphosphate, 8-azidoadenosine 5'-triphosphate, benzimidazole nucleoside 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.

[0129] Typically, mRNA synthesis involves the addition of a "cap" at the N-terminus (5' end) and a "tail" at the C-terminus (3' end). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" protects the mRNA from exonuclease degradation.

[0130] 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 terminal phosphate group from the 5' nucleotide, leaving two terminal phosphate groups; then guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase to produce a 5'5'5 triphosphate bond; 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.

[0131] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) includes a 3' poly-A (A) tail structure. The poly-A tail at the 3' end of the mRNA generally contains about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, the antibody encoding mRNA (e.g., mRNA encoding heavy and light chains) includes a 3' poly (C) tail structure. Suitable poly-C tails on the 3' end of the mRNA generally include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A tail or may replace the poly-A tail.

[0132] In some embodiments, mRNA (e.g., mRNA encoding heavy and light chains) includes 5' and / or 3' untranslated regions. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as an iron response element. In some embodiments, the length of the 5' untranslated region can be between about 50 and 500 nucleotides (e.g., a length of about 50 to 400 nucleotides, a length of about 50 to 300 nucleotides, a length of about 50 to 200 nucleotides, or a length of about 50 to 100 nucleotides).

[0133] In some embodiments, the 5' region of the mRNA (e.g., mRNA encoding the heavy and light chains) includes a sequence encoding a signal peptide, such as those described herein. In a specific embodiment, a signal peptide derived from human growth hormone (hGH) is integrated in the 5' region. Typically, the signal peptide coding sequence is directly or indirectly linked to the N-terminus of the heavy or light chain coding sequence.

[0134] The present technology can be used to deliver any antibody known in the art and antibodies raised against a desired antigen using standard methods. The present invention can be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or mixtures, human or humanized antibodies, chimeric antibodies or bispecific antibodies.

[0135] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides disclosed herein are fully human. Fully human antibodies can be made using techniques known in the art and methods 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 an antigenic attack, but whose endogenous loci have been inactivated. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents (6,150,584; 6,458,592; 6,420,140), the entire contents of which are incorporated by reference.

[0136] Treatment and uses

[0137] As described herein, the antibodies, variants, derivatives or antibody-drug conjugates of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0138] The present disclosure also relates to antibody-based therapeutic methods, which involve administering antibodies, fragments or antibody-drug conjugates described in the present disclosure to patients, such as animals, mammals and humans, to treat one or more diseases or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives as described herein).

[0139] The antibodies disclosed herein can also be used to treat or inhibit cancer. As described above, CCR8 can be overexpressed in tumor cells, particularly liver cancer, gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer. Inhibition of CCR8 has been shown to be useful for treating tumors.

[0140] Therefore, in some embodiments, a method for treating cancer in a patient in need is provided. In one embodiment, the method requires administering an effective amount of an antibody, fragment, or antibody-drug conjugate of the present disclosure to the patient. In certain embodiments, at least one cancer cell (such as a stromal cell) in the patient overexpresses CCR8.

[0141] The present disclosure also provides cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used, and the cells are transduced with a vector encoding a CAR containing an anti-CCR8 antibody disclosed herein, or contacted with a CAR containing an anti-CCR8 antibody disclosed herein (or can be engineered to express the anti-CCR8 antibody described in the present disclosure). After such contact or transformation, the cells can be introduced into the body of a cancer patient in need of treatment. Cancer patients may suffer from any type of cancer disclosed herein. The cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0142] In some embodiments, the cells are isolated from the cancer patient himself. In some embodiments, the cells are provided by a donor or come from a cell bank. When the cells are isolated from the cancer patient, adverse immune responses can be minimized.

[0143] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal 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, lung cancer, and cutaneous T-cell lymphoma.

[0144] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or predicted with the antibodies or variants or derivatives thereof of the present disclosure include, but are not limited to, progression and / or metastasis of malignancies and related diseases such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute granulocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroblastic leukemias)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, Osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, liver cancer, bile duct carcinoma, 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, cerebrovascular tumor, melanoma, neuroblastoma and retinoblastoma.

[0145] CCR8 is expressed in monocytes and Th2 lymphocytes as well as in the brain, spleen and thymus. It is a receptor for the chemokine CCL1, and CCL1 has a chemotactic effect on Th2 cells. CCL1 has also been shown to be involved in the recruitment of eosinophils. The antibodies disclosed herein can be used to inhibit CCR8 activity; inhibit CCL1 activity and inhibit or treat (therapeutic or preventive) disorders mediated by CCR8 and / or CCL1, including but not limited to inflammatory disorders and allergic disorders. The antibodies disclosed herein can also be advantageously used to inhibit disorders mediated by eosinophils and by monocytes, T lymphocytes and other immune system cells expressing CCR8, including inflammatory disorders and allergic disorders mediated by these cells (e.g., asthma, atopic dermatitis and allergic rhinitis)), as well as T cell leukemia and lymphoma.

[0146] In addition, for cells that do not express the major co-receptors CCR5 and CXCR4, CCR8 together with CD4 becomes a co-receptor for HIV infection by certain HIV-1 strains. Therefore, in one embodiment of the present invention, CCR8 monoclonal antibodies are used to block the interaction of CCR8 with HIV surface proteins, thereby preventing HIV infection of cells expressing CCR8.

[0147] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof, the patient's age, weight, general health, sex and diet, as well as the time of administration, the rate of excretion, the drug combination, and the severity of the specific disease being treated. The judgment of these factors by the medical caregiver is within the ordinary skill of the art. The dosage also depends 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 dosage can be determined according to pharmacological and pharmacokinetic principles well known in the art.

[0148] The method of administration of antibody, fragment or antibody-drug conjugate includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptide or composition can be applied by any convenient route, such as by infusion or push injection, by epithelial or mucocutaneous lining (e.g., oral mucosa, rectum and intestinal mucosa, etc.) absorption, and can be applied together with other bioactive agents. Therefore, the pharmaceutical composition containing antigen-binding polypeptide of the present disclosure can be oral, rectal, parenteral, intraventricular, intravaginal, intraperitoneal, or locally (such as by powder, ointment, drops or transdermal patch) administration, oral administration or as oral or nasal spray administration.

[0149] As used herein, the term "parenteral" refers to routes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0150] Administration can be systemic or local. In addition, the antibodies of the present disclosure can be introduced into the central nervous system by any suitable route, including intravenous injection and intrathecal injection; intravenous injection can be achieved by intravenous catheter, for example, connected to a reservoir (such as an Ommaya reservoir). Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations with an atomizer.

[0151] It may be desirable to administer the antigen-binding polypeptide or composition of the invention locally to the area in need of treatment; this may be accomplished, for example but not limited to, by local infusion during surgery, by topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous, or gel-like material, including membranes, such as silicone rubber membranes or fibers. Preferably, when administering proteins of the present disclosure, including antibodies, care must be taken to use materials that are not absorbed by the protein.

[0152] The effective dosage of the disclosed antibodies, fragments or antibody-drug conjugates in the treatment, inhibition and prevention of inflammation, immune or malignant diseases, disorders or conditions can be determined by standard clinical techniques. In addition, in vitro assays can be selected to help determine the optimal dosage range. The precise dosage used in the formulation also depends 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 the circumstances of each patient. The effective dose can be inferred from dose-response curves derived from in vitro or animal model test systems.

[0153] In general, the dosage of the antibody, fragment or antibody-drug conjugate of the present disclosure to the patient is usually 0.001 mg / kg to 100 mg / kg (based on the patient's body weight), or 0.01 mg / kg to 20 mg / kg (based on the patient's body weight), or 0.5 mg / kg to 10 mg / kg (based on the patient's body weight). In general, due to the immune response to exogenous polypeptides, human antibodies have a longer half-life in the human body than antibodies of other species. Therefore, lower doses of human antibodies and less frequent administration are generally possible. In addition, the uptake and tissue penetration of antibodies (e.g., into the brain) can be enhanced by modification (e.g., lipidation), thereby reducing the dosage and frequency of administration of the antibodies described in the present disclosure.

[0154] In other embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can 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.

[0155] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or preventive regimens, such as radiation therapy.

[0156] Composition

[0157] The present disclosure also provides a pharmaceutical composition. This composition comprises an effective amount of an antibody, fragment or antibody-drug conjugate, and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0158] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government, or listed in the United States Pharmacopeia or other recognized pharmacopoeia, for use in animals, especially humans. In addition, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary.

[0159] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline, aqueous glucose solution and aqueous glycerol solution can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water and ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer such as acetate, citrate or phosphate. 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 regulating osmotic pressure such as sodium chloride or glucose are all contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository together with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade 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 an antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of a carrier to provide a form of appropriate administration to the patient. The formulation should be suitable for the mode of administration. The parent formulation can be contained in an ampoule, disposable syringe, or multiple-dose vial made of glass or plastic.

[0160] In one embodiment, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to the human being according to conventional procedures. Usually, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to alleviate the pain at the injection site. Usually, each component is provided separately or mixed together in a unit dose form, for example, as a dry lyophilized powder or anhydrous concentrate, contained in a sealed container (for example, an ampoule or a pouch indicating the active dose). When the composition is administered by infusion, an infusion bottle equipped with sterile medicinal water or saline can be used to distribute. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix each component before administration.

[0161] 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. DETAILED DESCRIPTION

[0162] Example 1 Generation and testing of mouse anti-human CCR8 antibodies

[0163] This example describes the generation of mouse anti-human CCR8 monoclonal antibodies using hybridoma technology.

[0164] Antigen: Plasmid DNA encoding human CCR8 or CHO-K1 cell line expressing human CCR8 (CHO-K1 hCCR8 cells). Immunization with plasmid DNA was performed by hydrodynamic injection into the tail vein. Cellular immunization was performed by intraperitoneal injection (ip) of CHO-K1hCCR8 cell line.

[0165] Immunization: To generate monoclonal antibodies targeting human CCR8, Balb / c mice, C57BL / 6 mice, and SJL mice were immunized with full-length CCR8 DNA or CHO-K1 hCCR8 cell lines. To monitor immune responses, titrated sera from mice were screened by flow cytometry as described below, typically 4-6 weeks after immunization. Sera were screened for binding to antibodies from a variety of CCR8 cell lines as well as corresponding negative control cell lines that do not express CCR8. CCR8-specific and nonspecific antibody responses were measured for 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.

[0166] Cell fusion and hybridoma screening: Spleens were isolated from terminally boosted mice as described above. Hybridomas were generated by fusion with immortalized mouse myeloma cells by electric field-based electrofusion. Fusion cells were cultured in 96-flat-bottom microplates for hybridoma screening. Supernatants were screened by FACS using the HEK293 hCCR8 cell line, the CHO-K1 cyno CCR8 cell line, and a negative control cell line.

[0167] Subcloning and Screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parental cell. Subclones were screened in the same manner as primary clones, and in addition, culture supernatants of positive clones were subjected to an additional round of confirmatory screening using the β-arrestin hCCR8 CHO-K1 cell line in order to identify blocking antibodies.

[0168] 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.

[0169] Table 1. Antibody variable region sequences

[0170]

[0171]

[0172] Table 1A. CDR sequences (with optional mutations to avoid PTMs, Kabat numbering)

[0173]

[0174]

[0175] Bold / underlined residues indicate mutations made to avoid PTMs

[0176]

[0177] Sequence inspection revealed that antibodies 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4 and 115C5E3B8 were homologous to each other, and 163H9D5, 187B5F10, 195H8D10 and 200C9B9 were also homologous to each other. They are referred to as group A and group B, respectively, and their CDRs have the following consensus sequences.

[0178] Table 1B. Common CDR sequences of antibody groups

[0179]

[0180]

[0181] Example 2 Binding activity of anti-hCCR8 chimeric antibodies

[0182] To evaluate the binding activity of hybridoma clones 84D1-2H3, 86D4E12A5, 96G3-1F10, 99D1-1E11, 101D5G10G4, 115C5E3B8, 163H9D5, 187B5F10, 195H8D10 and 200C9B9, chimeric mAbs of these clones were subjected to FACS detection using HEK293 hCCR8 cell line, CHO-K1 cyno CCR8 cell line and CHO-K1 hCCR4 cell line.

[0183] Briefly, HEK293 hCCR8 cell line, CHO-K1 cyno CCR8 cell line or CHO-K1hCCR4 cell line were first incubated with 5-fold serial dilutions and 8 doses of the above 10 chimeric antibodies and hIgG1 control starting from 100 nM for 30 min at 4°C. After washing with FACS buffer, PE goat anti-human IgG Fc secondary antibody (eBioscience TM , Invitrogen) was added to each well and incubated at 4°C for 30 min. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was evaluated by MACSQuant Analyzer 16.

[0184] like Figure 1 As shown in Figure A, the anti-hCCR8 antibodies specifically bound to human CCR8 in a dose-dependent manner. Figure 1 As shown in Figure B, all anti-hCCR8 antibodies except 163H9D5 also bound to cynomolgus monkey CCR8, albeit to varying degrees. Figure 1 As shown in C, none of these antibodies bound to the closest family member, CCR4, further indicating that these antibodies are specific for CCR8.

[0185] Example 3 Anti-CCR8 chimeric antibody blocks hCCL1-induced signal transduction

[0186] It has been reported that the CCR8 ligand CCL1 is overexpressed and secreted into the tumor microenvironment by tumor-associated macrophages, cancer-associated fibroblasts, or other cell types. By activating its receptor CCR8, it causes proliferation, apoptosis resistance, and migration of immunosuppressive regulatory T cells, thereby further inhibiting anti-tumor immunity. Therefore, the ability of chimeric anti-hCCR8 antibodies to inhibit CCL1-mediated downstream signaling was tested in a cell-based assay.

[0187] In the β-arrestin assay, CCR8 is fused in-frame to the transcription factor Gal4 and linked via a protease site. Upon CCL1 binding, the β-arrestin fused to the protease is recruited to the intracellular end, and the tagged protease then cleaves the transcription factor, which translocates to the nucleus and activates the expression of Gal4-dependent luciferase. Briefly, β-arrestin hCCR8 CHO-K1 cells were plated at a density of 20,000 per well in a 96-well assay plate and incubated at 37°C, 5% CO2. After 24 hours of incubation, each chimeric antibody was serially diluted and added to the set rows of the assay plate. Human CCL1 (R&D system) was then added at a final concentration of 10 ng / ml and incubated for another 12-16 hours. The working detection solution (Promega's ONE-Glo TM luciferase assay system) was added to all wells of the assay plate, and the assay plate was incubated at room temperature in the dark for 3 minutes. The assay was performed on a multifunctional microplate reader ( 2105) to read the luminescence.

[0188] Data were plotted using GraphPad Prism software to obtain half-maximal inhibitory concentration (IC50) values ​​and are summarized in Figure 2 In A. Figure 2 As shown in A, all antibodies except 84D1-2H3 showed varying degrees of antagonism against hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells.

[0189] In addition, the blocking of hCCL1-mediated pathways by anti-hCCR8 monoclonal antibodies was tested by calcium (Ca2+) flux assay on ChemiScreenTM CCR8 chemokine receptor stable cell line (Chem1-hCCR8, Discover X), because hCCL1 induced calcium flux by engaging CCR8 on Chem1 cells. Chem-1hCCR8 cells were seeded in 384-well assay plates at a density of 5000 / well and cultured at 37°C, 5% CO2 for at least 18 hours. Dye working buffer (Screen QuestTM Fluo-8 No wash calcium assay kit, AAT Bioquest) and different anti-HCCR8 monoclonal antibodies were added to the cells and incubated at 37°C, 5% CO2 for 1 hour, followed by the addition of 19nM hCCL1 (R&D system). Ca2+ flux fluorescence signals were measured by Max-Min using FLIPRTM TETRA (Molecular Device). IC50 was calculated using Graphpad Prism.

[0190] like Figure 2 As shown in B, all antibodies except 84D1-2H3 showed varying degrees of antagonism on hCCL1-induced Ca2+ flux of hCCR8 in Chem1 cells, which was consistent with the β-arrestin assay.

[0191] In addition, chemotaxis assays were performed to evaluate the inhibitory activity of 86D4E12A5, 96G3-1F10, 187B5F10, 195H8D10, and 200C9B9 on their ability to block CCL1-induced migration of CCR8-expressing cells. BaF3 hCCR8 cell suspensions and diluted antibody samples were added to 96-well plates and incubated for 1 hour at 37°C, 5% CO2. Using a 5 μm pore size chemotaxis filter, a solution of hCCL1 (80 μl) at a concentration of 40 ng / ml was transferred to the corresponding wells at the bottom of the chemotaxis chamber and the assembly chamber. The cell / antibody mixture (100 μl) was then transferred to the top of the chamber and incubated at 37°C for 3 hours. Finally, the contents of the lower part of the chemotaxis chamber were transferred to a new 96-well plate, and 10 μL of resazurin was added to the plate. After incubation at 37°C for 24 hours, the reading was performed at 544 / 590 nM in a fluorescent microplate reader.

[0192] Figure 2 C shows that 86D4E12A5, 96G3-1F10, 187B5F10, 195H8D10, and 200C9B9 all inhibited the migration of BaF3 hCCR8 induced by hCCL1.

[0193] Example 4 Antibodies induce ADCC signaling in ADCC reporter gene bioassays

[0194] This example compares ADCC signaling of CCR8 antibodies in vitro.

[0195] In this experiment, HEK293hCCR8 cells were used as target cells, Jurkat cells stably expressing high affinity FcγRIIIa [CD16a (176V)] and a luciferase reporter driven by a NFAT response element were used as effector cells, and the antibodies tested were anti-hCCR8 antibodies with WT hIgG1Fc or hIgG1S239D / I332E Fc. When acting on target cells and effector cells simultaneously, the effector cells will transduce intracellular signals to produce NFAT-mediated luciferase, which can be quantified by a luminescence reader. In this experiment, target cells and effector cells were mixed at a 1:1 E:T ratio, increasing concentrations of antibodies were added, and incubated in a 96-well assay plate at 37°C, 5% CO2 for 6 hours. ADCC activity was determined by bioluminescence readings.

[0196] result( Figure 3 A) shows that chimeric anti-hCCR8 antibody with wild-type hIgG1 induced ADCC signaling in a dose-dependent manner.

[0197] like Figure 3 As shown in B, under the same experimental conditions, the chimeric anti-hCCR8 antibody with an enhanced Fc backbone (hIgG1 S239D / I332E) induced stronger ADCC signaling.

[0198] Example 5 Antibody-induced ADCC of NK92 CD16a as effector cells against HEK293 hCCR8 cells

[0199] Anti-hCCR8 antibodies with enhanced Fc (hIgG1 S239D / I332E) showed stronger ADCC reporter activity in the reporter assay, so their ability to mediate killing of target cells (HEK293hCCR8 cell line) by the natural killer cytotoxic cell line NK-92 overexpressing CD16a (176V) was tested.

[0200] This experiment uses ADCC was measured using the EuTDA cytotoxicity kit (PerkinElmer). Target cells (HEK293HCCR8 cells) were diluted to a density of 1,000,000 cells / ml with culture medium and labeled with a fluorescence-enhancing ligand for 25 minutes at 37°C. After extensive washing 3-5 times, the labeled target cells were mixed with NK92 CD16a (176V) cells at a ratio of 1:10 and a series of concentrations of anti-hCCR8 antibodies were added to microplates for co-culture. After 2 hours of incubation, 20 μL of the supernatant was transferred to a new plate, 200 μL of europium solution was added, and incubated for 15 minutes at room temperature using a DELFIA Plateshake. The signal correlated with the number of lysed cells.

[0201] like Figure 4 As shown, chimeric anti-hCCR8 antibodies (hIgG1S239D / I332E) with enhanced Fc function can induce ADCC effects against CCR8-positive cells by natural killer cell lines.

[0202] Example 6 Efficacy in MC38 tumor model

[0203] This example uses a syngeneic mouse model to test the in vivo anti-tumor efficacy of functional molecules. The Fc of the anti-CCR8 antibody was designed to be mIgG2a to mimic the stronger ADCC effect of human IgG1.

[0204] MC38 cells resuspended in PBS were subcutaneously (sc) inoculated into the right flank of B-hCCR8 humanized C57BL / 6 mice at a concentration of 5×105 cells in a volume of 0.1 ml. When the average tumor volume reached about 82 mm3, animals were randomly assigned to experimental groups according to tumor volume, with 7 animals in each group. The anti-CCR8 antibodies tested included 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). The antibodies tested were administered by intraperitoneal injection twice a week. Mouse body weight and tumor volume were measured twice a week.

[0205] like Figure 5 As shown, all anti-human CCR8 antibodies with enhanced ADCC activity showed significant tumor growth inhibition at the dose levels of 3 mg / kg and 6 mg / kg. Among these antibodies, 195H8D10 and 200C9B9 showed stronger anti-tumor activity.

[0206] Example 7 Humanization of hCCR8 Antibody

[0207] Humanized monoclonal antibodies were prepared using the variable region genes of antibodies 195H8D10 and 200C9B9. The amino acid sequences of VH and VK of 195H8D10 / 200C9B9 were compared with the available human Ig gene sequence database to find the best overall matching human germline Ig sequence.

[0208] For the light chain of 195H8D10, IGKV2-28*01 was the most suitable germline, and for the heavy chain of 195H8D10, IGHV3-72*01 was selected as the humanized framework. Then the humanized 195H8D10 CDR-grafted antibody was designed, in which CDR-L1, L2 and L3 were grafted onto the framework sequence of IGKV2-28*01, and CDR-H1, H2 and H3 were grafted onto the framework sequence of IGHV3-72*01. A 3D model was then generated to determine the amino acids in the original mouse framework region that are critical for antibody binding and conformation. Based on the analysis results, back mutations were performed on the grafted antibodies, thus, 4 additional humanized heavy chains and 4 additional light chains were generated.

[0209] For the light chain of 200C9B9, IGKV2-28*01 was the most suitable germline, and for the heavy chain of 200C9B9, IGHV3-72*01 was selected as the humanized framework. Then the humanized 200C9B9 CDR-grafted antibody was designed, in which CDRL1, L2 and L3 were grafted onto the framework sequence of IGKV2-28*01, and CDRH1, H2 and H3 were grafted onto the framework sequence of IGHV3-72*01. A 3D model was then generated to determine the amino acids in the original mouse FR region sequence that are critical for antibody binding and conformation. Based on the analysis results, back mutations were performed on the 200C9B9 CDR-grafted antibody, resulting in 4 additional humanized heavy chains and 4 additional light chains.

[0210] The resulting humanized sequences are listed in Tables 2-3.

[0211] Table 2. Humanization of 195H8D10

[0212]

[0213]

[0214] Table 2A. Humanized antibodies from 195H8D10

[0215]

[0216] Table 3. Humanization of 200C9B9

[0217]

[0218]

[0219] Table 3A. Humanized antibodies from 200C9B9

[0220]

[0221] Example 8 Binding activity of humanized antibodies

[0222] Flow cytometry was used to confirm the binding activity of the humanized antibodies using the CHO-K1 hCCR8 cell line and the CHO-K1 cyno CCR8 cell line.

[0223] Briefly, CHO-K1 hCCR8 cells and CHO-K1 cyno CCR8 cells were first incubated with different concentrations of humanized 195H8D10 and 200C9B9 antibodies at 4°C for 30 min. PE goat anti-human IgG Fc secondary antibody (eBioscience TM , Invitrogen) was added to each well and incubated at 4°C for 30 min. The samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was evaluated by MACSQuant Analyzer 16.

[0224] like Figure 6 As shown in A and B, the humanized 195H8D10 / 200C9B9 antibody is comparable to the corresponding chimeric antibody in human CCR8 binding.

[0225] also, Figure 6 C and D show that the humanized 195H8D10 / 200C9B9 antibodies except Hu195H8D10-2, Hu195H8D10-4, Hu200C9B9-2 and Hu200C9B9-4 are comparable to the corresponding chimeric antibodies in cynomolgus monkey CCR8 binding.

[0226] Example 9 Humanized Antibodies Induce ADCC Signaling in ADCC Reporter Gene Bioassay

[0227] This example compares ADCC signaling of humanized CCR8 antibodies in vitro.

[0228] In this experiment, as described in the previous example, CHO-K1 hCCR8 cells were used as target cells, Jurkat CD16a (176V) NFAT luciferase receptor cells were used as effector cells, and the antibodies tested were humanized 195H8D10 and 200C9B9 antibodies. This experiment was performed in a 96-well assay plate by incubating target cells, effector cells and test antibodies at 37°C, 5% CO2 for 6 hours according to the manufacturer's instructions. ADCC signal was determined by bioluminescence reading.

[0229] like Figure 7 As shown in A and B, the humanized 195H8D10 / 200C9B9 antibody induced ADCC signaling comparable to the corresponding chimeric antibody in an ADCC reporter gene assay.

[0230] Example 10 Humanized Antibodies Inhibit hCCL1-Induced β-Arrestin Recruitment

[0231] The humanized 195H8D10 / 200C9B9 antibody was tested again to confirm the antagonism of CCL1-mediated downstream signaling on CHO-K1hCCR8 cells.

[0232] 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 culture 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.

[0233] like Figure 8 As shown in A and B, all humanized 195H8D10 / 200C9B9 antibodies showed comparable levels of antagonism to the corresponding chimeric antibodies in the hCCL1-induced β-arrestin assay.

[0234] Example 11 PTM Elimination Confirmation of 195H8D10 and 200C9B9

[0235] It was observed that 195H8D10 and 200C9B9 VL CDR1 contained NG residues (Kabat numbering), which are at risk of post-translational modification (PTM), posing challenges to future production. Therefore, this example mutated NG on VL to QG or NA to prevent PTM. The sequences of potential PTM elimination sites are listed in Table 4.

[0236] Table 4. PTM elimination sites of 195H8D10 and 200C9B9

[0237]

[0238] like Fig. 9 As shown in Figure A, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 have comparable binding to the corresponding parental antibodies in human CCR8 binding.

[0239] like Fig. 9 As shown in B, 195H8D10-C2 and 200C9B9-C2 maintained the cynomolgus monkey binding ability compared to the corresponding antibodies.

[0240] like Fig. 9 As shown in FIG. 4C , in an ADCC reporter assay, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 induced ADCC signaling comparable to that of the corresponding parental antibodies.

[0241] like Fig. 9 As shown in D, in CHO-K1 cells, 195H8D10-C1 / C2 and 200C9B9-C1 / C2 showed comparable levels of antagonism to the corresponding parental antibodies in hCCL1-induced β-arrestin recruitment to hCCR8.

[0242] Therefore, this example confirms the sequences of humanized 195H8D10 and 200C9B9 that eliminate the potential PTM site (VL CDR1 NG-NA), and the sequences are listed in Table 5.

[0243] Table 5. Humanized 195H8D10 with elimination of potential PTM sites

[0244]

[0245] Table 5A. 195H8D10 humanized antibody with potential PTM sites eliminated

[0246]

[0247]

[0248] Table 6. Humanized 200C9B9 with elimination of potential PTM sites

[0249]

[0250] Table 6A. 200C9B9 humanized antibodies with PTM eliminated

[0251]

[0252]

[0253] Example 12 Confirmation of PTM elimination of humanized 195H8D10 and 200C9B9

[0254] To further confirm the performance of the humanized 195H8D10 / 200C9B9 antibody after elimination of PTM, the binding, ADCC activity, functional blocking activity, and tumor growth inhibitory effects of Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies were tested.

[0255] like Fig.10As shown in FIG. 8A , Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies are comparable to the corresponding parental antibodies in human CCR8 binding.

[0256] like Fig.10 As shown in FIG. 6B , Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies induced ADCC signaling comparable to the corresponding parental antibodies in an ADCC reporter assay.

[0257] like Fig.10 As shown in C, in CHO-K1 cells, Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies showed comparable levels of antagonism to the corresponding parental antibodies in hCCL1-induced recruitment of β-arrestin to hCCR8.

[0258] like Fig.10 As shown in D, hu195H8D10-7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies showed comparable levels of antagonism against hCCL1-induced Ca2+ flux in Chem1 cells, which is consistent with the β-arrestin assay.

[0259] In addition, a syngeneic mouse model was used to test the in vivo antitumor efficacy of Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies. The same mouse Fc was used for ADCC-mediated killing of regulatory T cells in B-hCCR8 mice. Briefly, MC38 cells resuspended in PBS were plated at 5 × 10 5 The cells were inoculated subcutaneously (sc) into the right flank of B-hCCR8 humanized C57BL / 6 mice. When the average tumor volume reached approximately 85 mm 3 At 1 , animals were randomly assigned to experimental groups according to tumor volume, with 7 animals in each group. Anti-CCR8 antibody (6 mg / kg) was the test antibody and was administered twice a week by intraperitoneal injection. Mouse body weight and tumor volume were measured twice a week.

[0260] like Fig.11 As shown, Hu195H8D10-1 / 7NG-NA and Hu200C9B9-3 / 7NG-NA antibodies with enhanced ADCC activity exhibited significant tumor growth inhibition (TGI). Among these antibodies, Hu200C9B9-7 NG-NA monotherapy showed stronger inhibition of tumor growth, with an inhibition rate of about 70% at the end of the study.

[0261] Example 13 Comparison of Hu200C9B9-7 NG-NA and Benchmark Antibodies

[0262] 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.

[0263] All CCR8 antibodies were fused with the same Fc (human IgG1κ). Binding activity, ADCC activity, and functional blocking activity were compared in vitro. Fig.12 As shown in A, hu200C9B9-7NG-NA is a strong binder of hCCR8, with binding capacity comparable to its benchmark determined by FACS. To determine specificity, binding assays were performed based on parental HEK293 without target. Results ( Fig.12 B) shows that Hu200C9B9-7 NG-NA does not bind to parental HEK293 at all, while benchmark antibodies developed by Gilead and BMS have a certain degree of non-specific binding to parental HEK293 cells.

[0264] ADCC signaling of Hu200C9B9-7 NG-NA and benchmark was further compared as described previously in the Examples. Fig.12 C shows that all anti-hCCR8 antibodies with wild-type hIgG1 induced ADCC signaling in a dose-dependent manner in HEK293HCCR8 and Jurkat CD16a(176V)NFAT-luciferase cell co-culture assays.

[0265] In addition, Hu200C9B9-7NG-NA and benchmark antibodies were tested for blockade of hCCL1-mediated pathways by performing β-arrestin recruitment assays and calcium (Ca2+) flux assays. Hu200C9B9-7NG-NA showed comparable levels of antagonism to benchmark antibodies in hCCL1-induced β-arrestin recruitment to hCCR8 in CHO-K1 cells ( Fig.12 D), while Hu200C9B9-7NG-NA could inhibit hCCL1-mediated Ca2+ flux in Chem-1hCCR8 cells better than the two benchmark antibodies ( Fig.12 E).

[0266] The preclinical efficacy of hu200C9B9-7NG-NA and benchmark was evaluated in a syngeneic mouse model in which the murine CCR8 gene was replaced by its human counterpart and MC38 was vaccinated as described in the previous example. Using the same mouse Fc, hu200C9B9-7NG-NA, 1-K17.044, and 4A19 monotherapy all showed significant inhibition of tumor growth, with inhibition rates of 61.2%, 53.2%, and 59.1%, respectively ( Fig.13 A). Ex vivo analysis showed that all of these antibodies effectively reduced CCR8+ regulatory T cells and increased mCD45+ lymphocytes at the tumor site ( Fig.13 B), while retaining peripheral regulatory T cell subsets and mCD45+ lymphocytes ( Fig.13 C).

[0267] Example 14 Fc selection of Hu200C9B9-7 NG-NA

[0268] Fc engineering is a promising approach to enhance the ADCC effect for better antitumor efficacy of monoclonal antibodies (mAbs).

[0269] To find the optimal ADCC enhancement method for therapeutic antibodies, Fc-DLE (triple mutation S239D / A330L / I332E introduced into two Fc chains), Fc-DE (double mutation S239D / I332E introduced into two Fc chains), Fc-afucosylated (Fc-Afucosylated), Fc-asymmetric (i.e., different substitutions were introduced into 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 (Fc-WT) antibodies were prepared. The binding affinity of these Fc variants or carbohydrate mutant Fc to FcγRIIIa isotype and inhibitory FcγRIIIb was evaluated with hIgG1 antibodies, and the ratio of activating FcγR binding to inhibitory FcγR binding (A / I) was calculated.

[0270] The binding of antibodies with different Fc to recombinant hCD16a-176V, hCD16a-176F and hCD32b proteins was tested with Biacore using the capture method. Proteins were captured using a CM5-anti-His chip. Serial dilutions of antibodies were injected on 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. It was found that the Fc variant with the DLE mutation had high affinity for both CD16a isotypes, while the affinity for CD32b was the lowest. Therefore, the Fc variant with the DLE mutation achieved the highest A / I ratio.

[0271] Table 7. Affinity and A / I ratios measured by Biacore

[0272]

[0273]

[0274] To further evaluate the ability of CCR8 antibodies in ADCC signaling, anti-CCR8 antibodies with Fc-DLE, Fc-DE, Fc-asymmetric, Fc-WT (using the same Fab portion) were compared. ADCC activity was determined by incubation of JurkathCD16a NFAT or Jurkat hCD16a hCD32b NFAT effector cells with CHO-K1 hCCR8 cells. Results ( Fig.14 A) shows that anti-hCCR8 antibodies with Fc-DLE, Fc-DE, Fc-non-fucosylation, and Fc-asymmetry induce potent ADCC activity compared to WT hIgG1. In a co-culture system of Jurkat hCD16a hCD32b cells expressing inhibitory receptors, anti-hCCR8 antibodies with Fc-DLE, Fc-non-fucosylation, and Fc-asymmetry exhibited stronger ADCC activity compared to Fc-DE and WT hIgG1. This result is consistent with the A / I ratio ( Fig.14 B).

[0275] In addition, antibody-mediated killing of HEK293 cells expressing CCR8 was tested. PBMCs from healthy donors co-incubated with HEK293 hCCR8 cells (PBMC to target cell ratio of 50:1) were treated with serially diluted anti-CCR8 antibodies with Fc-DLE, Fc-DE, Fc-non-fucosylated, Fc-WT and hIgG1 DLE controls at 37°C overnight. Cell lysis was determined by the level of LDH released in the supernatant after co-culture. The results showed that anti-hCCR8 antibodies with engineered Fc, especially anti-hCCR8 antibodies with DLE mutation, mediated the most effective killing of CCR8-expressing target cells ( Fig.14 C).

[0276] The present disclosure is not limited to the scope of the specific embodiments described, which are intended to be a single illustration of the various 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 may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, it is intended that modifications and variations of the present disclosure be encompassed as long as they are within the scope of the appended claims and their equivalents.

[0277] 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, which is specific for human C-C motif chemokine receptor 8 (CCR8) protein and 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, wherein: The VH CDR1 comprises the amino acid sequence shown in SEQ ID NO:22; the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:58; the VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:35, 36, 37 or 38; the VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:59; the VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:49, 50 or 51; and the VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:

60.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, VHCDR3, VL CDR1, VL CDR2 and VL CDR3 comprise the following amino acid sequences, 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; NO: 22, 30, 38, 44, 51 and 56; or SEQ ID NO: 22, 71, 38, 44, 51 and 56.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the VH and VL comprise the following amino acid sequences, respectively: 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.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The antibody or its antigen-binding fragment 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; preferably, the antibody or its antigen-binding fragment is humanized; more preferably, it comprises an Fc fragment with enhanced antibody-dependent cellular cytotoxicity (ADCC).

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein the Fc fragment is a human IgG1 fragment having one or more substitutions according to EU numbering 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.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the IgG1 Fc fragment: (a) comprises substitutions of S239D, A330L and I332E; (b) comprises substitutions of S239D and I332E; (c) is non-fucosylated; or (d) comprises one or more substitutions of L234Y, L235Q, G236W, S239M, H268D, D270E and S298A in one Fc chain and one or more substitutions of D270E, K326D, A330M and K334E in a second Fc chain.

7. A multispecific antibody comprising the antigen-binding fragment of any one of claims 1 to 6 and one or more antibodies or antigen-binding fragments having binding specificity to a target antigen other than CCR8.

8. One or more polynucleotides encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. A cell comprising the polynucleotide of claim 8.

10. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

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