WO2023193732A1 - 一种抗ccr8抗体或其抗原结合片段 - Google Patents

一种抗ccr8抗体或其抗原结合片段 Download PDF

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WO2023193732A1
WO2023193732A1 PCT/CN2023/086391 CN2023086391W WO2023193732A1 WO 2023193732 A1 WO2023193732 A1 WO 2023193732A1 CN 2023086391 W CN2023086391 W CN 2023086391W WO 2023193732 A1 WO2023193732 A1 WO 2023193732A1
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seq
amino acid
acid sequence
antigen
antibody
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PCT/CN2023/086391
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French (fr)
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周冲
秦春铃
姜晓玲
殷刘松
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盛禾(中国)生物制药有限公司
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Publication of WO2023193732A1 publication Critical patent/WO2023193732A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to an anti-CCR8 antibody or an antigen-binding fragment thereof.
  • Chemokine receptors are 7 transmembrane G protein-coupled receptors (GPCRs) expressed on the surface of some specific cells.
  • GPCRs G protein-coupled receptors
  • the chemokine receptor binds to the extracellular ligand chemokine, triggering the influx of calcium ions to produce a cell chemotactic response, thereby inducing cells to move to specific parts of the organism.
  • the combination of CCR8 and its ligand CCL1/CCL18 provides positive feedback to regulate the immunosuppressive function of the tumor microenvironment.
  • CCR8 (chemokine(C-C motif)receptor 8) is a chemokine receptor highly expressed in tumor-infiltrating Tregs. Its structure is similar to G protein-coupled receptors and it is a seven-transmembrane protein. CCR8 is specifically expressed on tumor-infiltrating regulatory T cells (Treg), but is basically not expressed on peripheral blood Treg, regulating the immunosuppressive function of the tumor microenvironment. CCR8 causes cancer cell proliferation, migration and apoptosis resistance by activating the CCR8 receptor on cancer cells. In addition, CCL1 can recruit Tregs to the tumor niche and cause CD4+ T cells to convert into Tregs.
  • Treg tumor-infiltrating regulatory T cells
  • CCL1 can recruit FOXp3+CCR8+Treg cells to infiltrate into tumor tissues and exert immunosuppressive functions. It can also induce the upregulation of CCR8 expression on the surface of FOXp3+Treg cells, induce Ca 2+ flow, and induce stat3-dependent Foxp3, CD39, and 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 effects by depleting tumor-infiltrating FOXp3+CCR8+Treg cells or blocking the CCL1/CCR8 pathway.
  • the anti-CCR8 antibody developed in the present invention can specifically recognize human CCR8 protein, can block the signaling pathway between CCR8 and CCL1/CCL18, can specifically recognize CCR8-expressing cells, and has target-specific ADCC activity against CCR8-expressing target cells.
  • An anti-CCR8 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 , the light chain variable region includes light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,
  • HCDR1 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 2, 10, 18, 26, 41, 45, 49, 53, or selected from the amino acid sequence of SEQ ID NO: 2, 10 , any one of the amino acid sequences of 18, 26, 41, 45, 49, and 53 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Sequences with 98%, 99% or above identity, or selected from the group consisting of one or more (preferably 2 or 3) amino acid sequence of conservative amino acid mutations (preferably substitution, insertion or deletion);
  • HCDR2 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 3, 11, 19, 27, 42, 46, 50, 54, or selected from the amino acid sequence of SEQ ID NO: 3, 11 , any one of the amino acid sequences of 19, 27, 42, 46, 50, and 54 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Sequences with 98%, 99% or above identity, or selected from the group consisting of one or more (preferably 2 or 3) amino acid sequence of conservative amino acid mutations (preferably substitution, insertion or deletion);
  • HCDR3 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 4, 12, 20, 28, 43, 47, 51, 55, or selected from the amino acid sequence of SEQ ID NO: 4, 12 , any one of the amino acid sequences of 20, 28, 43, 47, 51, and 55 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Sequences with 98%, 99% or above identity, or selected from the group consisting of one or more (preferably 2 or 3) amino acid sequence of conservative amino acid mutations (preferably substitution, insertion or deletion);
  • LCDR1 of the light chain variable region is selected from the amino acid sequence of SEQ ID NO: 6, 14, 22, 30, 57, 61, 65, 69, 73, or selected from the amino acid sequence of SEQ ID NO: 6, 14,
  • the amino acid sequences of 22, 30, 57, 61, 65, 69, 73 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , a sequence with 99% or more identity, or selected from the group consisting of one or more (preferably 2 or 3) Amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);
  • LCDR2 of the light chain variable region selected from SEQ ID NO: 7, 15, 23, 31, 58, 62, 66, 70, 74 amino acid sequence, or selected from the group consisting of at least 80%, 85%, 90%, 91%, 92 with the amino acid sequence of SEQ ID NO: 7, 15, 23, 31, 58, 62, 66, 70, 74 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or selected from the group consisting of SEQ ID NO: 7, 15, 23, 31, 58, 62, 66 , 70, 74 amino acid sequence compared with the amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitution, insertion or deletion); and/or
  • LCDR3 of the light chain variable region is selected from the amino acid sequence of SEQ ID NO: 8, 16, 24, 32, 59, 63, 67, 71, 75, or selected from the amino acid sequence of SEQ ID NO: 8, 16,
  • the amino acid sequences of 24, 32, 59, 63, 67, 71, 75 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , a sequence with 99% or more identity, or selected from the group consisting of one or more (preferably 2 or 3) amino acid sequence of conservative amino acid mutations (preferably substitution, insertion or deletion).
  • the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the anti-CCR8 antibody or its antigen-binding fragment, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from the following (1)- The amino acid sequence of any one of (14):
  • 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
  • SEQ ID NO: 7 shown LCDR2, LCDR3 shown in SEQ ID NO: 8;
  • 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, SEQ ID NO: 15 shown LCDR2, LCDR3 shown in SEQ ID NO: 16;
  • 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, SEQ ID NO: 31 shown LCDR2, LCDR3 shown in SEQ ID NO: 32;
  • 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
  • SEQ ID NO: 58 shown LCDR2, LCDR3 shown in SEQ ID NO: 59;
  • 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
  • SEQ ID NO: 66 shown LCDR2, LCDR3 shown in SEQ ID NO: 67;
  • 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
  • SEQ ID NO: 58 shown LCDR2, LCDR3 shown in SEQ ID NO: 59;
  • 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
  • SEQ ID NO: 62 shown LCDR2, LCDR3 shown in SEQ ID NO: 63;
  • 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
  • SEQ ID NO: 70 shown LCDR2, LCDR3 shown in SEQ ID NO: 71;
  • 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
  • SEQ ID NO: 74 shown LCDR2, LCDR3 shown in SEQ ID NO: 75;
  • 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
  • SEQ ID NO: 66 shown LCDR2, LCDR3 shown in SEQ ID NO: 67;
  • 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
  • SEQ ID NO: 66 shown LCDR2, LCDR3 shown in SEQ ID NO: 67.
  • the anti-CCR8 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region has any amino acid sequence given by SEQ ID NO: 1, 9, 17, 25, 33, 34, 35, 36, 40, 44, 48, 52,
  • any amino acid sequence of SEQ ID NO: 1, 9, 17, 25, 33, 34, 35, 36, 40, 44, 48, 52 it has one or more (preferably 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10) amino acid sequence of conservative amino acid mutations (preferably substitution, insertion or deletion);
  • the light chain variable region has any amino acid sequence given by SEQ ID NO: 5, 13, 21, 29, 37, 38, 39, 56, 60, 64, 68, 72,
  • the heavy chain variable region and light chain variable region of the anti-CCR8 antibody or antigen-binding fragment thereof are selected from any one of the following amino acid sequences (1)-(26):
  • the antibody or antigen-binding fragment thereof includes 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.
  • the anti-CCR8 antibody or antigen-binding fragment thereof further comprises an Fc region, and the Fc region is selected from mouse IgG1, IgG2a, IgG2b and/or IgG3, or is selected from rat IgG1, IgG2a , IgG2b and/or IgG2c.
  • the anti-CCR8 antibody or antigen-binding fragment thereof further comprises an Fc region
  • the Fc region is selected from human IgG1, IgG2, IgG3 and/or IgG4 or combined with human IgG1, IgG2, IgG3, IgG4
  • An Fc region amino acid sequence having one or more amino acid mutations preferably substitutions, insertions or deletions.
  • the Fc region can be modified, for example, in a Knob-in-Hole format.
  • the present invention also provides nucleic acid molecules encoding any of the above-mentioned anti-CCR8 antibodies or antigen-binding fragments thereof.
  • the present invention also provides recombinant vectors comprising the above nucleic acid molecules.
  • the present invention also provides recombinant cells, which contain the above-mentioned nucleic acid molecules and/or the above-mentioned recombinant vectors and are capable of expressing the anti-CCR8 antibody or antigen-binding fragment thereof.
  • the present invention also provides a multifunctional fusion protein comprising any of the above-mentioned anti-CCR8 antibodies or antigen-binding fragments thereof.
  • the multifunctional fusion protein also contains one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the antigen that binds the second antibody or antigen-binding portion thereof is selected from the group consisting of tumors Associated antigens (TAAs) or immune checkpoints.
  • TAAs tumors Associated antigens
  • any one of the above-described anti-CCR8 antibodies or antigen-binding fragments thereof, or any one of the multi-functional fusion proteins described above is used in the preparation of medicaments for treating and/or preventing and/or diagnosing diseases. use.
  • the use is achieved by one or more of tumor immunotherapy, cell therapy, and gene therapy.
  • any of the above-mentioned anti-CCR8 antibodies or antigen-binding fragments thereof, or any of the above-mentioned multifunctional fusion proteins in the preparation of drugs for treating cancer.
  • cancer is breast cancer, ovarian cancer, lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, colorectal cancer, gastric cancer, bladder cancer, pancreatic cancer, colon cancer, cervical cancer Cancer or related tumors.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the above-mentioned anti-CCR8 antibodies or antigen-binding fragments thereof and an acceptable carrier, diluent or excipient.
  • the present invention also provides a pharmaceutical composition, which contains any of the above-mentioned multifunctional fusion proteins and an acceptable carrier, diluent or excipient.
  • the anti-CCR8 antibody developed in the present invention can specifically recognize human CCR8 protein, can block the signaling pathway between CCR8 and CCL1/CCL18, can specifically recognize CCR8-expressing cells, and has target-specific ADCC activity against CCR8-expressing target cells.
  • CDR complementarity determining region in the antibody variable region
  • HCDR complementarity determining region in the variable region of the antibody heavy chain
  • LCDR complementarity determining region in the variable region of the antibody light chain
  • FR Antibody framework region, that is, the amino acid residues in the antibody variable region except CDR residues;
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting.
  • the term "antibody” refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis.
  • the antibody can be isolated from natural resources such as plasma or serum in which the antibody naturally occurs, or from the culture supernatant of hybridoma cells that produce the antibody, from animal immune serum, or from phage library screening. Alternatively, it may be partially or completely synthesized by using techniques such as genetic recombination.
  • Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes.
  • Human immunoglobulins are known to include nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
  • isotypes the antibodies of the invention may include IgGl, IgG2, IgG3 and/or IgG4.
  • antibody and “immunoglobulin” are used interchangeably, and as used herein, part of the antibody is an immunoglobulin consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). molecular.
  • 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 3 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 the light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of 3 CDRs and 4 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 respectively form the antigen-binding site.
  • antigen-binding fragment of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen that the full-length antibody binds and/or competes with the full-length antibody for the antigen. Specific binding, which is also known as the "antigen-binding moiety".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • 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, diabodies (diabody), linear antibody (linear antibody), Nanobody (such as technology from Ablynx), domain antibody (such as technology from Domantis), and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide .
  • CDR complementarity determining region
  • polypeptide refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation).
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "exogenous” in the sense that they are “heterologous", that is, foreign to the host cell utilized, such as a human polypeptide produced by a bacterial cell. This article discloses the polypeptide as Amino acid residue sequence. Those sequences are written from left to right in amino terminus to carboxyl terminus direction.
  • amino acid residue sequences are named with three-letter or single-letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), 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).
  • the Kabat system is used for the numbering of amino acid positions (such as amino acid residues in the Fc region) and target regions (such as CDRs) in the anti-CCR8 antibody of the present invention.
  • Percent (%) amino acid sequence "identity" with respect to a reference polypeptide sequence is defined as the amino acids in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity. Percentage of residues. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program Bag.
  • monoclonal antibody refers to a uniform antibody that targets only a specific antigenic epitope. In contrast to common polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen.
  • the modifier "monoclonal” indicates the uniform character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • Monoclonal antibodies of the invention are preferably produced by recombinant DNA methods or obtained by screening methods described elsewhere herein.
  • murine antibody in the present invention refers to a monoclonal antibody prepared according to the knowledge and skill in the art.
  • a subject is prepared by injecting an antigen and then isolating a hybridoma expressing an antibody with the desired sequence or functional properties.
  • mouse immune libraries are screened to obtain the desired of mouse antibodies.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies.
  • hybridomas that secrete murine specific monoclonal antibodies are first established, then the variable region genes are cloned from mouse hybridoma cells, and then the constant region genes of the human antibodies are cloned as needed.
  • the mouse variable region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector.
  • the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • humanized antibody refers to an antibody that includes at least one humanized antibody chain (ie, at least one humanized light or heavy chain).
  • humanized antibody chain i.e., “humanized immunoglobulin chain” refers to an antibody chain (i.e., a light chain or a heavy chain, respectively) having a variable region that includes substantial portions of a human antibody variable framework regions and complementarity determination.
  • a region (CDR) substantially derived from a non-human antibody eg, at least one CDR, two CDRs, or three CDRs.
  • the humanized antibody chain further includes a constant region (eg, in the case of a light chain, one constant region or a portion thereof, in the case of a heavy chain, preferably three constant regions).
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes the offspring of a parent cell, regardless of whether the offspring is identical in morphology or genetic composition to the original parent cell, as long as the selected gene of interest is present in the offspring.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Figures 1a and 1b show the binding activity of mouse anti-CCR8 antibodies to CHOK1-CCR8 cells.
  • Figure 2 shows the binding activity of chimeric anti-CCR8 antibodies to CHOK1-CCR8 cells.
  • Figure 3 shows the ADCC effect results of chimeric anti-CCR8 antibodies.
  • Figure 4 shows the inhibition of mouse anti-CCR8 antibody on the release of ⁇ -arestin from Tango-H_CCR8-CHO-K1 cells activated by CCL1.
  • Figure 5 shows the in vivo efficacy data of mouse-derived anti-CCR8 antibodies.
  • Figures 6a and 6b show the binding activity of humanized anti-CCR8 antibodies to CHOK1-CCR8 cells.
  • Figures 7a and 7b show the ADCC effect results of humanized anti-CCR8 antibodies.
  • Figure 8 shows the inhibition of ⁇ -arestin release by CCL1-activated Tango-H_CCR8-CHO-K1 cells by humanized anti-CCR8 antibodies.
  • CCR8-CHO-K1 cells overexpressing human CCR8 were constructed by lentiviral transduction of CHO cells.
  • CCR8-284 cells were used as immunogens, and 1 ⁇ 10 7 total cells were used to inoculate and immunize Balb/c mice.
  • the second vaccination is carried out half a month after the first vaccination, and then again every other month. Negative serum was collected from the mice 3 days before immunization, and 50 ⁇ L of blood was taken from the tail 6 days after each immunization.
  • B lymphocytes and lymph node cells used in the experiment were obtained from Balb/c mice that had been immunized four times with CCR8-293 cells.
  • the spleen and lymph nodes were placed in a cell sieve, and then the cell sieve was placed in a 50 mL centrifuge tube.
  • Add DMEM dropwise to the spleen grind it to make a spleen cell suspension, centrifuge at 1600 rpm for 10 minutes, and remove the supernatant.
  • Myeloma cells SP2/0 (ATCC) were passaged one day before fusion so that the cells were in the logarithmic growth phase during the experiment. After mixing splenocytes and SP2/0 at a ratio of 2:1, centrifuge at 1600 rpm for 10 minutes. Wash the mixed cells twice with the fusion solution and centrifuge at 1600 rpm for 10 minutes. According to the final cell density of 1 ⁇ 10 7 cells/mL, add fusion solution to suspend the cells. Within 5 minutes, move the cell suspension to the fusion chamber of the electrofusion instrument (BTX; ECM 2001) for fusion. After fusion is completed, move the cells from the fusion chamber to complete medium containing HAT and incubate at 37°C for 60 minutes. After incubation, the cells were plated in a 96-well plate containing feeder cells and cultured at 37°C and 5% CO2 .
  • BTX electrofusion instrument
  • the fusion supernatant was initially screened.
  • Hybridoma cells were cultured in T75 cell culture flask until the cell coverage rate was 80-90%. Discard the cell supernatant from the two bottles, add 30 mL hybridoma-SFM, and culture at 37°C and 5% CO2 . Cultivate for 2-3 days and observe the cell status and medium color. If the color of the medium turns yellow, add 30 mL of new hybridoma-SFM. Cultivate for 6-7 days, collect the culture supernatant after low-speed centrifugation, and perform purification.
  • the concentration of the antibody was taken as the logarithm as the abscissa, and the Sigmoidaldose-response (VariableSlope) method (GraphPad Prism software, GraphPad Software, San Diego, California) was used to perform nonlinear regression to obtain the binding activity curve of the antibody to CCR8-CHO cells. The results are shown in Figure 1a and Figure 1b.
  • the selected antibodies Ms-1, Ms-2, Ms-3, Ms-4, Ms-5, Ms-6, Ms-7, Ms-8, Ms-9, and 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 yang ginseng, and far better than that of yin ginseng.
  • hybridomas with good binding activity to the engineered cell CCR8-CHO obtained through FACs detection and screening were sequenced, and the heavy chain variable regions and light chain variable regions of Ms-1, Ms-2, Ms-8 and Ms-15 were obtained respectively. .
  • amino acid sequences of HCDR1, HCDR2 and HCDR3 of the Ms-1 heavy chain variable region are respectively: SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4;
  • the amino acid sequence of the Ms-1 heavy chain variable region is SEQ ID NO: 1;
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the Ms-1 light chain variable region are respectively: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
  • the amino acid sequence of the Ms-1 light chain variable region is SEQ ID NO: 5.
  • amino acid sequences of HCDR1, HCDR2 and HCDR3 of the Ms-2 heavy chain variable region are respectively: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12;
  • amino acid sequence of the Ms-2 heavy chain variable region is SEQ ID NO: 9;
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the Ms-2 light chain variable region are: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;
  • the amino acid sequence of the Ms-2 light chain variable region is SEQ ID NO: 13.
  • amino acid sequences of HCDR1, HCDR2 and HCDR3 of the Ms-8 heavy chain variable region are respectively: SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20;
  • amino acid sequence of the Ms-8 heavy chain variable region is SEQ ID NO: 17;
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the Ms-8 light chain variable region are respectively: SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24;
  • the amino acid sequence of the Ms-8 light chain variable region is SEQ ID NO: 21.
  • HCDR1, HCDR2 and HCDR3 of the Ms-15 heavy chain variable region are respectively: SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28;
  • amino acid sequence of the Ms-15 heavy chain variable region is SEQ ID NO: 25;
  • amino acid sequences of LCDR1, LCDR2 and LCDR3 of the Ms-15 light chain variable region are respectively: SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32;
  • the amino acid sequence of the Ms-15 light chain variable region is SEQ ID NO: 29.
  • the antibody fragment sequenced in Example 7 was gene synthesized and constructed into a human IgG framework. Then, molecular cloning technology was used to insert the antibody fragment into the PCDNA3.1 vector to construct a mammalian cell expression plasmid, and liposome transfection was used. dyeing method, introduce the host cell strain CHO cells, use cell Fed-batch to obtain the fermentation supernatant, take the fermentation broth supernatant for affinity chromatography purification, and finally purify the constructed chimeric antibodies: CH-1, CH-2, CH-8 and CH-15.
  • amino acid sequences of the CDRs and variable domains of chimeric antibodies CH-1, CH-2, CH-8 and CH-15 respectively correspond to those of Ms-1, Ms-2, Ms-8 and Ms-15 in Example 7. Amino acid sequences of CDRs and variable domains.
  • amino acid sequences of the heavy chain constant regions of chimeric antibodies CH-1, CH-2, CH-8 and CH-15 are all the same, as shown in SEQ ID NO: 76, and the amino acid sequences of the light chain constant regions are as shown in SEQ ID NO: 77 shown.
  • the FACs method was used to detect the binding activity of the chimeric antibody.
  • the secondary antibody was PE F(ab')2 goat anti-human IgG Fc (Biolegend, Cat: 398004).
  • the detection steps were the same as in Example 6. The results are shown in Figure 2.
  • chimeric antibodies CH-1, CH-2, CH-8 and CH-15 all have good binding activity to CCR8-CHO-K1 cells, which is comparable to the activity of Yangshen.
  • Example 10 ADCC activity of chimeric antibodies on target cells
  • CCR8-CHOK1 cells overexpressing human CCR8 were used as target cells, centrifuged at room temperature for 4 minutes at 1000 rpm and resuspended in RPMI1640 basic medium (containing 5% FBS), then plated at 1 ⁇ 10 4 cells/well, 50 ⁇ L/well.
  • 96-well plate use RPMI1640 basic medium (containing 5% FBS) to dilute the antibody, with a starting concentration of 10 ⁇ g/mL, and then 10-fold gradient dilution, a total of 7 concentration gradients, 100 ⁇ L/well; resuspend NK cells at 50 ⁇ L/ The wells were added to the corresponding wells, in which the target cells were CCR8-CHOK1 and the time-to-target ratio was 3:1.
  • M target cell maximum lysis well
  • ST target cell spontaneous release well
  • SE effector cell spontaneous release well
  • BV total volume correction blank well
  • BM medium blank control well
  • the IgG1 isotype control did not show killing of CCR8-CHOK1 cells, while the chimeric antibodies CH-1, CH-2, CH-8 and CH-15 and Yangshen showed lysis and death of CCR8-CHO-K1 cells. , and is concentration dependent.
  • CCR8 is the only known receptor for CCL1, and CCL1 binds to CCR8 on the cell surface, causing downstream signal transduction.
  • Anti-CCR8 antibodies can inhibit this signal transduction pathway by blocking CCR8. Therefore, to test the ability of anti-CCR8 antibodies to block the CCL1-CCR8 pathway, ⁇ -arrestin was used as the target, using Tango-H_CCR8-CHO-K1 (Yoshimitsu, GM-C13190). Briefly, the activator Switch-On Reagent (Jiman, cat: GM-040501A), which induces CCR8 expression, was added to Tango-H_CCR8-CHO-K1 cells.
  • the cells were collected by centrifugation at 200 g. Resuspend the cells in the analysis buffer in ONE-Glo TM Luciferase Assay System (Biolegend), add the cells to a 96-well assay plate with a cell concentration of 1.5 ⁇ 10 5 cells/mL and a volume of 100 ⁇ L, and incubate overnight in a cell culture incubator. Use assay buffer to dilute the antibody with a starting concentration of 15 ⁇ g/mL, then 5-fold gradient dilution, a total of 9 concentration gradients, and incubate for 1 hour at 50 ⁇ L/well.
  • ONE-Glo TM Luciferase Assay System Biolegend
  • CCL1 Biolegend
  • 50 ⁇ L CCL1 (Biolegend) was added as an activator with a final concentration of 0.06 ⁇ g/mL.
  • the fluorescence value was read with a microplate reader. Take the logarithm of the concentration of the constructed antibody as the abscissa, and use the Sigmoidaldose-response (VariableSlope) method (GraphPad Prism Software, GraphPad Software, San Diego, California) to perform nonlinear regression.
  • the target antibody binds to CCL1 and CCR8, it triggers downstream signaling molecules. Inhibition curve of ⁇ -arrestin.
  • IgG1 is the isotype control group and cannot inhibit the signal value of ⁇ -arrestin activated by CCL1. All candidate antibodies of CCL1 and CCL showed antagonistic activity and the activity was concentration-dependent.
  • Example 12 In vivo efficacy experiment of mouse-derived antibodies
  • MC38 colon cancer MC38 cell subcutaneous tumor model, and evaluate the anti-tumor efficacy of antibodies Ms-2 and Ms-15 in vivo.
  • Resuscitate MC38 cells perform cell culture, and prepare cell suspension after digestion. When the cells were cultured to the logarithmic growth phase, the cells were collected, and the tumor cell suspension was injected subcutaneously into Balb/C nude mice. Each animal was inoculated with 100 ⁇ L of cell suspension, containing 5 ⁇ 10 5 cells. The tumor growth was observed.
  • the animals were randomly divided into groups according to tumor volume and given CCR8 antibody treatment.
  • the experimental indicator is to examine whether tumor growth is inhibited, delayed or cured.
  • Tumor diameter was measured three times a week using vernier calipers.
  • the divided tumor-bearing mice were given intraperitoneal injection of antibodies and PBS, twice a week, 10.0 mg/kg each time, for a total of 5 times.
  • TGI (%) [1-(average tumor volume at the end of treatment in a certain treatment group - average tumor volume at the beginning of treatment in the treatment group)/(average tumor volume at the end of treatment in the solvent control group) Volume - average tumor volume in the solvent control group at the beginning of treatment)] ⁇ 100%.
  • Mouse colon cancer MC38 cell subcutaneous tumor model The tumor growth curve of hCCR8 tumor-bearing mice given antibodies is shown in Figure 5, where the abscissa represents the number of days after the start of treatment, and the ordinate represents the tumor volume.
  • candidate Antibodies Ms-2 and Ms-15 have a high tumor inhibitory effect, and their effect is significantly better than that of the PBS group and the positive control group, and their tumor inhibition rate TGI (%) is greater than 50%.
  • variable region of the chimeric antibody is humanized.
  • the design principle is to not introduce protein modification sites such as glycosylation, deamidation, isomerization, etc., and not to introduce integrin binding sites, cysteine, and framework regions.
  • the reverse mutation of important amino acids should maintain the original physical, chemical and biochemical activities. The specific method is as follows:
  • the IgBLAST tool uses the IgBLAST tool to align the mouse sequences Ms-15 and Ms-2 with the human Germline sequence respectively, and replace the FR with the human Germline sequence with the highest sequence similarity.
  • the Ms-2 heavy chain design template selects the IGHV1 category
  • the light chain design template selects the IGKV3 category.
  • the Ms-15 heavy chain design template selected the IGHV3 category
  • the light chain design template selected the IGKV3 category to obtain the sequence of the humanized antibody.
  • several important amino acids that affect the affinity of the antibody are reverse mutated, that is, mutated into the original mouse-derived FR site.
  • the humanization percentage is the similarity ratio between the designed sequence Framework and the Germline sequence Framework. Compare the designed humanized sequence with the human Germline sequence, and select the sequence with a humanization percentage of the antibody above 90%.
  • the heavy chain sequence of chimeric antibody CH-2 was designed into four humanized sequences, namely Hu-2-H1, Hu-2-H2, Hu-2-H3 and Hu-2-H4, among which HCDR1, HCDR2 and
  • the amino acid sequences of HCDR3 are SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
  • 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.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
  • amino acid sequences of the variable regions of the above four heavy chains and three light chains are shown in Table 1 respectively.
  • the heavy chain of the chimeric antibody CH-15 is designed as a humanized sequence.
  • the CDR2 contains a DS (risk of aspartic acid isomerization).
  • the DS in the humanized sequence is mutated into ES, and a reverse mutation is designed. of another heavy chain.
  • the heavy chain CDR3 also contains an RGD (integrin binding site).
  • RGD was mutated into RGE and RAD based on the reverse mutation sequence, adding two chains, so there are four chains in total, namely Hu-15 -H1p1, Hu-15-H2p1, Hu-15-H2p1p2 and Hu-15-H2p1p3.
  • the CDR1 of the CH-15 light chain contains an NG (glycosylation risk).
  • NG glycosylation risk
  • the light chain was designed with a total of 5 chains, namely Hu-15- L1, Hu-15-L2, Hu-15-L3, Hu-15-L3p1 and Hu-15-L3p2.
  • amino acid sequences of the variable regions and CDRs of the above four heavy chains and five light chains are shown in Table 3 respectively.
  • the binding activity of the humanized antibody was evaluated according to the steps of Example 6, and the results are shown in Figures 6a and 6b. It can be seen from the results that the humanized antibody has good binding activity to CHOK1-CCR8 cells. The binding activity is comparable to that of chimeric antibodies and BMK (derived from the 1K17 sequence of US20210277129), and is far better than that of unrelated antibodies.
  • the ADCC activity evaluation of the humanized antibody was carried out according to the steps of Example 10. The results are shown in Figure 7a and As shown in 7b. It can be seen from the results that humanized antibodies Hu-2-Z4, Hu-2-Z6, Hu-2-Z9, Hu-15-Z1, Hu-15-Z3 and Hu-15-Z6 all have good ADCC activity , its cell lysis rate is comparable to that of chimeric antibodies and BMK (derived from the 1K17 sequence of US20210277129), and is far superior to unrelated antibodies.
  • the antagonist activity of the CCR-CCL1 signaling pathway of the humanized antibody was evaluated according to the steps of Example 11, and the results are shown in Figure 8. The results showed that all the candidate antibodies of Yang and Shen showed antagonistic activity in a concentration-dependent manner, while the IgG isotype control was unable to inhibit the signal value of CCL1 activating ⁇ -arrestin.

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Abstract

本发明公开了一种抗CCR8抗体或其抗原结合片段。本发明的抗CCR8抗体能够特异性识别人CCR8蛋白，能阻断CCR8与CCL1/CCL18的信号通路，能够特异性识别表达CCR8的细胞，对表达CCR8的靶标细胞具有靶点特异的ADCC活性。

Description

一种抗CCR8抗体或其抗原结合片段 技术领域
本发明属于肿瘤免疫疗法和分子免疫学领域，具体涉及一种抗CCR8抗体或其抗原结合片段。
背景技术
趋化因子受体(Chemokine receptor)是表达在一些特定细胞表面的7跨膜G蛋白偶联受体(GPCRs)。趋化因子受体与细胞外的配体趋化因子结合，引发钙离子内流而产生细胞趋化反应，从而诱导细胞到生物体的特定部位。CCR8及其配体CCL1/CCL18的结合正反馈调节肿瘤微环境的免疫抑制功能。
CCR8(chemokine(C-C motif)receptor 8)是一种肿瘤浸润的Treg中高表达趋化因子受体，结构与G蛋白耦连受体相似，属于七次跨膜蛋白。CCR8在肿瘤浸润的调节性T细胞(Treg)上特异性表达，而在外周血Treg上基本不表达，调节肿瘤微环境免疫抑制功能。CCR8通过激活癌细胞上的CCR8受体，引起癌细胞的增殖、迁移和凋亡抵抗。另外，CCL1可募集Treg到肿瘤生态位，并导致CD4+T细胞转化为Treg。
CCR8已知受体有四个：CCL1、CCL8、CCL16和CCL18，其中主要受体-趋化因子CCL1，在炎症发生位置会表达上调。CCL1能够招募FOXp3+CCR8+Treg细胞浸润到肿瘤组织中，发挥免疫抑制功能，同时可以诱导FOXp3+Treg细胞表面CCR8表达上调，诱发Ca2+流动，诱导stat3依赖性的Foxp3、CD39、IL-10和颗粒酶B表达上调，进而增强这些肿瘤浸润的Treg细胞的免疫抑制活性。因此，靶向CCR8的药物可以通过耗竭肿瘤浸润的FOXp3+CCR8+Treg细胞，或阻断CCL1/CCR8途径，以增强抗肿瘤免疫效应。
发明内容
本发明开发的抗CCR8抗体能够特异性识别人CCR8蛋白，能阻断CCR8与CCL1/CCL18的信号通路，能够特异性识别表达CCR8的细胞，对表达CCR8的靶标细胞具有靶点特异的ADCC活性。
本发明采用的技术方案如下：
一种抗CCR8抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中，
(a)重链可变区的HCDR1，选自SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列，或选自与SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(b)重链可变区的HCDR2，选自SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列，或选自与SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(c)重链可变区的HCDR3，选自SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列，或选自与SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(d)轻链可变区的LCDR1，选自SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列，或选自与SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(e)轻链可变区的LCDR2，选自SEQ ID NO：7、15、23、31、58、62、66、 70、74的氨基酸序列，或选自与SEQ ID NO：7、15、23、31、58、62、66、70、74的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：7、15、23、31、58、62、66、70、74的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；和/或
(f)轻链可变区的LCDR3，选自SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列，或选自与SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
在可选的实施方案中，所述的抗CCR8抗体或其抗原结合片段重链可变区的HCDR1、HCDR2、HCDR3，和轻链可变区的LCDR1、LCDR2、LCDR3选自如下(1)-(14)中任一项氨基酸序列：
(1)SEQ ID NO：2所示的HCDR1，SEQ ID NO：3所示的HCDR2，SEQ ID NO：4所示的HCDR3，SEQ ID NO：6所示的LCDR1，SEQ ID NO：7所示的LCDR2，SEQ ID NO：8所示的LCDR3；
(2)SEQ ID NO：10所示的HCDR1，SEQ ID NO：11所示的HCDR2，SEQ ID NO：12所示的HCDR3，SEQ ID NO：14所示的LCDR1，SEQ ID NO：15所示的LCDR2，SEQ ID NO：16所示的LCDR3；
(3)SEQ ID NO：18所示的HCDR1，SEQ ID NO：19所示的HCDR2，SEQ ID NO：20所示的HCDR3，SEQ ID NO：22所示的LCDR1，SEQ ID NO：23所示的LCDR2，SEQ ID NO：24所示的LCDR3；
(4)SEQ ID NO：26所示的HCDR1，SEQ ID NO：27所示的HCDR2，SEQ ID NO：28所示的HCDR3，SEQ ID NO：30所示的LCDR1，SEQ ID NO：31所示的LCDR2，SEQ ID NO：32所示的LCDR3；
(5)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ ID NO：43所示的HCDR3，SEQ ID NO：57所示的LCDR1，SEQ ID NO：58所示的LCDR2，SEQ ID NO：59所示的LCDR3；
(6)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ  ID NO：43所示的HCDR3，SEQ ID NO：61所示的LCDR1，SEQ ID NO：62所示的LCDR2，SEQ ID NO：63所示的LCDR3；
(7)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ ID NO：43所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
(8)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：57所示的LCDR1，SEQ ID NO：58所示的LCDR2，SEQ ID NO：59所示的LCDR3；
(9)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：61所示的LCDR1，SEQ ID NO：62所示的LCDR2，SEQ ID NO：63所示的LCDR3；
(10)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
(11)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：69所示的LCDR1，SEQ ID NO：70所示的LCDR2，SEQ ID NO：71所示的LCDR3；
(12)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：73所示的LCDR1，SEQ ID NO：74所示的LCDR2，SEQ ID NO：75所示的LCDR3；
(13)SEQ ID NO：49所示的HCDR1，SEQ ID NO：50所示的HCDR2，SEQ ID NO：51所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
(14)SEQ ID NO：53所示的HCDR1，SEQ ID NO：54所示的HCDR2，SEQ ID NO：55所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3。
在可选的实施方案中，所述的抗CCR8抗体或其抗原结合片段，包括重链可变区和轻链可变区，其中，
(a)所述重链可变区具有SEQ ID NO：1、9、17、25、33、34、35、36、40、44、48、52给出的任一氨基酸序列，
或与SEQ ID NO：1、9、17、25、33、34、35、36、40、44、48、52给出的氨基酸序列具有至少50％、60％、70％、80％、85％、90％、95％或以上同一性的序列，
或与SEQ ID NO：1、9、17、25、33、34、35、36、40、44、48、52的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
(b)所述轻链可变区具有SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72给出的任一氨基酸序列，
或与SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72给出的氨基酸序列具有至少50％、60％、70％、80％、85％、90％、95％或以上同一性的序列，
或与SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
在可选的实施方案中，所述的抗CCR8抗体或其抗原结合片段的重链可变区和轻链可变区选自如下(1)-(26)中的任一氨基酸序列：
(1)SEQ ID NO：1和SEQ ID NO：5；
(2)SEQ ID NO：9和SEQ ID NO：13；
(3)SEQ ID NO：17和SEQ ID NO：21；
(4)SEQ ID NO：25和SEQ ID NO：29；
(5)SEQ ID NO：33和SEQ ID NO：37；
(6)SEQ ID NO：33和SEQ ID NO：38；
(7)SEQ ID NO：33和SEQ ID NO：39；
(8)SEQ ID NO：34和SEQ ID NO：37；
(9)SEQ ID NO：34和SEQ ID NO：38；
(10)SEQ ID NO：34和SEQ ID NO：39；
(11)SEQ ID NO：35和SEQ ID NO：37；
(12)SEQ ID NO：35和SEQ ID NO：38；
(13)SEQ ID NO：35和SEQ ID NO：39；
(14)SEQ ID NO：36和SEQ ID NO：37；
(15)SEQ ID NO：36和SEQ ID NO：38；
(16)SEQ ID NO：36和SEQ ID NO：39；
(17)SEQ ID NO：40和SEQ ID NO：56；
(18)SEQ ID NO：40和SEQ ID NO：60；
(19)SEQ ID NO：40和SEQ ID NO：64；
(20)SEQ ID NO：44和SEQ ID NO：56；
(21)SEQ ID NO：44和SEQ ID NO：60；
(22)SEQ ID NO：44和SEQ ID NO：64；
(23)SEQ ID NO：44和SEQ ID NO：68；
(24)SEQ ID NO：44和SEQ ID NO：72；
(25)SEQ ID NO：48和SEQ ID NO：64；
(26)SEQ ID NO：52和SEQ ID NO：64。
在可选的实施方案中，所述抗体或其抗原结合片段包括鼠源抗体或其抗原结合片段，嵌合抗体或其抗原结合片段，和/或人源化抗体或其抗原结合片段。
在可选的实施方案中，所述的抗CCR8抗体或其抗原结合片段还包含Fc区，所述Fc区选自小鼠IgG1、IgG2a、IgG2b和/或IgG3，或选自大鼠IgG1、IgG2a、IgG2b和/或IgG2c。
在可选的实施方案中，所述的抗CCR8抗体或其抗原结合片段还包含Fc区，所述Fc区选自人IgG1、IgG2、IgG3和/或IgG4或者与人IgG1、IgG2、IgG3、IgG4具有一个或多个氨基酸突变(优选置换、插入或缺失)的Fc区氨基酸序列。在可选的实施方案中，可以对Fc区进行改造，例如Knob-in-Hole等形式。
本发明还提供了核酸分子，其编码上述任一所述的抗CCR8抗体或其抗原结合片段。
本发明还提供了重组载体，其包含上述核酸分子。
本发明还提供了重组细胞，其包含上述核酸分子和/或上述重组载体，并能够表达所述抗CCR8抗体或其抗原结合片段。
本发明还提供了一种多功能融合蛋白，其包含上述任一所述的抗CCR8抗体或其抗原结合片段。
在可选的实施方案中，所述的多功能融合蛋白还包含一个或多个与其他抗原特异性结合的第二抗体或其抗原结合部分。
在可选的实施方案中，所述结合第二抗体或其抗原结合部分的抗原选自肿瘤 相关抗原(TAA)或免疫检查点。
在可选的实施方案中，上述任一所述的抗CCR8抗体或其抗原结合片段、任一所述的多功能融合蛋白在制备用于治疗和/或预防和/或诊断疾病的药物中的用途。
在可选的实施方案中，其中所述用途通过肿瘤免疫疗法、细胞疗法和基因疗法中的一种或多种来实现。
在可选的实施方案中，上述任一所述的抗CCR8抗体或其抗原结合片段、任一所述的多功能融合蛋白在制备治疗癌症的药物中的用途。
在可选的实施方案中，其中所述癌症为乳腺癌、卵巢癌、肺癌、肝癌、黑色素瘤、恶性胶质瘤、头颈癌、结肠直肠癌、胃癌、膀胱癌、胰腺癌、结肠癌、宫颈癌或相关肿瘤。
本发明还提供了一种药物组合物，其包含上述任一所述的抗CCR8抗体或其抗原结合片段和可接受的载体、稀释剂或赋形剂。
本发明还提供了一种药物组合物，其包含上述任一所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
有益效果
本发明开发的抗CCR8抗体能够特异性识别人CCR8蛋白，能阻断CCR8与CCL1/CCL18的信号通路，能够特异性识别表达CCR8的细胞，对表达CCR8的靶标细胞具有靶点特异的ADCC活性。
术语
为了帮助理解本文中阐述的发明，现提供以下缩写解释及术语定义。
在本文中使用以下缩写：
CDR：抗体可变区中的互补决定区；
HCDR：抗体重链可变区中的互补决定区；
LCDR：抗体轻链可变区中的互补决定区；
FR：抗体构架区，即抗体可变区中除CDR残基以外的氨基酸残基；
ELISA：酶联免疫吸附测定；
FACS：荧光激活细胞分选术。
在本说明书中，术语“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中、动物免疫血清中、噬菌体文库筛选进行重建得到分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选的抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgGl、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3和/或IgG4。
术语“抗体”和“免疫球蛋白”可互换使用，本文中所使用的部分抗体由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成，或者只有轻链恒定区(CL)。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。
术语抗体的“抗原结合片段”是指抗体的多肽片段，例如全长抗体的多肽片段，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)、线性抗体(linear antibody)、纳米抗体(如技术来自Ablynx)、结构域抗体(如技术来自Domantis)、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
术语“多肽”是指任何长度的氨基酸链，而与修饰(例如磷酸化或糖基化)无关。术语多肽包括蛋白质及其片段。多肽可以是“外源的”，意指它们是“异源的”，即是所利用的宿主细胞外来的，例如由细菌细胞产生的人多肽。本文将多肽公开为 氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名，如下所示：丙氨酸(Ala，A)、精氨酸(Arg，R)、天冬酰胺(Asn，N)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酰胺(Gln，Q)、谷氨酸(Glu，E)、甘氨酸(Gly，G)、组氨酸(His，H)、异亮氨酸(Ile，I)、亮氨酸(Leu，L)、赖氨酸(Lys，K)、甲硫氨酸(Met，M)、苯丙氨酸(Phe，F)、脯氨酸(Pro，P)、丝氨酸(Ser，S)、苏氨酸(Thr，T)、色氨酸(Trp，W)、酪氨酸(Tyr，Y)和缬氨酸(Val，V)。
本领域中有多种方法/系统来定义和描述CDR，这些系统和/或定义已经开发和精制多年，包括Kabat、Chothia、IMGT、AbM和Contact。Kabat是最常用的，基于序列变异性定义CDR；Chothia基于结构循环区域的位置基于序列变异性定义CDR；IMGT系统基于可变域结构内的序列变异性和位置定义CDR；AbM是基于牛津分子公司的AbM抗体建模软件进行定义，是Kabat和Chothia之间的折衷；Contact基于对复杂晶体结构的分析定义CDR，在多个方面与Chothia类似。
本发明的抗CCR8抗体中氨基酸位置的编号(例如Fc区的氨基酸残基)和目标区域(例如CDR)，使用Kabat系统。
关于参照多肽序列的百分比(％)氨基酸序列“同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST，BLAST-2，Clustal W，Megalign(DNASTAR)软件或FASTA程序包。
术语“单克隆抗体”指均一的仅针对某一特定抗原表位的抗体。与典型地包括针对不同抗原决定簇(表位)的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原决定簇。修饰语“单克隆”表示抗体的均一特征，不解释为需要通过任何特定方法产生的抗体。本发明的单克隆抗体优选通过重组DNA方法产生，或通过本文其它地方描述的筛选方法获得。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的单克隆抗体。在某些实施方案中，制备时用抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在某些实施方案中，通过小鼠免疫文库筛选得到所需要 的鼠源抗体。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。在某些实施方案中，建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
术语“人源化抗体”是指包括至少一条人源化抗体链(即，至少一条人源化轻链或重链)的抗体。术语“人源化抗体链”(即“人源化免疫球蛋白链”)是指具有可变区的抗体链(即分别为轻链或重链)，所述可变区包括人抗体实质性的可变框架区和互补性决定。基本上来自非人抗体的区域(CDR)(例如，至少一个CDR，两个CDR或三个CDR)。在一些实施方案中，人源化抗体链还包括恒定区(例如，在轻链的情况下，一个恒定区或其一部分，在重链的情况下，优选三个恒定区)。
术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达。此类载体在本文中称为“表达载体”。
附图说明
图1a和1b为鼠源抗CCR8抗体与CHOK1-CCR8细胞的结合活性。
图2为嵌合抗CCR8抗体与CHOK1-CCR8细胞的结合活性。
图3为嵌合抗CCR8抗体的ADCC效应结果。
图4为鼠源抗CCR8抗体对CCL1激活Tango-H_CCR8-CHO-K1细胞释放β-arestin的抑制。
图5为鼠源抗CCR8抗体的体内药效数据。
图6a和6b为人源化抗CCR8抗体与CHOK1-CCR8细胞的结合活性。
图7a和7b为人源化抗CCR8抗体的ADCC效应结果。
图8为人源化抗CCR8抗体对CCL1激活Tango-H_CCR8-CHO-K1细胞释放β-arestin的抑制。
具体实施方式
实施例1：动物免疫
以慢病毒转导CHO细胞方法构建过表达人CCR8的CCR8-CHO-K1细胞，以CCR8-284细胞作为免疫原，以1×107个总细胞接种免疫Balb/c小鼠。首次免疫半月后进行二次免疫，之后每隔一个月再次免疫。小鼠在免疫前3天采取阴性血清，每次免疫后6天剪尾取血50μL。将阴性血清及免疫血清，按比例稀释(1:0.1K，1:1K，1:10K，1:100K，1:1000K，1:10000K)，用过表达CCR8的CCR8-CHO-K1细胞采用Cell-ELISA法进行血清效价检测。当效价结果满足要求，在>1：10K的稀释度下检测到抗人CCR8抗体时，可收获大鼠脾脏和淋巴结。
实施例2：细胞融合
实验用B淋巴细胞和淋巴结细胞取自经CCR8-293细胞四次免疫的Balb/c小鼠，将脾脏和淋巴结置于细胞筛中，再将细胞筛置于50mL离心管内。吸取DMEM滴加在脾脏上，进行研磨，制成脾细胞悬液，离心，1600rpm，10分钟，去上清。用2mL红细胞裂解液重悬B细胞，室温裂解2分钟，加30mL DMEM，混匀离心1600rpm，10分钟，计数。
骨髓瘤细胞SP2/0(ATCC)于融合前一天将细胞传代，使实验时细胞处于对数生长期。将脾细胞和SP2/0按2:1的比率进行混合后，离心1600rpm，10分钟。将混合的细胞用融合液清洗两遍，离心1600rpm，10分钟。按细胞终密度为1×107个细胞/mL，加融合液将细胞悬浮，5分钟内，将细胞悬液移至电融合仪(BTX；ECM 2001)融合室内融合。融合完成后将细胞从融合室内移至含有HAT的完全培养基内，37℃孵育60分钟。孵育后将细胞铺在已含有饲养细胞的96孔板内，37℃，5％CO2培养。
实施例3：ELISA方法初步筛选阳性克隆
培养7天后对融合上清进行初筛。将CCR8-CHO-K1细胞铺到96酶标板中 (1.5×104个细胞/孔)，培养36h后，用PBST手洗两次。用4％的多聚甲醛固定，2％BSA封闭。倒掉封闭液，洗板机洗涤，3次。将融合上清取100μL/孔加入已封闭的酶标板内，37℃温育1h，弃去孔中液体。洗板机洗涤，3次。用0.5％BSA将羊抗鼠二抗-HRP(Abcam；Ab6789)稀释，100μL/孔，37℃温育。洗板机PBST洗涤，6次，并在平板纸上拍干。向板孔内加入索莱宝显色液(Solarbio；PR1200)，100μL/孔。并用铝箔纸包好，37℃避光显色。加入1mol/L HCl终止显色反应，在酶标仪上450nm读数，并对数据进行分析。选取检测上清结果OD450>1.0的细胞株作为初筛候选阳性细胞株，吸弃阳性细胞株的培养上清，添加新的HAT完全培养基。
实施例4：FACS方法进一步筛选阳性克隆
取CCR8-CHO-K1细胞转至离心管1000rpm离心5分钟。分别将100μL的3×105个稳定表达细胞等分至独立的管中，并添加100μL融合上清。将细胞在4℃下孵育60分钟，然后用过量FACS缓冲液洗涤两次。将细胞重新悬浮于100μL FACS缓冲液中，并将羊抗鼠二抗-FITC(Abcam；ab6785)添加到样品中，孵育30分钟并用过量FACS缓冲液洗涤两次。将细胞在固定缓冲液中固定，随后通过流式细胞术进行分析。FACS方法筛选出与CCR8-CHO-K1细胞特异性结合的抗体。
采用两轮有限稀释法进行杂交瘤细胞的单克隆化，采用ELISA方法检测，选取OD450>1.0的单克隆作为定株候选细胞株进行传代，无单抗的克隆选取OD450>1.0的克隆进行下一次亚克隆。
实施例5：候选细胞株抗体小样生产
将杂交瘤细胞在T75细胞培养瓶内，培养至细胞覆盖率为80-90％。将2瓶细胞上清弃去，加入30mL hybridoma-SFM，37℃，5％CO2培养。培养2-3天，观察细胞状态和培养基颜色，如培养基颜色变黄，可添加30mL新的hybridoma-SFM。培养6-7天，低速离心后收集培养上清，进行纯化。
实施例6：候选抗体与细胞的结合活性
取待测抗体按照50μg/mL为初始终浓度，5倍稀释，稀释6个梯度。取出培养箱内CCR8-CHO-K1细胞，将细胞悬液转至15mL离心管中，离心，PBS重悬 计数。留出空白对照组(Blank)、阴性对照组(NC)、实验组、阳参组(阳参BMK来源于US20210277129的1K17序列)和无关抗体组。按照约3×105个细胞/孔，将细胞悬液铺于96孔板中。
离心(1000rpm，5分钟)后用PBS清洗，再离心，重复两次去除培养基残留。弃去上清，实验组、无关抗体组分别加入100μL的一抗溶液、无关抗体溶液，重悬细胞后，室温孵育1h。空白对照组、阴性对照组使用等量的PBS进行孵育。
1h后离心，加入PBS清洗两次。弃上清后，除空白对照组加入100μL PBS外，其余样品组分别加入100μL荧光二抗稀释液(鼠源二抗来自Abcam ab6785)，室温避光孵育0.5h后，离心加入PBS清洗两次。弃去上清后，加入120μL的PBS重悬并按顺序进行流式细胞检测其平均荧光强度。将抗体的浓度取对数后作为横坐标，选用Sigmoidaldose-response(VariableSlope)方式(GraphPadPrism软件，GraphPadSoftware，SanDiego，California)进行非线性回归，得到抗体对CCR8-CHO细胞的结合活性曲线。结果见图1a和图1b。
由图1a和图1b可知，所选抗体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和Ms-15与CCR8-CHO均具有较好的结合活性，结合活性优于阳参，且远远优于阴参。
实施例7：单克隆抗体的测序
对通过FACs检测筛选得到与工程细胞CCR8-CHO结合活性好的杂交瘤进行测序，分别得到Ms-1、Ms-2、Ms-8和Ms-15的重链可变区与轻链可变区。
(1)Ms-1重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别为：SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4；
Ms-1重链可变区的氨基酸序列为SEQ ID NO：1；
Ms-1轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别为：SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8；
Ms-1轻链可变区的氨基酸序列为SEQ ID NO：5。
(2)Ms-2重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别为：SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12；
Ms-2重链可变区的氨基酸序列为SEQ ID NO：9；
Ms-2轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别为：SEQ  ID NO：14、SEQ ID NO：15和SEQ ID NO：16；
Ms-2轻链可变区的氨基酸序列为SEQ ID NO：13。
(3)Ms-8重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别为：SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20；
Ms-8重链可变区的氨基酸序列为SEQ ID NO：17；
Ms-8轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别为：SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24；
Ms-8轻链可变区的氨基酸序列为SEQ ID NO：21。
(4)Ms-15重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别为：SEQ ID NO：26、SEQ ID NO：27和SEQ ID NO：28；
Ms-15重链可变区的氨基酸序列为SEQ ID NO：25；
Ms-15轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别为：SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：32；
Ms-15轻链可变区的氨基酸序列为SEQ ID NO：29。
实施例8：嵌合抗体的构建与表达
将实施例7中测序得到的抗体片段进行基因合成，构建到人IgG框架中，而后利用分子克隆技术，将抗体片段插入PCDNA3.1载体中，构建成哺乳动物细胞表达质粒，利用脂质体转染方式，导入宿主细胞株CHO细胞，利用细胞Fed-batch获得发酵上清液，取发酵液上清进行亲和层析纯化，最终纯化得到构建的嵌合抗体：CH-1、CH-2、CH-8和CH-15。
嵌合抗体CH-1、CH-2、CH-8和CH-15的CDR和可变结构域的氨基酸序列分别对应实施例7中Ms-1、Ms-2、Ms-8和Ms-15的CDR和可变结构域的氨基酸序列。
嵌合抗体CH-1、CH-2、CH-8和CH-15的重链恒定区的氨基酸序列都相同，如SEQ ID NO：76所示，轻链恒定区的氨基酸序列如SEQ ID NO：77所示。
实施例9：嵌合抗体的结合活性
采用FACs方法检测嵌合抗体的结合活性，二抗为PE F(ab')2羊抗人IgG Fc(Biolegend，Cat:398004)，检测步骤同实施例6，结果如图2所示。
由图2可知，嵌合抗体CH-1、CH-2、CH-8和CH-15与CCR8-CHO-K1细胞均有很好的结合活性，跟阳参活性相当。
实施例10：嵌合抗体对靶细胞的ADCC活性
使用过表达人CCR8的CCR8-CHOK1细胞作为靶细胞，1000rpm室温离心4分钟并使用RPMI1640基础培养基(含5％FBS)重悬后，以1×104个细胞/孔、50μL/孔铺于96孔板；使用RPMI1640基础培养基(含5％FBS)稀释抗体，起始浓度为10μg/mL，而后10倍梯度稀释，共7个浓度梯度，100μL/孔；重悬NK细胞，以50μL/孔加入对应孔中，其中靶细胞为CCR8-CHOK1时效靶比为3：1。同时设置靶细胞最大裂解孔(M)、靶细胞自发释放孔(ST)、效应细胞自发释放孔(SE)、总体积校正空白孔(BV)和培养基空白对照孔(BM)。静置10分钟后，1000rpm室温离心4分钟，于5％CO2、37℃二氧化碳细胞培养箱中孵育4h。提前45分钟在M、B-V孔加入裂解液，混匀，孵育结束后1000rpm室温离心4分钟。吸取50μL上清至LDH分析板，加入50μL/孔分析缓冲液(assay buffer)溶解的底物，室温避光反应30分钟，加入50μL/孔终止液，静置10分钟，于490nm进行读数，计算细胞死亡率。
将构建抗体的浓度取对数后作为横坐标，选用Sigmoidaldose-response(VariableSlope)方式(GraphPadPrism软件，GraphPadSoftware，SanDiego，California)进行非线性回归，得到目标抗体对靶细胞的ADCC活性曲线。
由图3可知，IgG1同型对照未显示对CCR8-CHOK1细胞的杀伤，嵌合抗体CH-1、CH-2、CH-8和CH-15与阳参显示对CCR8-CHO-K1细胞的裂解死亡，且呈浓度依赖性。
实施例11：抗体对CCL1的拮抗活性
CCR8是CCL1的唯一已知受体，CCL1与细胞表面的CCR8结合，引起下游信号转导。抗CCR8抗体可以通过阻断CCR8抑制该信号转导途径。因此检测抗CCR8抗体对CCL1-CCR8通路的阻断能力，使用β-arrestin作为靶标，采用 Tango-H_CCR8-CHO-K1(吉满，GM-C13190)。简言之，使用诱导CCR8表达的激活剂Switch-On Reagent(吉满，cat:GM-040501A)加入Tango-H_CCR8-CHO-K1细胞中，持续培养48h后，消化后，200g离心收集细胞，使用ONE-GloTM Luciferase Assay System(Biolegend)中的分析缓冲液重悬细胞，按照细胞浓度为1.5×105个细胞/mL、100μL体积加入96孔测定板，于细胞培养箱中孵育过夜。使用分析缓冲液稀释抗体，起始浓度为15μg/mL，而后5倍梯度稀释，共9个浓度梯度，50μL/孔孵育1h。而后加入50μLCCL1(Biolegend)作为激活剂，终浓度0.06μg/mL，孵育7h后，酶标仪读取荧光值。将构建抗体的浓度取对数后作为横坐标，选用Sigmoidaldose-response(VariableSlope)方式(GraphPadPrism软件，GraphPadSoftware，SanDiego，California)进行非线性回归，得到目标抗体对CCL1与CCR8结合后，引发下游信号分子β-arrestin的抑制曲线。
结果如图4所示，IgG1为同型对照组，无法抑制CCL1激活β-arrestin的信号值，阳参与候选抗体均显示拮抗活性，且呈浓度依赖活性。
实施例12：鼠源抗体的体内药效实验
构建人源化CCR8的C57小鼠(百奥赛图，C57BL/6-CCR8tm1(CCR8)/Bcgen)结肠癌MC38细胞皮下瘤模型，对抗体Ms-2和Ms-15进行体内抗肿瘤药效评价。复苏MC38细胞，进行细胞培养，消化后制成细胞悬液。细胞培养至对数生长期时，收集细胞，将肿瘤细胞悬液注射到Balb/C裸鼠皮下，每只接种100μL细胞悬液，包含5×105个细胞。观察肿瘤生长情况，当皮下生长的肿瘤生长至100mm3左右时，将动物按肿瘤体积随机分组，并给予CCR8抗体治疗。实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周三次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。分别给予分组后的荷瘤小鼠腹腔注射抗体和PBS，每周给药2次，每次10.0mg/kg，共给药5次。
化合物的抑瘤疗效用TGI(％)评价。TGI(％)的计算公式为：TGI(％)＝【1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。
小鼠结肠癌MC38细胞皮下瘤模型荷瘤hCCR8小鼠给予抗体的肿瘤生长曲线如图5所示，其中横坐标表示开始治疗后的天数，纵坐标表示肿瘤体积。候选 抗体Ms-2和Ms-15有较高的肿瘤抑制作用，其效果显著优于PBS组和阳性对照组，其肿瘤抑瘤率TGI(％)大于50％。
实施例13：人源化单克隆抗体评价
将嵌合抗体的可变区进行人源化改造，设计原则为不引入糖基化、脱酰胺、异构化等蛋白修饰位点，不引入整合素结合位点、半胱氨酸，框架区重要氨基酸的回复突变应保持原有的理化生化活性。具体方法如下：
使用IgBLAST工具分别将鼠源序列Ms-15、Ms-2分别与人Germline序列比对，将FR更换为序列相似性最高的人Germline序列。Ms-2重链设计模板选取IGHV1大类，轻链设计模板选取IGKV3大类。Ms-15重链设计模板选取IGHV3大类，轻链设计模板选取IGKV3大类，得到人源化抗体的序列。而后在此人源化基础上，将影响抗体亲和力的几个重要氨基酸进行回复突变，即突变为原鼠源的FR位点。人源化百分比为设计序列Framework与Germline序列Framework的相似度比值，将设计好的人源化序列与人源Germline序列进行比对，选择抗体的人源化程度百分比在90％以上的序列。
将嵌合抗体CH-2的重链序列设计为4个人源化序列，分别为Hu-2-H1、Hu-2-H2、Hu-2-H3和Hu-2-H4，其中HCDR1、HCDR2和HCDR3的氨基酸序列分别为SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12。
将嵌合抗体CH-2的轻链序列设计为3个人源化序列，分别为Hu-2-L1、Hu-2-L2和Hu-2-L3，其中LCDR1、LCDR2和LCDR3的氨基酸序列分别为SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16。
上述4条重链和3条轻链的可变区的氨基酸序列分别如表1所示。
表1

将上述4条人源化重链和3条人源化轻链组合为12条抗体，如表2所示。
表2
将嵌合抗体CH-15的重链设计为1个人源化序列，CDR2含一个DS(天冬氨酸异构化风险)，将人源化序列中DS突变成ES，并另行设计回复突变的另一条重链。重链CDR3同时包含一个RGD(整合素结合位点)，设计时在回复突变序列的基础上分别将RGD突变成RGE和RAD，增加两条链，因此共计4条链，分别为Hu-15-H1p1、Hu-15-H2p1、Hu-15-H2p1p2和Hu-15-H2p1p3。
CH-15轻链的CDR1含一个NG(糖基化风险)，设计时在回复突变最多的基础上分别将NG突变成QG和NA，轻链共设计5条链，分别为Hu-15-L1、Hu-15-L2、Hu-15-L3、Hu-15-L3p1和Hu-15-L3p2。
上述4条重链和5条轻链的可变区和CDR的氨基酸序列分别如表3所示。
表3

将上述4条重链和5条轻链组成10条抗体如表4所示。
表4
基因合成表3中的氨基酸序列，并表达上述人源化序列组合，按照前述方法进行构建表达，制备表4中的人源化抗体。
按照实施例6的步骤进行人源化抗体的结合活性评价，结果如图6a和6b所示。由结果可知，人源化抗体与CHOK1-CCR8细胞都有很好的结合活性，结合活性跟嵌合抗体和BMK(来源于US20210277129的1K17序列)相当，远远优于无关抗体。
按照实施例10的步骤进行人源化抗体的ADCC活性评价，结果如图7a和 7b所示。由结果可知，人源化抗体Hu-2-Z4、Hu-2-Z6、Hu-2-Z9、Hu-15-Z1、Hu-15-Z3和Hu-15-Z6都有较好的ADCC活性，其细胞裂解率跟嵌合抗体和BMK(来源于US20210277129的1K17序列)相当，远远优于无关抗体。
按照实施例11的步骤进行人源化抗体的CCR-CCL1信号通路的拮抗剂活性评价，结果如图8所示。结果表示，阳参与候选抗体均显示拮抗活性，且呈浓度依赖活性，而IgG同型对照无法抑制CCL1激活β-arrestin的信号值。

Claims (19)

  1. 一种抗CCR8抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其特征在于，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3，其中，
    (a)重链可变区的HCDR1，选自SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列，或选自与SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：2、10、18、26、41、45、49、53的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (b)重链可变区的HCDR2，选自SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列，或选自与SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：3、11、19、27、42、46、50、54的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (c)重链可变区的HCDR3，选自SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列，或选自与SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：4、12、20、28、43、47、51、55的任一项氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (d)轻链可变区的LCDR1，选自SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列，或选自与SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：6、14、22、30、57、61、65、69、73的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (e)轻链可变区的LCDR2，选自SEQ ID NO：7、15、23、31、58、62、66、70、74的氨基酸序列，或选自与SEQ ID NO：7、15、23、31、58、62、66、70、74的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：7、15、23、31、58、62、66、70、74的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；和/或
    (f)轻链可变区的LCDR3，选自SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列，或选自与SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或选自与SEQ ID NO：8、16、24、32、59、63、67、71、75的氨基酸序列相比具有一个或多个(优选2个或3个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
  2. 根据权利要求1所述的抗CCR8抗体或其抗原结合片段，其特征在于，所述重链可变区的HCDR1、HCDR2、HCDR3，和轻链可变区的LCDR1、LCDR2、LCDR3选自如下(1)-(14)中任一项氨基酸序列：
    (1)SEQ ID NO：2所示的HCDR1，SEQ ID NO：3所示的HCDR2，SEQ ID NO：4所示的HCDR3，SEQ ID NO：6所示的LCDR1，SEQ ID NO：7所示的LCDR2，SEQ ID NO：8所示的LCDR3；
    (2)SEQ ID NO：10所示的HCDR1，SEQ ID NO：11所示的HCDR2，SEQ ID NO：12所示的HCDR3，SEQ ID NO：14所示的LCDR1，SEQ ID NO：15所示的LCDR2，SEQ ID NO：16所示的LCDR3；
    (3)SEQ ID NO：18所示的HCDR1，SEQ ID NO：19所示的HCDR2，SEQ ID NO：20所示的HCDR3，SEQ ID NO：22所示的LCDR1，SEQ ID NO：23所示的LCDR2，SEQ ID NO：24所示的LCDR3；
    (4)SEQ ID NO：26所示的HCDR1，SEQ ID NO：27所示的HCDR2，SEQ ID NO：28所示的HCDR3，SEQ ID NO：30所示的LCDR1，SEQ ID NO：31所示的LCDR2，SEQ ID NO：32所示的LCDR3；
    (5)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ ID NO：43所示的HCDR3，SEQ ID NO：57所示的LCDR1，SEQ ID NO：58所示的LCDR2，SEQ ID NO：59所示的LCDR3；
    (6)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ ID NO：43所示的HCDR3，SEQ ID NO：61所示的LCDR1，SEQ ID NO：62所示的LCDR2，SEQ ID NO：63所示的LCDR3；
    (7)SEQ ID NO：41所示的HCDR1，SEQ ID NO：42所示的HCDR2，SEQ ID NO：43所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
    (8)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：57所示的LCDR1，SEQ ID NO：58所示的LCDR2，SEQ ID NO：59所示的LCDR3；
    (9)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：61所示的LCDR1，SEQ ID NO：62所示的LCDR2，SEQ ID NO：63所示的LCDR3；
    (10)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
    (11)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：69所示的LCDR1，SEQ ID NO：70所示的LCDR2，SEQ ID NO：71所示的LCDR3；
    (12)SEQ ID NO：45所示的HCDR1，SEQ ID NO：46所示的HCDR2，SEQ ID NO：47所示的HCDR3，SEQ ID NO：73所示的LCDR1，SEQ ID NO：74所示的LCDR2，SEQ ID NO：75所示的LCDR3；
    (13)SEQ ID NO：49所示的HCDR1，SEQ ID NO：50所示的HCDR2，SEQ ID NO：51所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3；
    (14)SEQ ID NO：53所示的HCDR1，SEQ ID NO：54所示的HCDR2，SEQ ID NO：55所示的HCDR3，SEQ ID NO：65所示的LCDR1，SEQ ID NO：66所示的LCDR2，SEQ ID NO：67所示的LCDR3。
  3. 根据权利要求1或2所述的抗CCR8抗体或其抗原结合片段，包括重链可变区和轻链可变区，其中，
    (a)所述重链可变区具有SEQ ID NO：1、9、17、25、33、34、35、36、40、 44、48、52给出的任一氨基酸序列，
    或与SEQ ID NO：1、9、17、25、33、34、35、36、40、44、48、52给出的氨基酸序列具有至少50％、60％、70％、80％、85％、90％、95％或以上同一性的序列，
    或与SEQ ID NO：1、9、17、25、33、34、35、36、40、44、48、52的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；
    (b)所述轻链可变区具有SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72给出的任一氨基酸序列，
    或与SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72给出的氨基酸序列具有至少50％、60％、70％、80％、85％、90％、95％或以上同一性的序列，
    或与SEQ ID NO：5、13、21、29、37、38、39、56、60、64、68、72的任一氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
  4. 根据权利要求1-3任一项所述的抗CCR8抗体或其抗原结合片段，其特征在于，所述重链可变区和轻链可变区选自如下(1)-(26)中的任一氨基酸序列：
    (1)SEQ ID NO：1和SEQ ID NO：5；
    (2)SEQ ID NO：9和SEQ ID NO：13；
    (3)SEQ ID NO：17和SEQ ID NO：21；
    (4)SEQ ID NO：25和SEQ ID NO：29；
    (5)SEQ ID NO：33和SEQ ID NO：37；
    (6)SEQ ID NO：33和SEQ ID NO：38；
    (7)SEQ ID NO：33和SEQ ID NO：39；
    (8)SEQ ID NO：34和SEQ ID NO：37；
    (9)SEQ ID NO：34和SEQ ID NO：38；
    (10)SEQ ID NO：34和SEQ ID NO：39；
    (11)SEQ ID NO：35和SEQ ID NO：37；
    (12)SEQ ID NO：35和SEQ ID NO：38；
    (13)SEQ ID NO：35和SEQ ID NO：39；
    (14)SEQ ID NO：36和SEQ ID NO：37；
    (15)SEQ ID NO：36和SEQ ID NO：38；
    (16)SEQ ID NO：36和SEQ ID NO：39；
    (17)SEQ ID NO：40和SEQ ID NO：56；
    (18)SEQ ID NO：40和SEQ ID NO：60；
    (19)SEQ ID NO：40和SEQ ID NO：64；
    (20)SEQ ID NO：44和SEQ ID NO：56；
    (21)SEQ ID NO：44和SEQ ID NO：60；
    (22)SEQ ID NO：44和SEQ ID NO：64；
    (23)SEQ ID NO：44和SEQ ID NO：68；
    (24)SEQ ID NO：44和SEQ ID NO：72；
    (25)SEQ ID NO：48和SEQ ID NO：64；
    (26)SEQ ID NO：52和SEQ ID NO：64；
  5. 根据权利要求1-4任一项所述的抗CCR8抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括鼠源抗体或其抗原结合片段，嵌合抗体或其抗原结合片段，和/或人源化抗体或其抗原结合片段。
  6. 根据权利要求1-4任一项所述的抗CCR8抗体或其抗原结合片段，其特征在于，其还包含Fc区，所述Fc区选自小鼠IgG1、IgG2a、IgG2b和/或IgG3，或选自大鼠IgG1、IgG2a、IgG2b和/或IgG2c。
  7. 根据权利要求1-4任一项所述的抗CCR8抗体或其抗原结合片段，其特征在于，其还包含Fc区，所述Fc区选自人IgG1、IgG2、IgG3和/或IgG4或者与人IgG1、IgG2、IgG3、IgG4具有一个或多个氨基酸突变(优选置换、插入或缺失)的Fc区氨基酸序列。
  8. 核酸分子，其编码权利要求1-7任一项所述的抗CCR8抗体或其抗原结合片段。
  9. 重组载体，其包含权利要求8的核酸分子。
  10. 重组细胞，其包含权利要求8的核酸分子和/或权利要求9的重组载体，并能够表达所述抗CCR8抗体或其抗原结合片段。
  11. 一种多功能融合蛋白，其包含权利要求1-7任一项所述的抗CCR8抗体或其抗原结合片段。
  12. 根据权利要求11所述的多功能融合蛋白，其还包含一个或多个与其他 抗原特异性结合的第二抗体或其抗原结合部分。
  13. 根据权利要求12所述的多功能融合蛋白，其中所述结合第二抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
  14. 权利要求1-7任一项所述的抗CCR8抗体或其抗原结合片段、权利要求11-13任一项所述的多功能融合蛋白在制备用于治疗和/或预防和/或诊断疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其中所述用途通过肿瘤免疫疗法、细胞疗法和基因疗法中的一种或多种来实现。
  16. 权利要求1-7任一项所述的抗CCR8抗体或其抗原结合片段、权利要求11-13任一项所述的多功能融合蛋白在制备治疗癌症的药物中的用途。
  17. 根据权利要求16所述的用途，其中所述癌症为乳腺癌、卵巢癌、肺癌、肝癌、黑色素瘤、恶性胶质瘤、头颈癌、结肠直肠癌、胃癌、膀胱癌、胰腺癌、结肠癌、宫颈癌或相关肿瘤。
  18. 一种药物组合物，其包含权利要求1-7任一项所述抗CCR8抗体或其抗原结合片段和可接受的载体、稀释剂或赋形剂。
  19. 一种药物组合物，其包含权利要求11-13任一项所述的多功能融合蛋白和可接受的载体、稀释剂或赋形剂。
PCT/CN2023/086391 2022-04-07 2023-04-06 一种抗ccr8抗体或其抗原结合片段 WO2023193732A1 (zh)

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WO2022042690A1 (zh) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Ccr8抗体及其应用

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WO2022004760A1 (ja) * 2020-06-30 2022-01-06 塩野義製薬株式会社 抗ｃｃｒ８抗体と化学療法剤の併用
WO2022042690A1 (zh) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Ccr8抗体及其应用

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