An anti-CCR8 antibody or its antigen-binding fragment

Abstract

The application discloses an anti-CCR8 antibody or an antigen binding fragment thereof. The anti-CCR8 antibody of the application can specifically recognize a human CCR8 protein, can block the signal pathway of CCR8 and CCL1 / CCL18, can specifically recognize a cell expressing CCR8, and has target-specific ADCC activity on a target cell expressing CCR8.

Description

Technical Field

[0001] This invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to an anti-CCR8 antibody or its antigen-binding fragment. Background Technology

[0002] Chemokine receptors are seven-transmembrane G protein-coupled receptors (GPCRs) expressed on the surface of certain cells. These receptors bind to extracellular ligands that act as chemokines, triggering a calcium ion influx and inducing a chemotactic response that directs cells to specific sites within the organism. The binding of CCR8 and its ligands CCL1 / CCL18 positively feedback-regulates the immunosuppressive function of the tumor microenvironment.

[0003] CCR8 (chemokine (CC motif) receptor 8) is a chemokine receptor highly expressed in tumor-infiltrating Tregs. Structurally similar to G protein-coupled receptors, it is a seven-transmembrane protein. CCR8 is specifically expressed on tumor-infiltrating regulatory T cells (Tregs) but is largely absent on peripheral blood Tregs, regulating the immunosuppressive function of the tumor microenvironment. CCR8 activates its receptor on cancer cells, inducing proliferation, migration, and apoptosis resistance. Additionally, CCL1 can recruit Tregs to the tumor niche and lead to the transformation of CD4+ T cells into Tregs.

[0004] There are four known receptors for CCR8: CCL1, CCL8, CCL16, and CCL18. Among them, the major receptor—the chemokine CCL1—is upregulated at sites of inflammation. CCL1 can recruit FOXp3+CCR8+ Treg cells to infiltrate tumor tissues, exerting immunosuppressive functions. Simultaneously, it can induce upregulation of CCR8 expression on the surface of FOXp3+ Treg cells, triggering cancer cell proliferation. 2+ The flow of CCR8 induces stat3-dependent upregulation of Foxp3, CD39, IL-10, and granzyme B expression, thereby enhancing the immunosuppressive activity of these tumor-infiltrating Treg cells. Therefore, drugs targeting CCR8 can enhance anti-tumor immune responses by depleting tumor-infiltrating FOXp3+CCR8+ Treg cells or by blocking the CCL1 / CCR8 pathway. Summary of the Invention

[0005] The anti-CCR8 antibody developed in this invention can specifically recognize human CCR8 protein, block the signaling pathway between CCR8 and CCL1 / CCL18, specifically recognize cells expressing CCR8, and has target-specific ADCC activity against target cells expressing CCR8.

[0006] The technical solution adopted in this invention is as follows:

[0007] An anti-CCR8 antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and said light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein...

[0008] (a) HCDR1 of the heavy chain variable region is selected from any one of the amino acid sequences of SEQ ID NO: 2, 10, 18, 26, 41, 45, 49, 53, or selected from a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any one of the amino acid sequences of SEQ ID NO: 2, 10, 18, 26, 41, 45, 49, 53, or selected from an amino acid sequence having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of the amino acid sequences of SEQ ID NO: 2, 10, 18, 26, 41, 45, 49, 53;

[0009] (b) The HCDR2 of the heavy chain variable region is selected from any one of the amino acid sequences of SEQ ID NO: 3, 11, 19, 27, 42, 46, 50, 54, or selected from a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any one of the amino acid sequences of SEQ ID NO: 3, 11, 19, 27, 42, 46, 50, 54, or selected from an amino acid sequence having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of the amino acid sequences of SEQ ID NO: 3, 11, 19, 27, 42, 46, 50, 54;

[0010] (c) HCDR3 of the heavy chain variable region is selected from any one of the amino acid sequences of SEQ ID NO: 4, 12, 20, 28, 43, 47, 51, 55, or selected from a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with any one of the amino acid sequences of SEQ ID NO: 4, 12, 20, 28, 43, 47, 51, 55, or selected from an amino acid sequence having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to any one of the amino acid sequences of SEQ ID NO: 4, 12, 20, 28, 43, 47, 51, 55;

[0011] (d) The LCDR1 of the light chain variable region is selected from the amino acid sequences of SEQ ID NO: 6, 14, 22, 30, 57, 61, 65, 69, 73, or selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with the amino acid sequences of SEQ ID NO: 6, 14, 22, 30, 57, 61, 65, 69, 73, or selected from amino acid sequences having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences of SEQ ID NO: 6, 14, 22, 30, 57, 61, 65, 69, 73;

[0012] (e) The LCDR2 of the light chain variable region is selected from the amino acid sequences of SEQ ID NO: 7, 15, 23, 31, 58, 62, 66, 70, 74, or selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the amino acid sequences of SEQ ID NO: 7, 15, 23, 31, 58, 62, 66, 70, 74, or selected from amino acid sequences having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences of SEQ ID NO: 7, 15, 23, 31, 58, 62, 66, 70, 74; and / or

[0013] (f) The LCDR3 of the light chain variable region is selected from the amino acid sequences of SEQ ID NO: 8, 16, 24, 32, 59, 63, 67, 71, 75, or selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity with the amino acid sequences of SEQ ID NO: 8, 16, 24, 32, 59, 63, 67, 71, 75, or selected from amino acid sequences having one or more (preferably two or three) conserved amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences of SEQ ID NO: 8, 16, 24, 32, 59, 63, 67, 71, 75.

[0014] In an optional embodiment, the anti-CCR8 antibody or its antigen-binding fragment heavy chain variable region HCDR1, HCDR2, HCDR3, and light chain variable region LCDR1, LCDR2, LCDR3 are selected from any one of the following amino acid sequences (1)-(14):

[0015] (1) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 3, HCDR3 shown in SEQ ID NO: 4, LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8;

[0016] (2) HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 15, and LCDR3 shown in SEQ ID NO: 16;

[0017] (3) HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 22, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24;

[0018] (4) HCDR1 shown in SEQ ID NO: 26, HCDR2 shown in SEQ ID NO: 27, HCDR3 shown in SEQ ID NO: 28, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, and LCDR3 shown in SEQ ID NO: 32;

[0019] (5) HCDR1 shown in SEQ ID NO: 41, HCDR2 shown in SEQ ID NO: 42, HCDR3 shown in SEQ ID NO: 43, LCDR1 shown in SEQ ID NO: 57, LCDR2 shown in SEQ ID NO: 58, and LCDR3 shown in SEQ ID NO: 59;

[0020] (6) HCDR1 shown in SEQ ID NO: 41, HCDR2 shown in SEQ ID NO: 42, HCDR3 shown in SEQ ID NO: 43, LCDR1 shown in SEQ ID NO: 61, LCDR2 shown in SEQ ID NO: 62, and LCDR3 shown in SEQ ID NO: 63;

[0021] (7) HCDR1 shown in SEQ ID NO: 41, HCDR2 shown in SEQ ID NO: 42, HCDR3 shown in SEQ ID NO: 43, LCDR1 shown in SEQ ID NO: 65, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO: 67;

[0022] (8) HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, HCDR3 shown in SEQ ID NO: 47, LCDR1 shown in SEQ ID NO: 57, LCDR2 shown in SEQ ID NO: 58, and LCDR3 shown in SEQ ID NO: 59;

[0023] (9) HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, HCDR3 shown in SEQ ID NO: 47, LCDR1 shown in SEQ ID NO: 61, LCDR2 shown in SEQ ID NO: 62, and LCDR3 shown in SEQ ID NO: 63;

[0024] (10) HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, HCDR3 shown in SEQ ID NO: 47, LCDR1 shown in SEQ ID NO: 65, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO: 67;

[0025] (11) HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, HCDR3 shown in SEQ ID NO: 47, LCDR1 shown in SEQ ID NO: 69, LCDR2 shown in SEQ ID NO: 70, and LCDR3 shown in SEQ ID NO: 71;

[0026] (12) HCDR1 shown in SEQ ID NO: 45, HCDR2 shown in SEQ ID NO: 46, HCDR3 shown in SEQ ID NO: 47, LCDR1 shown in SEQ ID NO: 73, LCDR2 shown in SEQ ID NO: 74, and LCDR3 shown in SEQ ID NO: 75;

[0027] (13) HCDR1 shown in SEQ ID NO: 49, HCDR2 shown in SEQ ID NO: 50, HCDR3 shown in SEQ ID NO: 51, LCDR1 shown in SEQ ID NO: 65, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO: 67;

[0028] (14) HCDR1 shown in SEQ ID NO: 53, HCDR2 shown in SEQ ID NO: 54, HCDR3 shown in SEQ ID NO: 55, LCDR1 shown in SEQ ID NO: 65, LCDR2 shown in SEQ ID NO: 66, and LCDR3 shown in SEQ ID NO: 67.

[0029] In an optional embodiment, the anti-CCR8 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein,

[0030] (a) The heavy chain variable region has any of the amino acid sequences given in SEQ ID NO: 1, 9, 17, 25, 33, 34, 35, 36, 40, 44, 48, 52.

[0031] Or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity with the amino acid sequences given in SEQ ID NO: 1, 9, 17, 25, 33, 34, 35, 36, 40, 44, 48, 52.

[0032] Or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to any of the amino acid sequences of SEQ ID NO: 1, 9, 17, 25, 33, 34, 35, 36, 40, 44, 48, 52;

[0033] (b) The light chain variable region has any of the amino acid sequences given in SEQ ID NO: 5, 13, 21, 29, 37, 38, 39, 56, 60, 64, 68, 72.

[0034] Or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or more identity with the amino acid sequences given in SEQ ID NO: 5, 13, 21, 29, 37, 38, 39, 56, 60, 64, 68, 72.

[0035] Or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to any of the amino acid sequences of SEQ ID NO: 5, 13, 21, 29, 37, 38, 39, 56, 60, 64, 68, 72.

[0036] In an optional embodiment, the heavy chain variable region and light chain variable region of the anti-CCR8 antibody or its antigen-binding fragment are selected from any of the following amino acid sequences (1)-(26):

[0037] (1) SEQ ID NO: 1 and SEQ ID NO: 5;

[0038] (2) SEQ ID NO: 9 and SEQ ID NO: 13;

[0039] (3) SEQ ID NO: 17 and SEQ ID NO: 21;

[0040] (4) SEQ ID NO: 25 and SEQ ID NO: 29;

[0041] (5) SEQ ID NO: 33 and SEQ ID NO: 37;

[0042] (6) SEQ ID NO: 33 and SEQ ID NO: 38;

[0043] (7) SEQ ID NO: 33 and SEQ ID NO: 39;

[0044] (8) SEQ ID NO: 34 and SEQ ID NO: 37;

[0045] (9) SEQ ID NO: 34 and SEQ ID NO: 38;

[0046] (10) SEQ ID NO: 34 and SEQ ID NO: 39;

[0047] (11) SEQ ID NO: 35 and SEQ ID NO: 37;

[0048] (12) SEQ ID NO: 35 and SEQ ID NO: 38;

[0049] (13) SEQ ID NO: 35 and SEQ ID NO: 39;

[0050] (14) SEQ ID NO: 36 and SEQ ID NO: 37;

[0051] (15) SEQ ID NO: 36 and SEQ ID NO: 38;

[0052] (16) SEQ ID NO: 36 and SEQ ID NO: 39;

[0053] (17) SEQ ID NO: 40 and SEQ ID NO: 56;

[0054] (18) SEQ ID NO: 40 and SEQ ID NO: 60;

[0055] (19) SEQ ID NO: 40 and SEQ ID NO: 64;

[0056] (20) SEQ ID NO: 44 and SEQ ID NO: 56;

[0057] (21) SEQ ID NO: 44 and SEQ ID NO: 60;

[0058] (22) SEQ ID NO: 44 and SEQ ID NO: 64;

[0059] (23) SEQ ID NO: 44 and SEQ ID NO: 68;

[0060] (24) SEQ ID NO: 44 and SEQ ID NO: 72;

[0061] (25) SEQ ID NO: 48 and SEQ ID NO: 64;

[0062] (26) SEQ ID NO: 52 and SEQ ID NO: 64.

[0063] In an optional implementation, the antibody or its antigen-binding fragment includes a murine antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, and / or a humanized antibody or its antigen-binding fragment.

[0064] In an optional embodiment, the anti-CCR8 antibody or its antigen-binding fragment further comprises an Fc region selected from mouse IgG1, IgG2a, IgG2b and / or IgG3, or rat IgG1, IgG2a, IgG2b and / or IgG2c.

[0065] In an optional embodiment, the anti-CCR8 antibody or its antigen-binding fragment further comprises an Fc region, wherein the Fc region is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence having one or more amino acid mutations (preferably substitutions, insertions, or deletions) with human IgG1, IgG2, IgG3, and IgG4. In an optional embodiment, the Fc region can be modified, for example, in a Knob-in-Hole manner.

[0066] The present invention also provides a nucleic acid molecule encoding any of the anti-CCR8 antibodies described above or their antigen-binding fragments.

[0067] The present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecules.

[0068] The present invention also provides recombinant cells comprising the above-described nucleic acid molecules and / or the above-described recombinant vector, and capable of expressing the anti-CCR8 antibody or its antigen-binding fragment.

[0069] The present invention also provides a multifunctional fusion protein comprising any of the anti-CCR8 antibodies described above or their antigen-binding fragments.

[0070] In an optional embodiment, the multifunctional fusion protein further comprises one or more second antibodies or their antigen-binding moieties that specifically bind to other antigens.

[0071] In an optional implementation, the antigen that binds to the second antibody or its antigen-binding portion is selected from tumor-associated antigens (TAAs) or immune checkpoints.

[0072] In an optional embodiment, the use of any of the above-described anti-CCR8 antibodies or their antigen-binding fragments, or any of the described multifunctional fusion proteins, in the preparation of medicaments for treating and / or preventing and / or diagnosing diseases.

[0073] In an alternative implementation, the use is achieved through one or more of tumor immunotherapy, cell therapy, and gene therapy.

[0074] In optional embodiments, the use of any of the above-described anti-CCR8 antibodies or their antigen-binding fragments, or any of the described multifunctional fusion proteins, in the preparation of medicaments for treating cancer.

[0075] In an optional implementation, the cancer is breast cancer, ovarian cancer, lung cancer, liver cancer, melanoma, malignant glioma, head and neck cancer, colorectal cancer, stomach cancer, bladder cancer, pancreatic cancer, colon cancer, cervical cancer, or related tumors.

[0076] The present invention also provides a pharmaceutical composition comprising any of the anti-CCR8 antibodies described above or their antigen-binding fragments and an acceptable carrier, diluent or excipient.

[0077] The present invention also provides a pharmaceutical composition comprising any of the multifunctional fusion proteins described above and an acceptable carrier, diluent or excipient.

[0078] Beneficial effects

[0079] The anti-CCR8 antibody developed in this invention can specifically recognize human CCR8 protein, block the signaling pathway between CCR8 and CCL1 / CCL18, specifically recognize cells expressing CCR8, and has target-specific ADCC activity against target cells expressing CCR8.

[0080] the term

[0081] To aid in understanding the invention described herein, the following explanations of abbreviations and definitions of terms are provided.

[0082] The following abbreviations are used in this article:

[0083] CDR: Complementation-determining region in the antibody variable region;

[0084] HCDR: Complementarity-determining region in the variable region of the antibody heavy chain;

[0085] LCDR: Complementation-determining region in the variable region of the antibody light chain;

[0086] FR: Antibody framework region, which is the amino acid residue in the antibody variable region other than CDR residues;

[0087] ELISA: Enzyme-linked immunosorbent assay;

[0088] FACS: Fluorescence-activated cell sorting.

[0089] In this specification, the term "antibody" refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis. Antibodies can be isolated from natural resources such as plasma or serum where the antibody is naturally present, or from the culture supernatant of antibody-producing hybridoma cells, animal immune serum, or by phage library screening. Alternatively, they can be partially or completely synthesized using techniques such as gene recombination. Preferred antibodies include, for example, antibodies to isotypes or subtypes of immunoglobulins. Human immunoglobulins are known to include nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgG1, IgA2, IgD, IgE, and IgM. Among these isotypes, the antibodies of the present invention may include IgG1, IgG2, IgG3, and / or IgG4.

[0090] The terms "antibody" and "immunoglobulin" are used interchangeably. The antibodies used in this article are immunoglobulin molecules composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding sites.

[0091] The term "antigen-binding fragment" in the context of an antibody refers to a polypeptide fragment of the antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; this fragment is also referred to as the "antigen-binding moiety." Antigen-binding fragments of antibodies can be generated through recombinant DNA technology or through enzymatic or chemical cleavage of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, linear antibodies, nanobodies (such as those from Ablynx), domain antibodies (such as those from Domantis), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0092] The term "peptide" refers to an amino acid chain of any length, regardless of modifications such as phosphorylation or glycosylation. The term peptide includes proteins and fragments thereof. Peptides can be "exogenous," meaning they are "heterogeneous," i.e., derived from a host cell, such as human peptides produced by bacterial cells. Peptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right, from the amino terminus to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named with three-letter or single-letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0093] Several methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the location of structural loop regions; the IMGT system defines CDRs based on sequence variability and location within variable domain structures; AbM is defined based on Oxford Molecular's AbM antibody modeling software and represents a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in several ways.

[0094] The amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDR) in the anti-CCR8 antibody of the present invention are numbered using the Kabat system.

[0095] Regarding the percentage (%) amino acid sequence identity of the reference polypeptide sequence, it is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after alignment and, where necessary, nicking to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package.

[0096] The term "monoclonal antibody" refers to a homogeneous antibody that targets only a specific antigenic epitope. In contrast to conventional polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody targets a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the homogeneous nature of the antibody and is not interpreted as requiring the antibody to be produced by any particular method. The monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.

[0097] The term "mouse antibody" in this invention refers to a monoclonal antibody prepared in accordance with the knowledge and skills in the art. In some embodiments, preparation involves injecting the test subject with an antigen, followed by isolating a hybridoma expressing an antibody having the desired sequence or functional characteristics. In some embodiments, the desired mouse antibody is obtained by screening a mouse immune library.

[0098] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. In some implementations, the creation of a chimeric antibody involves first establishing a hybridoma that secretes a murine-specific monoclonal antibody, then cloning the variable region gene from mouse hybridoma cells, followed by cloning the constant region gene of the human antibody as needed, linking the mouse variable region gene and the human constant region gene to form a chimeric gene, inserting it into a human vector, and finally expressing the chimeric antibody molecule in a eukaryotic or prokaryotic industrial system.

[0099] The term "humanized antibody" refers to an antibody comprising at least one humanized antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized antibody chain" (i.e., "humanized immunoglobulin chain") refers to an antibody chain having variable regions (i.e., light or heavy chains, respectively), said variable regions comprising substantially variable framework regions of human antibodies and complementarity-determining regions substantially derived from non-human antibodies (e.g., at least one CDR, two CDRs, or three CDRs). In some embodiments, the humanized antibody chain also includes constant regions (e.g., one constant region or a portion thereof in the case of the light chain, and preferably three constant regions in the case of the heavy chain).

[0100] The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence to express the selected target gene. This term includes the offspring of the parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the selected target gene is present in the offspring. Commonly used host cells include bacteria, yeast, and mammalian cells.

[0101] The term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell to which they are introduced. Some vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Attached Figure Description

[0102] Figure 1a and 1b The binding activity of murine anti-CCR8 antibody to CHOK1-CCR8 cells was measured.

[0103] Figure 2 To assess the binding activity of chimeric anti-CCR8 antibody to CHOK1-CCR8 cells.

[0104] Figure 3 The results show the ADCC effect of the chimeric anti-CCR8 antibody.

[0105] Figure 4 The murine anti-CCR8 antibody inhibited the release of β-arestin from CCL1-activated Tango-H_CCR8-CHO-K1 cells.

[0106] Figure 5 This provides in vivo efficacy data for murine anti-CCR8 antibodies.

[0107] Figure 6a and 6b The binding activity of humanized anti-CCR8 antibody to CHOK1-CCR8 cells.

[0108] Figure 7a and 7b The results of ADCC effect of humanized anti-CCR8 antibody.

[0109] Figure 8 The humanized anti-CCR8 antibody inhibited the release of β-arestin from CCL1-activated Tango-H_CCR8-CHO-K1 cells. Detailed Implementation

[0110] Example 1: Animal Immunization

[0111] CCR8-CHO-K1 cells overexpressing human CCR8 were constructed using lentiviral transduction of CHO cells. CCR8-284 cells were used as the immunogen, and 1×10⁻⁶ cells were injected. 7Balb / c mice were immunized with total cells. A second immunization was administered half a month after the first, followed by immunizations every month thereafter. Negative serum was collected from mice 3 days before immunization, and 50 μL of blood was collected by tail clipping 6 days after each immunization. Negative and immune sera were diluted proportionally (1:0.1K, 1:1K, 1:10K, 1:100K, 1:1000K, 1:1000K, 1:10000K) and serum titers were determined using Cell-ELISA with CCR8-CHO-K1 cells overexpressing CCR8. When the titer met the requirements and anti-human CCR8 antibodies were detected at dilutions >1:10K, rat spleens and lymph nodes were harvested.

[0112] Example 2: Cell Fusion

[0113] B lymphocytes and lymph node cells used in the experiment were obtained from Balb / c mice immunized four times with CCR8-293 cells. The spleen and lymph nodes were placed in a cell strainer, which was then placed in a 50 mL centrifuge tube. DMEM was added to the spleen, and the cells were ground to prepare a spleen cell suspension. The suspension was centrifuged at 1600 rpm for 10 minutes, and the supernatant was discarded. B cells were resuspended in 2 mL of erythrocyte lysis buffer, lysed at room temperature for 2 minutes, and then 30 mL of DMEM was added. After mixing, the cells were centrifuged at 1600 rpm for 10 minutes, and the cells were counted.

[0114] Myeloma cells SP2 / 0 (ATCC) were passaged one day prior to fusion to ensure they were in logarithmic growth phase at the time of the experiment. Spleen cells and SP2 / 0 cells were mixed at a 2:1 ratio and centrifuged at 1600 rpm for 10 minutes. The mixed cells were washed twice with fusion buffer and centrifuged at 1600 rpm for 10 minutes. The final cell density was 1 × 10⁻⁶ cells / mL. 7 Cells / mL were added to suspend the cells in fusion buffer. Within 5 minutes, the cell suspension was transferred to the fusion chamber of an electrofusion apparatus (BTX; ECM 2001) for fusion. After fusion, the cells were transferred from the fusion chamber to complete culture medium containing HAT and incubated at 37°C for 60 minutes. After incubation, the cells were seeded into 96-well plates containing feeder cells and cultured at 37°C with 5% CO2.

[0115] Example 3: Preliminary screening of positive clones using ELISA method

[0116] After 7 days of culture, the fusion supernatant was used for initial screening. CCR8-CHO-K1 cells were seeded into 96 μL ELISA plates (1.5 × 10⁻⁶). 4Cells / well were cultured for 36 hours, then hand-washed twice with PBST. The cells were fixed with 4% paraformaldehyde and blocked with 2% BSA. The blocking solution was discarded, and the plate was washed three times. 100 μL / well of the fusion supernatant was added to the blocked plate and incubated at 37°C for 1 hour. The liquid in the wells was discarded. The plate was washed three times. 100 μL / well of goat anti-mouse secondary antibody-HRP (Abcam; Ab6789) was diluted with 0.5% BSA and incubated at 37°C. The plate was washed six times with PBST and patted dry on a plate. 100 μL / well of Solarbio chromogenic solution (PR1200) was added to the wells. The plate was wrapped in aluminum foil and incubated at 37°C in the dark. The colorimetric reaction was terminated by adding 1 mol / L HCl. The readings were taken at 450 nm using a microplate reader, and the data were analyzed. Cell lines with OD450 > 1.0 in the supernatant were selected as candidate positive cell lines for initial screening. The culture supernatant of the positive cell lines was discarded and fresh HAT complete medium was added.

[0117] Example 4: FACS method for further screening of positive clones

[0118] Transfer CCR8-CHO-K1 cells to centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Then, add 100 μL of 3 × 10⁻⁶ cells to each tube. 5 Stable expressing cells were aliquoted into individual tubes, and 100 μL of fusion supernatant was added. Cells were incubated at 4°C for 60 min, followed by two washes with excess FACS buffer. Cells were resuspended in 100 μL of FACS buffer, and goat anti-mouse secondary antibody-FITC (Abcam; ab6785) was added to the sample. Incubation was performed for 30 min, followed by two washes with excess FACS buffer. Cells were fixed in fixation buffer and then analyzed by flow cytometry. Antibodies specifically binding to CCR8-CHO-K1 cells were screened using the FACS method.

[0119] Hybridoma cells were monocloned using a two-round limiting dilution method. ELISA was used to detect the cells, and monoclonal cells with OD450 > 1.0 were selected as candidate cell lines for passage. For clones without monoclonal antibodies, clones with OD450 > 1.0 were selected for the next subcloning.

[0120] Example 5: Small-scale antibody production from candidate cell lines

[0121] Hybridoma cells were cultured in T75 cell culture flasks until cell coverage reached 80-90%. The supernatant from both flasks was discarded, and 30 mL of hybridoma-SFM was added. The cells were incubated at 37°C with 5% CO2. After 2-3 days of culture, the cell status and culture medium color were observed. If the medium turned yellow, 30 mL of fresh hybridoma-SFM could be added. After 6-7 days of culture, the culture supernatant was collected by low-speed centrifugation and purified.

[0122] Example 6: Binding activity of candidate antibodies to cells

[0123] The antibody to be tested was initially and initially diluted 5-fold at a total concentration of 50 μg / mL, resulting in 6 dilutions. CCR8-CHO-K1 cells were removed from the incubator, and the cell suspension was transferred to 15 mL centrifuge tubes. After centrifugation, the cells were resuspended in PBS and counted. Blank control, negative control (NC), experimental group, serotonin group (serotonin BMK derived from the 1K17 sequence of US20210277129), and irrelevant antibody group were prepared. The cells were then diluted approximately 3 × 10⁻⁶ times. 5 Cells per well were used to spread the cell suspension in a 96-well plate.

[0124] After centrifugation (1000 rpm, 5 minutes), wash with PBS, then centrifuge again, repeating twice to remove residual culture medium. Discard the supernatant. Add 100 μL of primary antibody solution and irrelevant antibody solution to the experimental group and the irrelevant antibody group, respectively. Resuspend the cells and incubate at room temperature for 1 hour. The blank control group and the negative control group are incubated with an equal volume of PBS.

[0125] Centrifuge after 1 hour, wash twice with PBS. After discarding the supernatant, except for the blank control group which was added with 100 μL PBS, add 100 μL of fluorescent secondary antibody dilution buffer (mouse secondary antibody from Abcam ab6785) to each of the other sample groups. Incubate at room temperature in the dark for 0.5 hours, centrifuge, wash twice with PBS. After discarding the supernatant, resuspend in 120 μL PBS and perform flow cytometry to detect the average fluorescence intensity. The antibody concentration was logarithmized and used as the x-axis. A nonlinear regression was performed using the Sigmoidal dose-response (Variable Slope) method (GraphPadPrism software, GraphPad Software, San Diego, California) to obtain the antibody binding activity curve for CCR8-CHO cells. Results are shown below. Figure 1a and Figure 1b .

[0126] Depend on Figure 1a and Figure 1b It can be seen that the selected antibodies Ms-1, Ms-2, Ms-3, Ms-4, Ms-5, Ms-6, Ms-7, Ms-8, Ms-9, Ms-10, Ms-11, Ms-12, Ms-13, Ms-14 and Ms-15 all have good binding activity with CCR8-CHO, and the binding activity is better than that of positive ginseng and far better than that of negative ginseng.

[0127] Example 7: Sequencing of monoclonal antibodies

[0128] Hybridomas that showed good binding activity to engineered cell CCR8-CHO, selected by FACs detection, were sequenced to obtain the heavy chain variable region and light chain variable region of Ms-1, Ms-2, Ms-8 and Ms-15, respectively.

[0129] (1) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of Ms-1 heavy chain are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively;

[0130] The amino acid sequence of the variable region of the Ms-1 heavy chain is SEQ ID NO: 1;

[0131] The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the variable region of the Ms-1 light chain are: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

[0132] The amino acid sequence of the variable region of the Ms-1 light chain is SEQ ID NO: 5.

[0133] (2) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of Ms-2 heavy chain are: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

[0134] The amino acid sequence of the variable region of the Ms-2 heavy chain is SEQ ID NO: 9;

[0135] The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the variable region of the Ms-2 light chain are: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;

[0136] The amino acid sequence of the variable region of the Ms-2 light chain is SEQ ID NO: 13.

[0137] (3) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of Ms-8 heavy chain are: SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively;

[0138] The amino acid sequence of the variable region of the Ms-8 heavy chain is SEQ ID NO: 17;

[0139] The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the variable region of the Ms-8 light chain are: SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively;

[0140] The amino acid sequence of the variable region of the Ms-8 light chain is SEQ ID NO: 21.

[0141] (4) The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of Ms-15 heavy chain are: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, respectively;

[0142] The amino acid sequence of the variable region of the Ms-15 heavy chain is SEQ ID NO: 25;

[0143] The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the variable region of the Ms-15 light chain are: SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively;

[0144] The amino acid sequence of the variable region of the Ms-15 light chain is SEQ ID NO: 29.

[0145] Example 8: Construction and expression of chimeric antibodies

[0146] The antibody fragments obtained from sequencing in Example 7 were genetically synthesized and constructed into a human IgG framework. Then, using molecular cloning technology, the antibody fragments were inserted into the PCDNA3.1 vector to construct mammalian cell expression plasmids. The plasmids were introduced into the host cell line CHO cells using liposome transfection. The fermentation supernatant was obtained using cell Fed-batch and purified by affinity chromatography. Finally, the constructed chimeric antibodies CH-1, CH-2, CH-8, and CH-15 were obtained.

[0147] The amino acid sequences of the CDR and variable domain of the chimeric antibodies CH-1, CH-2, CH-8 and CH-15 correspond to the amino acid sequences of the CDR and variable domain of Ms-1, Ms-2, Ms-8 and Ms-15 in Example 7, respectively.

[0148] The heavy chain constant regions of the chimeric antibodies CH-1, CH-2, CH-8, and CH-15 are identical, as shown in SEQ ID NO: 76, and the amino acid sequences of the light chain constant regions are shown in SEQ ID NO: 77.

[0149] Example 9: Binding activity of chimeric antibodies

[0150] The binding activity of the chimeric antibody was detected using the FACs method. The secondary antibody was PE F(ab')2 goat anti-human IgG Fc (Biolegend, Cat:398004). The detection procedure was the same as in Example 6, and the results are as follows. Figure 2 As shown.

[0151] Depend on Figure 2 It can be seen that the chimeric antibodies CH-1, CH-2, CH-8 and CH-15 all have good binding activity with CCR8-CHO-K1 cells, which is comparable to that of ginseng.

[0152] Example 10: ADCC activity of chimeric antibodies on target cells

[0153] CCR8-CHOK1 cells overexpressing human CCR8 were used as target cells. Cells were centrifuged at 1000 rpm for 4 minutes at room temperature and resuspended in RPMI 1640 basal medium (containing 5% FBS) at 1×10⁻⁶ ppm. 4 50 μL / well of cells were seeded into 96-well plates. Antibody was diluted using RPMI 1640 basal medium (containing 5% FBS) at an initial concentration of 10 μg / mL, followed by 10-fold serial dilutions (7 concentration gradients), 100 μL / well. NK cells were resuspended and added to the corresponding wells at 50 μL / well, with a target cell ratio of 3:1 for CCR8-CHOK1 cells. Wells were also set up for maximum target cell lysis (M), spontaneous target cell release (ST), spontaneous effector cell release (SE), total volume correction blank (BV), and culture medium blank control (BM). After standing for 10 minutes, the cells were centrifuged at 1000 rpm for 4 minutes at room temperature and incubated for 4 hours in a 5% CO2, 37°C CO2 cell culture incubator. Lysis buffer was added to wells M and BV 45 minutes before incubation, mixed well, and centrifuged at 1000 rpm for 4 minutes at room temperature after incubation. Pipette 50 μL of supernatant into an LDH analysis plate, add 50 μL of assay buffer per well to dissolve the substrate, incubate at room temperature in the dark for 30 minutes, add 50 μL of stop solution per well, let stand for 10 minutes, and take readings at 490 nm to calculate cell death rate.

[0154]

[0155] The concentration of the constructed antibody was taken as the logarithm and used as the x-axis. A nonlinear regression was performed using the Sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) to obtain the ADCC activity curve of the target antibody on the target cells.

[0156] Depend on Figure 3 It was found that the IgG1 isotype control did not show any killing effect on CCR8-CHOK1 cells, while the chimeric antibodies CH-1, CH-2, CH-8 and CH-15, along with Yangshen, showed lysis and death of CCR8-CHO-K1 cells in a concentration-dependent manner.

[0157] Example 11: Antibody antagonistic activity against CCL1

[0158] CCR8 is the only known receptor for CCL1. CCL1 binds to CCR8 on the cell surface, triggering downstream signal transduction. Anti-CCR8 antibodies can inhibit this signal transduction pathway by blocking CCR8. Therefore, to test the blocking ability of anti-CCR8 antibodies on the CCL1-CCR8 pathway, β-arrestin was used as the target in Tango-H_CCR8-CHO-K1 cells (GM-C13190). In short, the activator Switch-On Reagent (GM-040501A), which induces CCR8 expression, was added to Tango-H_CCR8-CHO-K1 cells. After 48 hours of continuous culture, the cells were digested, centrifuged at 200g to collect the cells, and then processed using ONE-Glo TM Resuspend cells in the analysis buffer of the Luciferase Assay System (Biolegend) at a cell concentration of 1.5 × 10⁻⁶. 5 100 μL of cells / mL was added to a 96-well plate and incubated overnight in a cell culture incubator. The antibody was diluted with analytical buffer, starting at 15 μg / mL, followed by 5-fold serial dilutions for a total of 9 concentration gradients, with 50 μL / well incubated for 1 h. Then, 50 μL of CCL1 (Biolegend) was added as an activator, resulting in a final concentration of 0.06 μg / mL. After incubation for 7 h, fluorescence values ​​were read using a microplate reader. The logarithm of the antibody concentration was plotted on the x-axis, and a nonlinear regression was performed using the Sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) to obtain the inhibition curve of the target antibody on the downstream signaling molecule β-arrestin triggered by the binding of CCL1 and CCR8.

[0159] The results are as follows Figure 4 As shown, IgG1, the isotype control group, could not inhibit the signal value of CCL1 activating β-arrestin. Both positive and candidate antibodies showed antagonistic activity, and the activity was concentration-dependent.

[0160] Example 12: In vivo efficacy experiment of mouse antibody

[0161] Constructing humanized CCR8 C57 mice (Biocytogen, C57BL / 6-CCR8) tm1(CCR8)A subcutaneous MC38 cell tumor model of colon cancer (Bcgen) was established to evaluate the in vivo antitumor efficacy of antibodies Ms-2 and Ms-15. MC38 cells were resuscitated, cultured, and digested to prepare a cell suspension. Cells were collected at the logarithmic growth phase, and the tumor cell suspension was injected subcutaneously into Balb / C nude mice. Each mouse was inoculated with 100 μL of cell suspension containing 5 × 10⁶ cells / mL. 5 Individual cells. Observe tumor growth; when the subcutaneous tumor grows to 100mm... 3 Animals were randomly grouped according to tumor volume and treated with CCR8 antibody. The experimental endpoint was to assess whether tumor growth was inhibited, delayed, or cured. Tumor diameter was measured three times a week using calipers. The formula for calculating tumor volume was: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. Tumor-bearing mice were administered antibodies and PBS via intraperitoneal injection, respectively, twice weekly at a dose of 10.0 mg / kg, for a total of 5 weeks.

[0162] The antitumor efficacy of the compound was evaluated using TGI (%). The formula for calculating TGI (%) is: TGI (%) = [1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] × 100%.

[0163] The tumor growth curve of hCCR8 tumor-bearing mice with MC38 cell subcutaneous colon cancer model after antibody administration is shown in the figure. Figure 5 As shown, the horizontal axis represents the number of days after treatment began, and the vertical axis represents tumor volume. Candidate antibodies Ms-2 and Ms-15 exhibited high tumor-suppressive activity, significantly outperforming the PBS group and the positive control group, with a tumor inhibition rate (TGI) greater than 50%.

[0164] Example 13: Evaluation of Humanized Monoclonal Antibodies

[0165] The variable region of the chimeric antibody was humanized, with the design principle being to avoid introducing protein modification sites such as glycosylation, deamidation, and isomerization, as well as integrin binding sites and cysteine ​​residues. Reversion mutations of key amino acids in the framework region should maintain the original physicochemical and biochemical activities. The specific methods are as follows:

[0166] The mouse sequences Ms-15 and Ms-2 were aligned with human Germline sequences using the IgBLAST tool, and the FR (Frequency Reduction) sites were replaced with the human Germline sequence with the highest sequence similarity. For Ms-2, the heavy chain template was selected from the IGHV1 class, and the light chain template from the IGKV3 class. For Ms-15, the heavy chain template was selected from the IGHV3 class, and the light chain template from the IGKV3 class, resulting in the humanized antibody sequence. Then, based on this humanization, several important amino acids affecting antibody affinity were reverse-mutated, i.e., mutated to the original mouse FR sites. The humanization percentage was calculated as the similarity ratio between the designed sequence framework and the Germline sequence framework. The designed humanized sequence was compared with the human Germline sequence, and sequences with a humanization percentage of over 90% were selected.

[0167] The heavy chain sequence of the chimeric antibody CH-2 was designed as four humanized sequences, namely Hu-2-H1, Hu-2-H2, Hu-2-H3 and Hu-2-H4, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0168] The light chain sequence of the chimeric antibody CH-2 was designed into three humanized sequences, namely Hu-2-L1, Hu-2-L2 and Hu-2-L3, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

[0169] The amino acid sequences of the variable regions of the four heavy chains and three light chains are shown in Table 1.

[0170] Table 1

[0171]

[0172]

[0173] The above 4 humanized heavy chains and 3 humanized light chains were combined into 12 antibodies, as shown in Table 2.

[0174] Table 2

[0175] Antibody name Heavy chain variable region sequence Light chain variable region sequence Hu-2-Z1 Hu-2-H1 Hu-2-L1 Hu-2-Z2 Hu-2-H1 Hu-2-L2 Hu-2-Z3 Hu-2-H1 Hu-2-L3 Hu-2-Z4 Hu-2-H2 Hu-2-L1 Hu-2-Z5 Hu-2-H2 Hu-2-L2 Hu-2-Z6 Hu-2-H2 Hu-2-L3 Hu-2-Z7 Hu-2-H3 Hu-2-L1 Hu-2-Z8 Hu-2-H3 Hu-2-L2 Hu-2-Z9 Hu-2-H3 Hu-2-L3 Hu-2-Z10 Hu-2-H4 Hu-2-L1 Hu-2-Z11 Hu-2-H4 Hu-2-L2 Hu-2-Z12 Hu-2-H4 Hu-2-L3

[0176] The heavy chain of the chimeric antibody CH-15 was designed as a humanized sequence. CDR2 contains a DS (aspartate isomerization risk). The DS in the humanized sequence was mutated to ES, and another heavy chain with a reversion mutation was designed. The heavy chain CDR3 also contains an RGD (integrin binding site). In the design, the RGD was mutated to RGE and RAD respectively based on the reversion mutation sequence, adding two chains, thus a total of 4 chains, namely Hu-15-H1p1, Hu-15-H2p1, Hu-15-H2p1p2, and Hu-15-H2p1p3.

[0177] The CDR1 of the CH-15 light chain contains an NG (glycosylation risk). During the design, based on the maximum number of reversion mutations, the NG was mutated to QG and NA respectively. A total of 5 light chains were designed, namely Hu-15-L1, Hu-15-L2, Hu-15-L3, Hu-15-L3p1 and Hu-15-L3p2.

[0178] The amino acid sequences of the variable regions and CDRs of the above 4 heavy chains and 5 light chains are shown in Table 3.

[0179] Table 3

[0180]

[0181]

[0182] The above 4 heavy chains and 5 light chains were combined to form 10 antibodies, as shown in Table 4.

[0183] Table 4

[0184] Antibody name Heavy chain variable region sequence Light chain variable region sequence Hu-15-Z1 Hu-15-H1p1 Hu-15-L1 Hu-15-Z2 Hu-15-H1p1 Hu-15-L2 Hu-15-Z3 Hu-15-H1p1 Hu-15-L3 Hu-15-Z4 Hu-15-H2p1 Hu-15-L1 Hu-15-Z5 Hu-15-H2p1 Hu-15-L2 Hu-15-Z6 Hu-15-H2p1 Hu-15-L3 Hu-15-Z7 Hu-15-H2p1 Hu-15-L3p1 Hu-15-Z8 Hu-15-H2p1 Hu-15-L3p2 Hu-15-Z9 Hu-15-H2p1p2 Hu-15-L3 Hu-15-Z10 Hu-15-H2p1p3 Hu-15-L3

[0185] The amino acid sequences in Table 3 were synthesized and expressed using the above humanized sequence combination. The humanized antibodies in Table 4 were prepared by constructing and expressing the sequences according to the aforementioned method.

[0186] The binding activity of the humanized antibody was evaluated according to the steps in Example 6, and the results are as follows: Figure 6a and 6b As shown in the results, the humanized antibody exhibits excellent binding activity with CHOK1-CCR8 cells, comparable to that of the chimeric antibody and BMK (derived from the 1K17 sequence of US20210277129), and far superior to the unrelated antibody.

[0187] The ADCC activity of the humanized antibody was evaluated according to the steps in Example 10, and the results are as follows: Figure 7a and 7bAs shown in the results, the humanized antibodies Hu-2-Z4, Hu-2-Z6, Hu-2-Z9, Hu-15-Z1, Hu-15-Z3, and Hu-15-Z6 all exhibited good ADCC activity. Their cell lysis rates were comparable to those of chimeric antibodies and BMK (derived from the 1K17 sequence of US20210277129), and far superior to those of unrelated antibodies.

[0188] The antagonistic activity of the humanized antibody against the CCR-CCL1 signaling pathway was evaluated according to the steps in Example 11, and the results are as follows: Figure 8 As shown in the figure. The results indicate that both positive and candidate antibodies exhibited antagonistic activity in a concentration-dependent manner, while the IgG isotype control failed to inhibit the signal value of CCL1 activating β-arrestin.

Claims

1. An anti-CCR8 antibody or antigen-binding fragment thereof, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 of the heavy chain variable region, and the LCDR1, LCDR2, LCDR3 of the light chain variable region are the following amino acid sequences: HCDR1 as shown in SEQ ID NO: 49, HCDR2 as shown in SEQ ID NO: 50, HCDR3 as shown in SEQ ID NO: 51, LCDR1 as shown in SEQ ID NO: 65, LCDR2 as shown in SEQ ID NO: 66, and LCDR3 as shown in SEQ ID NO:

67.

2. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 1, characterized in that, The heavy chain variable region and the light chain variable region are the following amino acid sequences: SEQ ID NO: 48 and SEQ ID NO:

64.

3. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, and / or a humanized antibody or antigen-binding fragment thereof.

4. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 1, wherein, It further comprises a Fc region selected from mouse IgGl, IgG2a, IgG2b or IgG3, or from rat IgGl, IgG2a, IgG2b or IgG2c.

5. The anti-CCR8 antibody or antigen-binding fragment thereof of claim 1, wherein, It further comprises a Fc region selected from human IgGl, IgG2, IgG3 or IgG4.

6. A nucleic acid molecule encoding the anti-CCR8 antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

7. A recombinant vector comprising the nucleic acid molecule of claim 6.

8. A recombinant cell comprising the nucleic acid molecule of claim 6 and / or the recombinant vector of claim 7, and capable of expressing the anti-CCR8 antibody or antigen-binding fragment thereof.

9. Use of the anti-CCR8 antibody or antigen-binding fragment thereof of any one of claims 1 to 5 for the manufacture of a medicament for the treatment and / or prevention of colon cancer.

10. The use according to claim 9, wherein the use is achieved by tumor immunotherapy.

11. A pharmaceutical composition comprising the anti-CCR8 antibody or antigen-binding fragment thereof of any one of claims 1 to 5 and an acceptable carrier, diluent or excipient.

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