WO2024120418A1 - Anticorps anti-ccr8 et utilisation associée - Google Patents

Anticorps anti-ccr8 et utilisation associée Download PDF

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WO2024120418A1
WO2024120418A1 PCT/CN2023/136644 CN2023136644W WO2024120418A1 WO 2024120418 A1 WO2024120418 A1 WO 2024120418A1 CN 2023136644 W CN2023136644 W CN 2023136644W WO 2024120418 A1 WO2024120418 A1 WO 2024120418A1
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seq
antibody
amino acid
acid sequence
variable region
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PCT/CN2023/136644
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English (en)
Chinese (zh)
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郭建
霍永庭
芦迪
肖亮
张轶博
欧颖烨
罗颖
张喆
李永锋
耿梦圆
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广东菲鹏制药股份有限公司
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Publication of WO2024120418A1 publication Critical patent/WO2024120418A1/fr

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  • the present disclosure relates to the field of biotechnology, and in particular, to an anti-CCR8 antibody and an application thereof.
  • Chemokine receptor 8 is a seven-transmembrane G protein-coupled receptor that belongs to the CC subfamily of chemokine receptors.
  • the present disclosure develops an anti-CCR8 antibody, which in some embodiments can enhance the body's anti-tumor immune response and improve the survival rate of tumor patients.
  • the present disclosure discloses an anti-human CCR8 antibody, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12; in some embodiments, wherein,
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.2;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.6;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.8;
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.10; or
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No.11, and
  • the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12;
  • the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system; in some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat numbering system. In some embodiments, the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the IMGT numbering system.
  • the anti-human CCR8 antibody as described in any of the above items, wherein
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.14
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.15
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.20
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.21
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.23
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.24
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.26
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.27;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.28
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.29
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.30
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.32
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.33
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.35
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.36;
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.37
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.38
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • the anti-human CCR8 antibody as described in any of the above items comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively.
  • the anti-human CCR8 antibody comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively, and the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27, respectively.
  • the anti-human CCR8 antibody as described in any of the above items is a murine antibody, a chimeric antibody or a humanized antibody.
  • the anti-human CCR8 antibody as described in any of the above items wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present disclosure provides an anti-human CCR8 antibody, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the anti-human CCR8 antibody as described in any of the above items, wherein the heavy chain variable region of the antibody comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region comprises an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.1
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO.2
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.4;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.6;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.8;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.11, and the light chain variable region comprises the amino acid sequence of SEQ ID NO.12;
  • the anti-human CCR8 antibody as described in any of the above items the antibody further comprises a constant region; in some embodiments, the heavy chain constant region of the antibody is selected from the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the constant region of ⁇ or ⁇ chain; in some embodiments, the species of the constant region is mouse or human; in some embodiments, the heavy chain constant region is a mouse IgG2a constant region, a mouse IgG1 constant region, a human IgG1 constant region; in some embodiments, the heavy chain constant region is a human IgG1 (DLE) or human IgG1 (DE) constant region; and/or the light chain constant region is a mouse ⁇ constant region or a human ⁇ constant region.
  • the heavy chain constant region includes the amino acid sequence of SEQ ID NO.39, 41 or 42, and the light chain constant region includes the amino acid sequence of SEQ ID NO.40 or 43.
  • the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.44, and the light chain comprises the amino acid sequence of SEQ ID NO.45; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.46, and the light chain comprises the amino acid sequence of SEQ ID NO.47; or the anti-human CCR8 antibody heavy chain comprises the amino acid sequence of SEQ ID NO.48, and the light chain comprises the amino acid sequence of SEQ ID NO.49.
  • the anti-human CCR8 antibody as described in any of the above items is a full-length antibody or an antigen-binding fragment selected from any one of F(ab’)2, Fab’-SH, Fab’, Fab, scFab, dsFv, (dsFv)2, Fv and scFv.
  • the invention discloses an antibody that competes with any of the above anti-human CCR8 antibodies for binding to human CCR8, or an antibody that binds to the same epitope as any of the above anti-human CCR8 antibodies.
  • the anti-human CCR8 antibody can specifically bind to human CCR8; in some embodiments, the anti-human CCR8 antibody can bind to Raji cells expressing human CCR8 with an EC50 value of ⁇ 5nM (e.g., ⁇ 4.50nM, ⁇ 3.00nM, ⁇ 2.00nM, ⁇ 1.00nM, ⁇ 0.9nM, ⁇ 0.8nM, ⁇ 0.7nM, ⁇ 0.6nM, ⁇ 0.5nM, ⁇ 0.4nM, ⁇ 0.3nM, ⁇ 0.2nM, ⁇ 0.1nM, ⁇ 0.09nM, ⁇ 0.08nM or less), wherein the EC50 value is determined by flow cytometry; in some embodiments, the EC50 value is determined by the method of Example 2 of the present application.
  • ⁇ 5nM e.g., ⁇ 4.50nM, ⁇ 3.00nM, ⁇ 2.00nM, ⁇ 1.00nM, ⁇ 0.9nM, ⁇ 0.8nM, ⁇ 0.7
  • the anti-human CCR8 antibody has the function of inhibiting tumor growth; in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (e.g., greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or greater); in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of the present disclosure;
  • the anti-human CCR8 antibody has ADCC activity; in some embodiments, the ADCC activity of the anti-human CCR8 antibody is determined by the method of Example 4 of the present disclosure.
  • the anti-human CCR8 antibody as described in any of the above items has the function of inhibiting tumor growth; in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is greater than or equal to 30% (e.g., greater than 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or greater); in some embodiments, the anti-human CCR8 antibody tumor inhibition rate is determined by the method of Example 5 of the present disclosure;
  • the anti-human CCR8 antibody as described in any of the above items has ADCC activity; in some embodiments, the ADCC activity of the anti-human CCR8 antibody is determined by the method of Example 4 of the present disclosure.
  • the present disclosure also provides a multispecific antibody, wherein the multispecific antibody comprises any of the anti-human CCR8 antibodies described above.
  • the antibody is a bispecific antibody.
  • the present disclosure also provides an antibody conjugate, which includes the anti-human CCR8 antibody described in any of the above.
  • the antibody conjugate also includes a therapeutic agent or an imaging agent coupled to the antibody; in some embodiments, the antibody conjugate also includes biotin or a biotin derivative coupled to the antibody; in some embodiments, the antibody conjugate also includes a solid phase carrier coupled to the antibody; in some embodiments, the antibody conjugate also includes a marker coupled to the antibody; in some embodiments, the marker is selected from at least one of a fluorescent dye, an enzyme, a radioactive isotope, a chemiluminescent agent, and a nanoparticle marker; in some embodiments, the marker is colloidal gold.
  • the present disclosure also provides a chimeric antigen receptor (CAR), which comprises an antigen binding domain, and the antigen binding domain comprises the anti-human CCR8 antibody described in any one of the above.
  • CAR chimeric antigen receptor
  • the present disclosure also provides a CAR-immune cell, wherein the CAR-immune cell expresses the chimeric antigen receptor described above.
  • the present disclosure also provides an isolated nucleic acid encoding the anti-human CCR8 antibody described in any one of the above.
  • the present disclosure also provides a cell comprising the nucleic acid of claim 8.
  • the present disclosure also provides a pharmaceutical composition, comprising: the anti-human CCR8 antibody, multispecific antibody, antibody conjugate, cell or nucleic acid described in any of the foregoing items.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present disclosure also provides the use of any of the above anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or pharmaceutical compositions in the preparation of products having at least one of the following uses: diagnosing diseases related to abnormal human CCR8 expression, treating diseases related to abnormal human CCR8 expression or detecting human CCR8 expression; in some embodiments, the disease related to abnormal human CCR8 expression is a tumor.
  • the tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the present disclosure provides a method for treating a disease, comprising administering to a subject in need thereof a therapeutically effective amount of any of the above anti-human CCR8 antibodies, multispecific antibodies, antibody conjugates, cells, nucleic acids or pharmaceutical compositions.
  • the disease is abnormal expression of human CCR8.
  • the disease is a tumor.
  • the tumor with abnormal human CCR8 expression is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the present disclosure also provides an anti-human CCR8 antibody, a multispecific antibody, an antibody conjugate, a cell, a nucleic acid or a pharmaceutical composition as described in any of the foregoing for use as a drug.
  • the drug is used to treat a disease.
  • the disease is a disease related to abnormal expression of human CCR8.
  • the disease related to abnormal expression of human CCR8 is a tumor.
  • the tumor with abnormal expression of human CCR8 is preferably breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, bladder cancer, gastric cancer, cervical cancer, colon cancer, sarcoma, liver cancer or lung cancer.
  • the treatment described in any of the above further comprises administering to the subject an additional therapeutic drug.
  • the additional therapeutic drug is a chemotherapeutic agent, such as a platinum complex, taxane, pemetrexed, gemcitabine, fluorouracil, irinotecan, etoposide or doxorubicin, etc.
  • the present disclosure also provides a method for detecting or measuring human CCR8, comprising the step of using the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items to detect or measure human CCR8.
  • the present disclosure also provides a kit, comprising the anti-human CCR8 antibody or antibody conjugate as described in any of the preceding items; in some embodiments, the kit is used to detect or measure human CCR8.
  • the present disclosure also provides a method for preparing the anti-human CCR8 antibody as described in any of the preceding items, comprising the steps of culturing the cell as described in any of the preceding items, and then isolating and purifying to obtain the anti-human CCR8 antibody.
  • the anti-human CCR8 antibodies disclosed in the present disclosure can specifically bind to human CCR8; in some embodiments, the antibodies have significant anti-tumor effects both in vitro and in vivo; in some embodiments, they also have good ADCC activity.
  • FIG1 is a graph showing the binding test results of hybridoma purified antibodies and Raji-hCCR8;
  • FIG2 is a graph showing the binding test results of the recombinant antibody and Raji-hCCR8;
  • FIG3 is a diagram showing the binding detection results of the recombinant antibody and Treg
  • FIG4 is a graph showing the binding test results of the recombinant antibody and Raji;
  • FIG5 is a graph showing the binding test results of the recombinant antibody and PBMC
  • FIG6 is a graph showing the binding test results of the recombinant antibody and Raji-hCCR4;
  • FIG7 is a graph showing the results of a recombinant antibody ADCC activity experiment
  • FIG8 is a graph showing the results of an experiment in which anti-CCR8 antibodies inhibit tumor growth.
  • amino acid refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs or amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine; common natural amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), 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
  • Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.
  • antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, chimeric antibodies, humanized antibodies, full-length antibodies, or antigen-binding fragments thereof (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Natural antibodies refer to naturally occurring immunoglobulin molecules.
  • natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains. From N to C-terminus, each heavy chain has a heavy chain variable region (VH, also known as a variable heavy domain), followed by a heavy chain constant region, and the natural IgG heavy chain constant region generally includes three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a light chain variable region (VL, also known as a variable light domain), followed by a light chain constant region (CL, also known as a light chain constant domain).
  • VH heavy chain variable region
  • CL light chain constant region
  • full length antibody or “intact antibody” refers to an antibody that comprises a structure substantially similar to a native antibody structure, or an antibody whose heavy chain comprises an Fc region.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in antibody binding to an antigen.
  • VH antibody heavy chain variable region
  • VL light chain variable region
  • FR conserved framework regions
  • CDR Complementarity determining region
  • VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N terminus) to the carboxyl terminus (also called the C terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs refer to more than two CDRs in the heavy chain and light chain of the antibody.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al.
  • the anti-human CCR8 antibody disclosed herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and/or the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present invention discloses an anti-human CCR8 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in SEQ ID No. 1, 3, 5, 7, 9 or 11, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No. 2, 4, 6, 8, 10 or 12.
  • the present disclosure discloses an anti-human CCR8 antibody, wherein A. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.1, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.2; B. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.3, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.4; C.
  • the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.5, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.6; D. the heavy chain variable region of the antibody includes HCDR1, HCDR2 and HCDR3 in SEQ ID No.7, and the light chain variable region of the antibody includes LCDR1, LCDR2 and LCDR3 in SEQ ID No.8; E. the heavy chain variable region of the antibody includes SEQ ID F. the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 9, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No.
  • the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3 in SEQ ID No. 11, and the light chain variable region of the antibody comprises LCDR1, LCDR2 and LCDR3 in SEQ ID No. 12.
  • the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the Kabat numbering system.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined according to the IMGT numbering system.
  • the anti-human CCR8 antibody disclosed herein wherein a. the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13, HCDR2 comprises the amino acid sequence of SEQ ID NO.14, and HCDR3 comprises the amino acid sequence of SEQ ID NO.15; and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16, LCDR2 comprises the amino acid sequence of SEQ ID NO.17, and LCDR3 comprises the amino acid sequence of SEQ ID NO.18; b.
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.19
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.20
  • HCDR3 comprises The amino acid sequence of SEQ ID NO.21
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18;
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.22
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.23
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.24
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.25
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.26
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.27; d.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.28
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.29
  • LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.30
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18; e.
  • HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.31
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.32
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.33 amino acid sequence
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.34
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.35
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.36; or f.
  • the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.13
  • HCDR2 comprises the amino acid sequence of SEQ ID NO.37
  • HCDR3 comprises the amino acid sequence of SEQ ID NO.38
  • the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO.16
  • LCDR2 comprises the amino acid sequence of SEQ ID NO.17
  • LCDR3 comprises the amino acid sequence of SEQ ID NO.18.
  • the anti-human CCR8 antibody of the present disclosure comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.25, SEQ ID NO.
  • the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.28 and SEQ ID NO.29, respectively, and SEQ ID NO.16, SEQ ID NO.30, respectively.
  • the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35, respectively; or the anti-human CCR8 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.13, SEQ ID NO.36 and SEQ ID NO.37, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively.
  • antigen-binding fragment or "antigen-binding domain” refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that is capable of specifically binding to an antigen to which the intact antibody binds.
  • antigen-binding fragments include, but are not limited to, Fv (consisting of VH and VL), Fab (consisting of one light chain and one heavy chain constant region 1 (CH1) and a heavy chain variable region), Fab' (consisting of a Fab region and a hinge region), Fab'-SH (the cysteine residue in the hinge region of the Fab' fragment carries a free thiol group), (Fab')2 (dimeric Fab'), scFv (single-chain antibody molecule, in which the light chain variable region is directly connected to the heavy chain variable region or connected by a linker), scFab (single-chain Fab), dsFv (disulfide-stabilized Fv fragment), (dsst al
  • the term "Fc region” is used to define the C-terminal region of the heavy chain of an antibody, including a native Fc region and a modified Fc region.
  • the Fc region for the antibodies described herein includes the Fc region of human IgG1, IgG2 (IgG2a, IgG2b), IgG3, and IgG4.
  • the Fc region is a human IgG1 comprising a DLE mutation (i.e., the Fc of human IgG1 undergoes S239D/A330L/I332E mutations, and the sequence is shown in SEQ ID No. 42).
  • the Fc region is a human IgG1 comprising a DE mutation (i.e., the Fc of human IgG1 undergoes S239D/I332E mutations).
  • the boundaries of the Fc region can also vary, such as the deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system).
  • the numbering convention of the Fc region is the EU numbering system, also known as the EU index.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from one species, while the other portion of the heavy and/or light chain is derived from another species.
  • humanized antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, it can be achieved by retaining the non-human CDR region and replacing the rest with a human antibody counterpart (i.e., the constant region and the framework region portion of the variable region).
  • human antibody humanized antibody
  • fully human antibody fully human antibody
  • completely human antibody refers to antibodies whose variable and constant regions are human sequences.
  • affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise indicated, as used herein, binding “affinity” refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art.
  • KD refers to a dissociation constant, which is obtained from the ratio of kd to ka (i.e., kd/ka) and is expressed as a molar concentration (M).
  • M molar concentration
  • effector function refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an Fc region of amino acid sequence mutation).
  • antibody effector functions include, but are not limited to, C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • the term “monoclonal antibody” refers to a group of substantially homogeneous antibodies, i.e., the amino acid sequences of the antibody molecules contained in the group are identical, except for the natural mutations that may be present in small amounts.
  • polyclonal antibody preparations are typically comprised of a variety of different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes.
  • “Monoclonal” represents the characteristics of antibodies obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any particular method.
  • the antibody provided by the present disclosure is a monoclonal antibody.
  • antigen refers to a molecule that can be bound by an antigen binding protein (eg, an antibody) selective binding agent.
  • An antigen may have one or more epitopes that can interact with different antigen binding proteins (eg, antibodies).
  • epitope refers to an area or region on an antigen that can specifically bind to an antibody.
  • An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-continuous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen by protein properties) and become spatially close.
  • the difference between a conformational epitope and a linear epitope is that the binding of the antibody to the conformational epitope is lost in the presence of a denaturing solvent.
  • a conformational epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
  • Screening for antibodies that bind to a specific epitope can be performed using routine methods in the art, such as alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.
  • An antibody that binds to the same epitope as a reference antibody or an antibody that competes for binding with a reference antibody means an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, or an antibody whose binding to the antigen is blocked by the reference antibody by 50% or more.
  • test antibody binds to the same epitope
  • routine experiments e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art
  • two antibodies are considered to bind to the same or overlapping epitope if an excess (e.g., a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold amount) of one antibody inhibits binding of the other antibody to the antigen by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
  • an antibody that is capable of binding to an antigen or an epitope within the antigen with a higher affinity than other antigens or epitopes.
  • an antibody binds to an antigen or an epitope within the antigen with an equilibrium dissociation constant (KD) of about 100 nM or less (e.g., about 10 nM, 1 nM, 0.1 nM, 0.01 nM or less).
  • KD equilibrium dissociation constant
  • the KD of an antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein).
  • KD can be measured using known methods, including but not limited to Biacore assays, Octet methods, microthermophoresis, HPLC-MS methods, and flow cytometry fluorescence sorting techniques; for example, by Measured by surface plasmon resonance assay.
  • the binding of the anti-human CCR8 antibody provided by the present disclosure to human CCR8 can also be expressed by a "half-maximal effective concentration" (EC50) value.
  • EC50 half-maximal effective concentration
  • the EC50 value can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometric fluorescence sorting technology, and other binding assays).
  • antibody-dependent cellular cytotoxicity is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages, and neutrophils) via Fc ⁇ receptors (Fc ⁇ Rs) expressed on effector cells.
  • lytic activity such as natural killer cells (NK), monocytes, macrophages, and neutrophils
  • Fc ⁇ Rs Fc ⁇ receptors
  • NK cells express Fc ⁇ RIIIa
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIIIa.
  • the ADCC activity of the antibodies provided herein can be assessed using in vitro assays using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on markers released from lysed cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins).
  • ADCP antibody-dependent cellular phagocytosis
  • complement-dependent cytotoxicity refers to a mechanism of inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death.
  • Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • CAR is a chimeric antigen receptor, which is an artificially modified receptor, for example, it is capable of anchoring specific molecules (such as antibodies) that recognize tumor cell surface antigens or viral antigens on immune cells (such as T cells), so that immune cells recognize tumor antigens or viral antigens and kill tumor cells or viruses.
  • T cells expressing CAR are called CAR-T (or CART).
  • chimeric antigen receptors sequentially include an extracellular domain, a transmembrane region, and an intracellular signaling domain.
  • the chimeric antigen receptor of the present disclosure can be constructed using transmembrane regions and intracellular signaling domains known in the art for constructing CAR.
  • the signal is transmitted to the cell through the hinge region and the transmembrane region, and the intracellular signal domain converts the signal into an activation signal, activating effector cells, and effector cells kill tumor cells by secreting perforin or producing cytokines, and the effector cells themselves also amplify, further expanding the immune killing effect.
  • the extracellular domain may include an antigen binding domain (also known as the part of the antibody that specifically binds to the antigen), which may be a single-chain variable fragment (scFv) that specifically binds to the antigen.
  • the single-chain variable fragment may serve as the extracellular domain of CAR, which determines the specificity of CAR-immune cells.
  • the hinge region is the extracellular domain of CAR that connects the single-chain antibody unit and the transmembrane domain. It usually maintains the stability required for robust CAR expression and activity in effector cells.
  • the hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28. The type and length of the hinge region have an important influence on the functional activity of CAR.
  • the transmembrane domain connects the extracellular domain of CAR to the intracellular signal transduction domain. Commonly used transmembrane domains are derived from CD4, CD8 ⁇ , CD28 and CD3 ⁇ .
  • the choice of transmembrane domain affects the degree of activation of the CAR structure in cell function.
  • the intracellular domain is composed of a co-stimulatory domain and a signal transduction domain.
  • the CAR is a chimeric antigen receptor, and its main components may also include a transmembrane domain and an intracellular domain.
  • the CAR may also include a co-stimulatory molecule.
  • "Co-stimulatory molecule” refers to a molecule that exists on the surface of an antigen presenting cell and can bind to a co-stimulatory molecule receptor on a Th cell to produce a co-stimulatory signal.
  • Isolated nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which cells typically contain the nucleic acid molecules, but the nucleic acid molecules are present outside the chromosome or at a chromosomal position different from its natural chromosomal position.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • sequence identity refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned (introducing gaps, if necessary, to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity).
  • percentage of sequence identity alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • a person skilled in the art can determine Applicable parameters for measuring alignment include any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • antibody conjugates include conjugates formed by conjugating anti-human CCR8 antibodies with therapeutic agents or imaging agents.
  • the antibody conjugate is formed by conjugating anti-human CCR8 antibodies with therapeutic agents via a linker; in an optional embodiment, the therapeutic agent is a cytotoxic agent; in an optional embodiment, the therapeutic agent can be selected from toxins, chemotherapeutic agents, antibiotics, radioactive isotopes and nucleolytic enzymes.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction
  • toxin refers to a substance that can have a deleterious effect on the growth or proliferation of cells (e.g., maytansine, calicheamicin, taxanes, vincristine, colchicine, auristatin, etc.).
  • Cyclotherapeutic agent refers to a chemical compound that can be used to treat cancer.
  • the antibody conjugate of the present disclosure further comprises biotin or a biotin derivative conjugated to the antibody.
  • the antibody conjugate further comprises a label coupled to the antibody.
  • the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.
  • the marker is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle marker.
  • a fluorescent dye an enzyme, a radioisotope, a chemiluminescent agent, and a nanoparticle marker.
  • the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc.
  • fluorescein dyes and their derivatives for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs
  • rhodamine dyes and their derivatives for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B
  • Cy series dyes and their derivatives for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc.
  • Alexa series dyes and their derivatives for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
  • protein dyes and their derivatives for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.
  • the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
  • the radioactive isotopes include but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.
  • the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, lophanine and its derivatives, and peroxyoxalate and its derivatives.
  • the sodium Rice particle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
  • the colloid includes but is not limited to colloidal metal, colloidal selenium, disperse dyes, dye-labeled microspheres and latex.
  • the colloidal metal includes but is not limited to colloidal gold or colloidal silver. In an optional embodiment, the colloidal metal is colloidal gold.
  • the antibody conjugate may further include a solid phase carrier coupled to the antibody.
  • the antibody is coupled to a solid phase carrier.
  • the solid phase carrier is selected from microspheres, plates and membranes.
  • the solid phase includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.
  • the solid phase carrier is a nitrocellulose membrane.
  • linker refers to a connecting unit connecting two polypeptide fragments, which usually has a certain flexibility, and the use of the linker will not cause the loss of the original function of the protein domain.
  • the linker can be a peptide linker, which contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 0-6.
  • the linker is selected from (GxS)y linkers, wherein x is selected from an integer of 1-5 and y is selected from an integer of 1-6.
  • the disclosed embodiments also provide a method for detecting CCR8, comprising: mixing the antibody as described in any of the preceding items, or the antibody conjugate as described in any of the preceding items, or the reagent or kit as described in any of the preceding items with a sample to be detected, so that the antibody contacts the CCR8 in the sample to be detected to form an immune complex.
  • the immune complex further comprises a second antibody, and the second antibody binds to the antibody.
  • the immune complex further comprises a second antibody, and the second antibody binds to CCR8.
  • the present disclosure also provides a method for preparing the antibody as described in any of the preceding items, comprising: a step of culturing the cells as described in any of the preceding items, and a step of purifying and recovering the antibody.
  • vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked.
  • plasmid refers to a circular double-stranded DNA loop to which additional DNA segments can be attached.
  • viral vector such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be attached to the viral genome.
  • AAV adeno-associated viral vector
  • Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating along with the host genome.
  • expression vector or "expression construct” refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and/or controls (together with the host cell) the expression of one or more heterologous coding regions to which it is operably linked.
  • Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region to which it is operably linked.
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without regard to the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Mutant offspring with the same function or biological activity as screened or selected in the initial transformed cells are included herein.
  • Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells and fungal cells.
  • Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells and HEK-293 cells.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • A549 cells e.g., 3T3 cells and HEK-293 cells.
  • Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membraneaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia pastoris.
  • Pichia pastoris Pichia pastoris
  • Pichia finlandica Pichia trehalophila
  • Pichia koclamae Pichia membraneaefaciens
  • Pichia minuta Ogataea minuta, Pichia lindneri
  • Pichia puntiae Pichia thermotolerans
  • Pichia salictaria Pichia guercuum
  • Saccharomyces cerevisiae Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucnowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa.
  • Pichia spp. any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucnowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa.
  • progeny As used in this application, “cell”, “cell line” and “cell culture” can be used interchangeably, and all such names include progeny.
  • the words “transformant” and “converted cell” include the primary subject cell and the culture derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, not all progeny have exactly the same DNA content. Included are mutant progeny that have the same function or biological activity as the original transformed cell from which it was screened.
  • composition refers to a mixture containing one or more active ingredients such as the antibodies described herein and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients.
  • pharmaceutically acceptable carrier refers to a component of a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • subject or “individual” includes humans and non-human animals.
  • Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles.
  • patient or “subject” are used interchangeably herein.
  • cyno or “cynomolgus” refers to cynomolgus monkeys (Macaca fascicularis).
  • the individual or subject is a human.
  • administering refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid.
  • sample refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject.
  • exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically removed tissues, organ cultures, or cell cultures.
  • biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory
  • Treatment refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology.
  • the desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.
  • the antibodies of the present disclosure are used to delay the development of the disease or slow the progression of the disease.
  • Effective amount is generally enough to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or potential causes, prevent symptoms and/or their potential causes from occurring and/or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state.
  • the effective amount is a therapeutically effective amount or a preventive effective amount.
  • “Therapeutically effective amount” is enough to treat a disease state or symptom, especially a state or symptom associated with the disease state, or otherwise prevent, hinder, delay or reverse the disease state or any other undesirable symptom associated with the disease in any way.
  • Preventive effective amount is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset (or recurrence) possibility of the disease state or related symptoms. Complete treatment or preventive effect may not occur after administering one dose, but may occur after administering a series of doses. Thus, the therapeutic or preventive effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” and a “prophylactically effective amount” may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.
  • mice Balb/c mice were used, and CHO (Chinese hamster ovary cells) cells (CHO-hCCR8) overexpressing human CCR8 and a fusion protein of amino acids 1-35 at the N-terminus of human CCR8 and mouse Fc (CCR8N-term-mFc) were used as immunogens for multiple immunizations. This was performed once every one to two weeks, for a total of 6-10 times. After the fourth immunization, blood was collected from the mice's orbits, and the serum titer of anti-human CCR8 antibodies was detected by ELISA and flow cytometry.
  • CHO Choinese hamster ovary cells
  • CCR8N-term-mFc mouse Fc
  • the mouse serum was diluted in multiples and incubated with Raji cell lines (human BURKITT'S lymphoma cells) and Raji cell lines (Raji-hCCR8) overexpressing human CCR8, and stained with PE-labeled goat anti-mouse Fc antibodies (Biolegend), and the serum titer was detected by flow cytometry.
  • CCR8N-term-hFc protein (a fusion protein of amino acids 1-35 at the N-terminus of human CCR8 and human Fc) was coated in a 96-well plate, and serum diluted in multiples was added for incubation, followed by the addition of HRP-labeled goat anti-mouse Fc antibody (Sigma), and the serum titer was detected by ELISA.
  • the mice with the highest titer were determined based on the results of flow cytometry and ELISA, and the mice were euthanized, spleen cells were collected, and fused with the SP20 cell line.
  • Monoclonal cells with strong binding activity to Raji-hCCR8 and no binding to Raji cell line were selected for expansion culture. After one week, the supernatant was collected and the antibodies in the supernatant were purified.
  • Count the Raji-hCCR8 and Raji cells adjust the density to 2E6/ml, and add 100 ⁇ L/well of cell suspension Place in a 96-well plate, centrifuge at 400 ⁇ g for 5 minutes, and discard the supernatant.
  • the binding test results of anti-CCR8 antibodies and Raji-hCCR8 are shown in Figure 1.
  • the experimental results show that the six hybridoma purified antibodies F022-7, F022-59, F022-63, F022-66, F022-68, and F022-75 exhibited strong binding activity with Raji-hCCR8.
  • the sequences of hybridoma purified antibodies of F022-7, F022-59, F022-63, F022-66, F022-68 and F022-75 were retrieved, and the sequencing results showed that the light chain constant regions of all hybridoma purified antibodies were of mouse ⁇ type (as shown in SEQ ID No.40), the heavy chain constant regions of F022-7, F022-63 and F022-75 were of mIgG2a type (as shown in SEQ ID No.41), and the heavy chain constant regions of F022-59, F022-66 and F022-68 were of mIgG1 type (as shown in SEQ ID No.39).
  • the heavy chain constant region of the hybridoma purified antibody was replaced with mouse mIgG2a, and the other sequences remained unchanged.
  • the antibody was cloned into the pCDNA3.4A expression plasmid, and the plasmid was transfected into the EXPI293 cell line. The supernatant was collected after 7 days and purified with protein A to obtain the recombinant antibody.
  • the recombinant antibodies were named F022-7-mIgG2a, F022-59-mIgG2a, F022-63-mIgG2a, F022-66-mIgG2a, F022-68-mIgG2a, and F022-75-mIgG2a, respectively.
  • the recombinant antibody "F022-59-mIgG2a” represents the antibody formed after the heavy chain constant region of the hybridoma purified antibody “F022-59” was replaced with “mIgG2a", and the others are similar.
  • the heavy chain constant region of the hybridoma purified antibody was replaced with human hIgG1Fc (DLE) (i.e., the Fc of human IgG1 was mutated with S239D/A330L/I332E, and the sequence is shown in SEQ ID No.42), and the light chain constant region was replaced with human ⁇ light chain constant region (as shown in SEQ ID No.43), while other sequences remained unchanged, and cloned into the pCDNA3.4A expression plasmid.
  • the plasmid was transfected into the EXPI293 cell line, and the supernatant was collected after 7 days.
  • the recombinant antibodies were obtained after purification with protein A.
  • the recombinant antibodies were named F02 2-7-hIgG1(DLE), F022-59-hIgG1(DLE), F022-63-hIgG1(DLE), F022-66-hIgG1(DLE), F022-68-hIgG1(DLE), F022-75-hIgG1(DLE), for example, the recombinant antibody "F022-59-hIgG1(DLE)” means the heavy chain constant region of the hybridoma purified antibody "F022-59” is replaced by human hIgG1(DLE) and the light chain constant region is replaced by human ⁇ light chain constant region, and the others are similar.
  • the variable region sequences of the antibodies are shown in Table 1, and the CDRs sequences are shown in Table 2:
  • amino acid sequence of the mouse mIgG1 heavy chain constant region (SEQ ID No.39):
  • amino acid sequence of the constant region of the mouse mIgG2a heavy chain (SEQ ID No.41):
  • F022-59, F022-59-mIgG2a, and F022-59-hIgG1(DLE) are as follows:
  • amino acid sequence of the light chain variable region of F022-59 (SEQ ID No.45):
  • amino acid sequence of the heavy chain variable region of F022-59-mIgG2a (SEQ ID No.46):
  • amino acid sequence of the light chain variable region of F022-59-mIgG2a (SEQ ID No.47):
  • amino acid sequence of the light chain variable region of F022-59-hIgG1(DLE) (SEQ ID No.49):
  • amino acid sequence of the positive control antibody 4A19-mIgG2a (obtained by connecting the 4A19 antibody variable region in the WO2021194942A1 patent to the mIgG2a constant region) is as follows:
  • Amino acid sequence of 4A19-mIgG2a heavy chain (SEQ ID No.50):
  • amino acid sequence of 4A19-mIgG2a light chain (SEQ ID No.51):
  • PBMC Peripheral blood mononuclear cells
  • Lymphoprep Axis-Shield
  • Tregs were isolated from PBMC using EasySep Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stem Cell) according to the instructions.
  • the isolated Tregs were mixed with Dynabeads Human T-Activator CD3/CD28 (Gibco) in a 1:1 ratio and cultured with complete medium (90% X-VIVO 15+10% FBS+500IU/ml IL-2, X-VIVO 15: Lonza, FBS: Gibco, IL-2: Tetracycline) for 5 to 8 days to activate and expand Treg cells.
  • the binding activity of the recombinant antibody to Raji-hCCR8, Treg (after activation), Raji, PBMC and Raji-hCCR4 (Raji cells overexpressing human CCR4) was detected by flow cytometry.
  • the experimental method was the same as in Example 2 above.
  • the mIgG2a subtype antibody was detected using a PE-labeled anti-mouse Fc secondary antibody (Biolegend), and the hIgG1 (DLE) subtype antibody was detected using an Alexa Fluor 647-labeled anti-human Fab secondary antibody (Jackson).
  • the experimental results are shown in Figures 2-6.
  • the recombinant antibodies all showed strong binding activity with Raji-hCCR8 (Figure 2) and Treg ( Figure 3). At the same time, the recombinant antibodies did not bind to Raji ( Figure 4), PBMC ( Figure 5) and Raji-hCCR4 ( Figure 6), which shows that the anti-CCR8 antibodies disclosed in the present invention have good
  • hCD16 molecules were expressed in NK-92 cells to detect antibody-mediated ADCC function.
  • Jurkat-hCCR8 cells Jurkat, human peripheral blood leukemia T cell line, ATCC
  • CFSE human peripheral blood leukemia T cell line
  • NK-92-hCD16 cells were added to a 96-well plate at a density of 1E5/well, followed by the addition of anti-CCR8 antibodies with hIgG1Fc (DLE) and incubation at 37 degrees for 6 hours. The 96-well plate was taken out and PI staining was added.
  • MC38 cells (3E5/mouse) were subcutaneously injected into hCCR8 knock-in mice (B-hCCR8, Biocytogen) to establish a tumor model.
  • F022-7-mIgG2a, F022-59-mIgG2a, and F022-75-mIgG2a antibodies were intravenously injected, and isotype-independent target antibodies were used as negative controls, and 4A19-mIgG2a was used as a positive control.
  • the dosage was 10 mg/kg, and the drug was administered twice a week for a total of 6 times.
  • the experimental results are shown in Table 3 and Figure 8 , and the recombinant antibodies have significant anti-tumor effects in vivo.

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Abstract

L'invention concerne un anticorps anti-CCR8 et une utilisation associée. L'anticorps anti-CCR8 comprend une région de détermination de complémentarité de chaîne lourde et une région de détermination de complémentarité de chaîne légère, ledit anticorps peut se lier spécifiquement à un antigène CCR8 humain et ledit anticorps peut être utilisé pour traiter des tumeurs.
PCT/CN2023/136644 2022-12-07 2023-12-06 Anticorps anti-ccr8 et utilisation associée WO2024120418A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
WO2020138489A1 (fr) * 2018-12-27 2020-07-02 塩野義製薬株式会社 Nouvel anticorps anti-ccr8
WO2022042690A1 (fr) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Anticorps anti-ccr8 et application correspondante
WO2022081718A1 (fr) * 2020-10-14 2022-04-21 Five Prime Therapeutics, Inc. Anticorps anti-récepteur de chimiokine c-c 8 (ccr8) et leurs procédés d'utilisation
CN114891117A (zh) * 2022-04-26 2022-08-12 深圳市体内生物医药科技有限公司 一种靶向ccr8的嵌合抗原受体t细胞及其制备方法和应用
CN114929278A (zh) * 2020-01-06 2022-08-19 瓦西尼斯公司 抗ccr8抗体及其用途
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110835371A (zh) * 2018-08-19 2020-02-25 普米斯生物技术(苏州)有限公司 抗ccr8单克隆抗体及其应用
WO2020138489A1 (fr) * 2018-12-27 2020-07-02 塩野義製薬株式会社 Nouvel anticorps anti-ccr8
CN114929278A (zh) * 2020-01-06 2022-08-19 瓦西尼斯公司 抗ccr8抗体及其用途
WO2022042690A1 (fr) * 2020-08-28 2022-03-03 和铂医药(上海)有限责任公司 Anticorps anti-ccr8 et application correspondante
WO2022081718A1 (fr) * 2020-10-14 2022-04-21 Five Prime Therapeutics, Inc. Anticorps anti-récepteur de chimiokine c-c 8 (ccr8) et leurs procédés d'utilisation
CN115052892A (zh) * 2020-10-16 2022-09-13 礼新医药科技(上海)有限公司 抗ccr8单克隆抗体及其用途
CN114891117A (zh) * 2022-04-26 2022-08-12 深圳市体内生物医药科技有限公司 一种靶向ccr8的嵌合抗原受体t细胞及其制备方法和应用

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