Monoclonal antibodies and bispecific antibodies against CCR8 and TIGIT

Abstract

The present application relates to an antibody or an antigen-binding fragment thereof capable of specifically binding to CCR8 or TIGIT, and an immunoconjugate, a pharmaceutical composition and a multispecific antibody comprising the same. Further, the present application also relates to a multispecific antibody capable of specifically binding to CCR8 and TIGIT, and uses thereof.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and more specifically, it relates to antibodies or antigen-binding fragments thereof capable of specifically binding to CCR8 or TIGIT, as well as immunoconjugates, pharmaceutical compositions, and multispecific antibodies containing the same. Furthermore, this application also relates to multispecific antibodies capable of specifically binding to CCR8 and TIGIT and their uses. Background Technology

[0002] PD-1 / PD-L1 drugs have made groundbreaking progress in tumor immunotherapy. However, the tumor microenvironment is infiltrated by a large number of immunosuppressive cells, resulting in low response rates and a high relapse rate in cancer patients after PD-1 / PD-L1 immunotherapy. Regulatory T cells (Tregs) play a crucial role in maintaining autoimmune homeostasis and mediating self-antigen tolerance, and are also an important cause of the formation of the tumor suppressor microenvironment. Therefore, targeting and eliminating Tregs infiltrating the tumor microenvironment is a direction for improving the tumor immunosuppressive microenvironment and overcoming the limitations of immune checkpoint inhibitors.

[0003] Chemokine receptor 8 (CCR8) is a seven-transmembrane G protein-coupled receptor belonging to the CC subfamily of chemokine receptors. Studies have shown that CCR8 is specifically highly expressed on the surface of Treg cells infiltrating various types of tumors (breast cancer, colorectal cancer, non-small cell lung cancer, pancreatic cancer, etc.), while its expression level is low in Treg cells from the thymus, spleen, and peripheral blood (Immunity, 2016, 45(5):1122-1134; Cell, 2017, 169(7):1342-1356.e16; Nature, 2018, 564(7735):268-272; Nat Med, 2018, 24(7):978-985; Sci Bull (Beijing), 2018, 63(15):972-981.). In addition, some literature reports that high expression of CCR8 on the surface of tumor-infiltrating Treg cells is associated with poor prognosis in cancer patients (Immunity 2016; 45:1135-1147; World J Gastroenterol. 2020 Aug 21; 26(31):4656-4668.). In preclinical tumor-bearing mouse models, anti-CCR8 monoclonal antibodies that retain Fc effector function can effectively delete Treg cells in tumor tissue, showing a significant tumor-suppressive effect (Cancer Res. 2021 Jun 1; 81(11):2983-2994.). Therefore, the development of monoclonal antibodies specifically targeting CCR8 has become one of the hot topics in tumor research and development. Currently, many companies are investing in this field, such as Bristol Myers Squibb's BMS-986340, Gilead Sciences' GS-1811, Lixin Pharmaceuticals' LM108, and Shionogi's S-531011, all of which have entered clinical development. However, according to some literature reports, CCR8 is not only expressed on the surface of tumor-infiltrating Treg cells, but also on the surface of resident T cells in normal skin tissue (JImmunol. 2018 Mar 1; 200(5):1639-1650; Cancer Res. 2021 Jun 1; 81(11):2983-2994.). Therefore, selecting more specific Treg surface markers or developing bispecific antibodies targeting dual markers on the Treg cell surface can improve the specificity for tumor-infiltrating Tregs and avoid damage to normal tissues caused by targeted clearance of Tregs.

[0004] T cell immunoglobulin with Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed on lymphocytes (T, NK, and Treg cells) and has recently attracted much attention as an emerging target for cancer immunotherapy. Studies have shown that TIGIT is also highly expressed on the surface of Treg cells and has a certain co-expression relationship with CCR8 on Treg cells (Sci Rep. 2022 Mar 30; 12(1):5377; J Clin Invest. 2015 Nov 2; 125(11):4053-62.).

[0005] Bispecific antibodies can simultaneously and specifically bind to two antigens or epitopes, exhibiting both specificity and bifunctionality. Therefore, there is still an urgent need in this field to develop a bispecific antibody targeting the CCR8 and TIGIT dual markers on Treg cells to improve antibody specificity and enhance tumor treatment efficacy. Summary of the Invention

[0006] The inventors of this application, through extensive research, have obtained antibodies or antigen-binding fragments thereof capable of specifically binding to TIGIT. Furthermore, bispecific antibodies capable of specifically binding to CCR8 and TIGIT, as well as defucosylated bispecific antibodies, have been prepared. The obtained bispecific antibodies possess ADCC and / or ADCP activities, and these antibodies exhibit high affinity and specificity for binding to CCR8 and TIGIT. In animal models, administration of the bispecific antibodies of this invention can significantly inhibit tumor growth.

[0007] Monoclonal antibodies

[0008] In a first aspect, this application provides an antibody or antigen-binding fragment thereof capable of specifically binding to TIGIT, said antibody or antigen-binding fragment comprising:

[0009] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0010] (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:1, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0011] (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:2, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0012] (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:3, or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0013] And / or,

[0014] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0015] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0016] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0017] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0018] In some implementations, the permutation in any one of (i)-(vi) is a conservative permutation.

[0019] In some implementations, the CDR described in any one of (i)-(vi) is defined according to the Kabat, IMGT or Chothia numbering system.

[0020] In some implementations, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system.

[0021] In some embodiments, the antibody or its antigen-binding fragment as described above comprises: three heavy chain CDRs as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, and VH CDR3 as shown in SEQ ID NO:3; and / or three light chain CDRs as shown in SEQ ID NO:5, VL CDR1 as shown in SEQ ID NO:6, and VL CDR3 as shown in SEQ ID NO:7.

[0022] In some embodiments, the antibody or its antigen-binding fragment further comprises the framework region of a human immunoglobulin.

[0023] In some embodiments, the antibody or its antigen-binding fragment, as described above, comprises:

[0024] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0025] (i) The sequence shown in SEQ ID NO:4;

[0026] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:4; or

[0027] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:4;

[0028] and / or

[0029] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0030] (iv) The sequence shown in SEQ ID NO:8;

[0031] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:8; or

[0032] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:8.

[0033] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0034] In some embodiments, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:4 and VL having the sequence shown in SEQ ID NO:8.

[0035] In a second aspect, this application provides an antibody or antigen-binding fragment thereof capable of specifically binding to CCR8, said antibody or antigen-binding fragment comprising:

[0036] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0037] (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:9 or 13, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0038] (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:10 or 14, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0039] (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:11 or 15, or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0040] And / or,

[0041] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0042] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:17, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it.

[0043] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:18, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and

[0044] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0045] In some implementations, the permutation in any one of (i)-(vi) is a conservative permutation.

[0046] In some implementations, the CDR described in any one of (i)-(vi) is defined according to the Kabat, IMGT or Chothia numbering system.

[0047] In some implementations, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system.

[0048] In some embodiments, the antibody or its antigen-binding fragment as described above comprises: three heavy chain CDRs as shown in SEQ ID NO:9, VH CDR1 as shown in SEQ ID NO:10, and VH CDR3 as shown in SEQ ID NO:11; and / or three light chain CDRs as shown in SEQ ID NO:17, VL CDR1 as shown in SEQ ID NO:18, and VL CDR3 as shown in SEQ ID NO:19.

[0049] In some embodiments, the antibody or its antigen-binding fragment as described above comprises: three heavy chain CDRs as shown in SEQ ID NO:13, VH CDR2 as shown in SEQ ID NO:14, and VH CDR3 as shown in SEQ ID NO:15; and / or three light chain CDRs as shown in SEQ ID NO:17, VL CDR1 as shown in SEQ ID NO:18, and VL CDR3 as shown in SEQ ID NO:19.

[0050] In some embodiments, the antibody or its antigen-binding fragment further comprises the framework region of a human immunoglobulin.

[0051] In some embodiments, the antibody or its antigen-binding fragment, as described above, comprises:

[0052] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0053] (i) The sequence shown in SEQ ID NO: 12 or 16;

[0054] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 12 or 16; or

[0055] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 12 or 16;

[0056] and / or

[0057] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0058] (iv) The sequence shown in SEQ ID NO:20;

[0059] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:20; or

[0060] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:20.

[0061] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0062] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0063] (1) VH having the sequence shown in SEQ ID NO:12 and VL having the sequence shown in SEQ ID NO:20;

[0064] (2) VH having the sequence shown in SEQ ID NO:16 and VL having the sequence shown in SEQ ID NO:20.

[0065] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region derived from mammalian (e.g., human, mouse) immunoglobulins.

[0066] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human or mouse. For example, a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; or a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region.

[0067] In some implementations, the heavy chain constant region has an LALA mutation and / or a knock-into-hole modification.

[0068] In some embodiments, the heavy chain constant region has a sequence as shown in any one of SEQ ID NO:29-34.

[0069] In some embodiments, the light chain of the antibody or its antigen-binding fragment contains a light chain constant region derived from mammalian (e.g., human, mouse) immunoglobulins (e.g., κ or λ).

[0070] In some embodiments, the light chain constant region has a sequence as shown in SEQ ID NO:35 or 36.

[0071] In some embodiments, the antibody or its antigen-binding fragment is hypofucosylated or nonfucosylated.

[0072] In some embodiments, the antibody or its antigen-binding fragment as described above is used, wherein the antigen-binding fragment is selected from Fab, Fab', or (Fab'). 2 Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0073] In some implementations, the antibody or its antigen-binding fragment, as described above, is a murine antibody, a chimeric antibody, a humanized antibody, or a multispecific antibody.

[0074] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments further has a feature selected from the following:

[0075] (1) Possesses ADCC activity; for example, induces the killing of cells (e.g., tumor cells) expressing CCR8 and / or TIGIT via ADCC; in some embodiments, the antibody or its antigen-binding fragment with low or no fucosylation has stronger ADCC activity; in such embodiments, the antibody prepared by modifying host cells (e.g., fucosylation knockout CHO cells) is a low or no fucosylation antibody;

[0076] (2) Possesses ADCP activity; for example, induces the killing of cells (e.g., tumor cells) expressing CCR8 and / or TIGIT via ADCP; in some embodiments, the antibody or its antigen-binding fragment containing a mutated heavy chain constant region has stronger ADCP activity; and / or,

[0077] (3) Inhibition of tumor growth; in some embodiments, the antibody or its antigen-binding fragment, when used in combination with other pharmaceutically active agents (e.g., anti-PD-L1 / TGF-βRII fusion protein), has a stronger ability to inhibit tumor growth.

[0078] In a third aspect, this application provides isolated nucleic acid molecules that encode antibodies or antigen-binding fragments thereof as described in the first or second aspect.

[0079] In a fourth aspect, this application provides a vector comprising the nucleic acid molecule as described in the third aspect. In some embodiments, the vector is a cloning vector or an expression vector.

[0080] In some embodiments, the vector of the present invention is selected from plasmids, granules, bacteriophages, and lentiviruses. In some embodiments, the vector is capable of expressing the antibody or antigen-binding fragment of the present invention in a subject (e.g., a mammal, such as a human).

[0081] In a fifth aspect, this application provides a host cell comprising a nucleic acid molecule as described in the third aspect or a vector as described in the fourth aspect.

[0082] The host cell can be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., *E. coli*). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as a CHO cell (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0083] In some implementations, the host cell is a mammalian cell.

[0084] In some embodiments, the host cell has low or no fucosylation activity, for example, selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosylation enzymes.

[0085] In a sixth aspect, this application provides a method for preparing an antibody or antigen-binding fragment thereof as described in the first or second aspect, comprising culturing a host cell as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0086] In some embodiments, the host cell has low or no fucosylation activity, for example, selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosylation enzymes.

[0087] Bispecific antibodies

[0088] In a seventh aspect, this application provides a multispecific antibody (e.g., a bispecific antibody) comprising an antibody or an antigen-binding fragment thereof as described in the first or second aspect.

[0089] In an eighth aspect, this application provides a multispecific antibody (e.g., a bispecific antibody) that specifically binds to CCR8 and TIGIT, comprising a first antigen-binding domain specific to CCR8 and a second antigen-binding domain specific to TIGIT.

[0090] In some embodiments, the second antigen-binding domain comprises an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0091] In some embodiments, the first antigen-binding domain comprises an antibody or an antigen-binding fragment thereof as described in the second aspect.

[0092] In some embodiments, the first antigen-binding domain is a Fab fragment or scFv, and the second antigen-binding domain is a Fab fragment or scFv.

[0093] In some embodiments, the first antigen-binding domain is a Fab fragment, and the second antigen-binding domain is a Fab fragment or scFv.

[0094] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are optionally connected via a connector. In some embodiments, the first antigen-binding domain is optionally connected via a connector to the N-terminus or C-terminus of the second antigen-binding domain.

[0095] In some embodiments, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In some embodiments, the peptide linker consists of 9 to 25 amino acid residues (e.g., 10-25, 10-20, 10-17, 10-15). In some embodiments, the peptide linker is selected from (GGGGS)3, GGGSGGGGS, (GGGGS)5, (EAAAK)2, A(AEAAK)3A, (EAAAK)5, or combinations thereof.

[0096] In some embodiments, the multispecific antibody as described above further comprises an Fc domain, which includes a first monomer and a second monomer.

[0097] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are optionally connected to the N-terminus of the first monomer and the second monomer of the Fc domain, respectively, via a connector.

[0098] In some embodiments, the Fc domain contains modifications to facilitate the dimerization of the first and second monomers.

[0099] In some embodiments, the modification includes an amino acid substitution in the CH3 domain of the Fc domain.

[0100] In some implementations, the Fc domain includes modifications to facilitate cFAE (Controlled Fab-arm Exchange).

[0101] In some embodiments, the modification includes an amino acid substitution in the CH3 domain of the Fc domain.

[0102] In some implementations, the modification includes a “knob” modification in one of the two monomers in the Fc domain and a “knob-into-hole” modification in the other of the two monomers in the Fc domain to form a “knob-into-hole” modification.

[0103] In some embodiments, the modification comprises a substitution selected from F405L and / or R411T in one of the two monomers in the Fc domain and a substitution selected from T370K and / or K409R in the other of the two monomers in the Fc domain, to form a modification that promotes cFAE.

[0104] In some embodiments, the two monomers of the Fc domain respectively contain amino acid sequences as shown in SEQ ID NO:29 and 30, or amino acid sequences as shown in SEQ ID NO:31 and 32, or amino acid sequences as shown in SEQ ID NO:33 and 34.

[0105] In some implementations, it is either low-fucosylated or non-fucosylated.

[0106] In some embodiments, the first antigen-binding domain is a Fab fragment, the second antigen-binding domain is a Fab fragment, and the multispecific antibody comprises:

[0107] (i) A first peptide chain comprising a VL of the first antigen-binding domain and a first light chain constant region (CL); in some embodiments, the first CL is kappa;

[0108] (ii) A second peptide chain comprising the VH of the first antigen-binding domain and a first heavy chain constant region (CH); in some embodiments, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0109] (iii) A third peptide chain comprising the VH of the second antigen-binding domain and the second heavy chain constant region (CH); in some embodiments, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0110] (iv) A fourth peptide chain comprising the VL of the second antigen-binding domain and the second light chain constant region (CL); in some embodiments, the second CL is kappa.

[0111] In some embodiments, the Fc domain monomers of the first CH and the second CH form a dimer. In some embodiments, the Fc domain includes modifications as defined above.

[0112] In some implementations, the first CH and the first CL, and the second CH and the second CL, can each form a dimer.

[0113] In some embodiments, the multispecific antibody comprises: a first peptide chain containing the sequence shown in SEQ ID NO:43, a second peptide chain containing the sequence shown in SEQ ID NO:37, a third peptide chain containing the sequence shown in SEQ ID NO:41, and a fourth peptide chain containing the sequence shown in SEQ ID NO:44.

[0114] In some embodiments, the multispecific antibody comprises: a first peptide chain containing the sequence shown in SEQ ID NO:43, a second peptide chain containing the sequence shown in SEQ ID NO:38, a third peptide chain containing the sequence shown in SEQ ID NO:41, and a fourth peptide chain containing the sequence shown in SEQ ID NO:44.

[0115] In some embodiments, the multispecific antibody comprises: a first peptide chain containing the sequence shown in SEQ ID NO:45, a second peptide chain containing the sequence shown in SEQ ID NO:39, a third peptide chain containing the sequence shown in SEQ ID NO:42, and a fourth peptide chain containing the sequence shown in SEQ ID NO:46.

[0116] In some embodiments, the multispecific antibody comprises: a first peptide chain containing the sequence shown in SEQ ID NO:45, a second peptide chain containing the sequence shown in SEQ ID NO:40, a third peptide chain containing the sequence shown in SEQ ID NO:42, and a fourth peptide chain containing the sequence shown in SEQ ID NO:46.

[0117] In some embodiments, the first antigen-binding domain is a Fab fragment, the second antigen-binding domain is an scFv, and the multispecific antibody comprises:

[0118] (i) A first peptide chain comprising a VL of the first antigen-binding domain and a light chain constant region (CL); in some embodiments, the CL is a kappa light chain constant region;

[0119] (ii) A second peptide chain comprising the VH of the first antigen-binding domain and the heavy chain constant region (CH); in some embodiments, the CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0120] and

[0121] (iii) A third peptide chain comprising the second antigen-binding domain and an Fc domain monomer; preferably, the Fc domain monomer is IgG, such as IgG1, IgG2, IgG3 or IgG4; preferably, the Fc domain monomer comprises a hinge region, CH2 and CH3; preferably, the second antigen-binding domain is connected to the N-terminus of the Fc domain monomer via a linker (e.g., a peptide linker comprising one or more glycine (G) and / or serine (S));

[0122] In some embodiments, the Fc domain monomer of the third peptide chain is capable of forming a dimer with the Fc domain of the heavy chain constant region (CH) of the second peptide chain; in some embodiments, the Fc domain includes modifications as defined above.

[0123] In some embodiments, the CL of the first peptide chain and the CH of the second peptide chain can form a dimer.

[0124] In some embodiments, the second antigen-binding domain is scFv and has a structure as shown in VH-L-VL or VL-L-VH, where L is a peptide linker.

[0125] In some embodiments, the L is a peptide linker containing one or more glycine (G) and / or one or more serine (S), for example, a flexible peptide containing (G4S)n.

[0126] In some embodiments, the second antigen-binding domain has a structure shown as VH-L-VL.

[0127] In a ninth aspect, this application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a multispecific antibody as described in the seventh or eighth aspect, or at least one peptide chain thereof.

[0128] In a tenth aspect, this application provides a vector comprising isolated nucleic acid molecules as described in the ninth aspect.

[0129] In some embodiments, the vector contains nucleotide sequences encoding each peptide chain of the multispecific antibody, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

[0130] In the eleventh aspect, this application provides a host cell comprising isolated nucleic acid molecules as described in the ninth aspect or a vector as described in the tenth aspect.

[0131] In some embodiments, the host cell has low or no fucosylation activity, for example, it may be a mammalian cell lacking the expression of a gene encoding a fucosylation enzyme (e.g., FUT8), such as a CHO cell.

[0132] In a twelfth aspect, this application provides a method for preparing a multispecific antibody as described in the seventh or eighth aspect, comprising culturing a host cell as described above under conditions that allow protein expression, and recovering the multispecific antibody from the cultured host cell culture.

[0133] In some embodiments, the host cell has low or no fucosylation activity, thereby producing low or no fucosylated antibodies. In some embodiments, the host cell may be a mammalian cell lacking the expression of a gene encoding a fucosylation enzyme (e.g., FUT8), such as a CHO cell.

[0134] Immunoconjugates

[0135] In a thirteenth aspect, this application provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof as described in the first or second aspect, or a multispecific antibody as described in the seventh or eighth aspect, and a therapeutic agent attached to said antibody or antigen-binding fragment thereof or multispecific antibody.

[0136] In some embodiments, the therapeutic agent is selected from cytotoxic agents.

[0137] In some embodiments, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

[0138] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0139] In some embodiments, the multispecific antibody of the present invention is optionally conjugated to the coupling portion via a linker.

[0140] In some embodiments, the conjugation portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select a suitable protein tag based on the desired purpose (e.g., purification, detection, or tracing). In some exemplary embodiments, the C-terminus of the bispecific antibody of the present invention is linked to a His tag (e.g., 6×His).

[0141] Pharmaceutical composition

[0142] In its fourteenth aspect, this application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first or second aspect, or a multispecific antibody as described in the seventh or eighth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0143] In some embodiments, the pharmaceutical composition also contains additional pharmaceutically active agents.

[0144] In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus.

[0145] Reagent test kit

[0146] In the fifteenth aspect, this application provides a kit comprising an antibody or antigen-binding fragment thereof as described in the first or second aspect, or a multispecific antibody as described in the seventh or eighth aspect.

[0147] In some embodiments, the antibody or its antigen-binding fragment carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0148] In some embodiments, the multispecific antibody carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0149] Methods and uses

[0150] In a sixteenth aspect, this application provides a method for inhibiting the growth of tumor cells expressing CCR8 and / or TIGIT and / or killing said tumor cells, comprising contacting said tumor cells with an effective amount of an antibody or antigen-binding fragment thereof as described in the first or second aspect, or a multispecific antibody as described in the seventh or eighth aspect, or an immunoconjugate as described in the thirteenth aspect, or a pharmaceutical composition as described in the fourteenth aspect.

[0151] In its seventeenth aspect, this application provides the use of an antibody or antigen-binding fragment thereof as described in the first or second aspect, or a multispecific antibody as described in the seventh or eighth aspect, or an immunoconjugate as described in the thirteenth aspect, or a pharmaceutical composition as described in the fourteenth aspect, in the preparation of a medicament, said medicament being used for:

[0152] (1) Enhance the activity of immune cells in vitro or in subjects;

[0153] (2) Enhance the immune response in subjects;

[0154] (3) Prevention and / or treatment of tumors in subjects; or

[0155] (4) Prevention and / or treatment of infection in subjects.

[0156] In some implementations, the tumor expresses CCR8 and / or TIGIT.

[0157] In some embodiments, the CCR8 and / or TIGIT are expressed on the surface of the tumor cells.

[0158] In some embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, and mycosis fungoides. (fungoids), Merkel cell carcinoma and other hematologic malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell rich lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumors, and brainstem gliomas.

[0159] In some implementations, the infection is selected from viral infections, bacterial infections, fungal infections, and parasitic infections.

[0160] In some implementations, the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.

[0161] In the eighteenth aspect, this application provides the use of antibodies or antigen-binding fragments thereof as described in the first or second aspect, or multispecific antibodies as described in the seventh or eighth aspect, in the preparation of a kit for detecting whether a tumor can be treated by antitumor therapies targeting CCR8 and / or TIGIT.

[0162] (1) Contact the sample containing the tumor cells with an antibody or antigen-binding fragment thereof as described in the first or second aspect or a multispecific antibody as described in the seventh or eighth aspect;

[0163] (2) Detect the formation of the antibody or its antigen-binding fragment or the complex between the multispecific antibody and CCR8 and / or TIGIT.

[0164] In some embodiments, the antibody or its antigen-binding fragment, or the multispecific antibody, carries a detectable label.

[0165] In some embodiments, the CCR8 and / or TIGIT are CCR8 and / or TIGIT of mammals (e.g., humans, monkeys).

[0166] In some embodiments, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, and mycosis fungoides. (fungoids), Merkel cell carcinoma and other hematologic malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell rich lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumors, and brainstem gliomas.

[0167] Definition of terms

[0168] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0169] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0170] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is determined using the Kabat, Chothia, or IMGT numbering systems.

[0171] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0172] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0173] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0174] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0175] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0176] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain comprising a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain when the full-length antibody is an IgE isotype. In some embodiments, the "full-length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0177] As used herein, the term "Fd" refers to an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546(1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and the term "F(ab')" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains. 2 The term "fragment" refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab' fragment" refers to a reductively linked F(ab'). 2 The fragment obtained after the disulfide bond of the two heavy chain segments in the fragment consists of a complete light chain and a heavy chain Fd fragment (composed of VH and CH1 domains).

[0178] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0179] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0180] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked together to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked together in parallel to form an antibody.

[0181] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0182] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0183] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0184] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0185] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). In some embodiments, the term "chimeric antibody" may include an antibody in which the variable regions of the heavy and light chains are derived from a first antibody, while the constant regions of the heavy and light chains are derived from a second antibody.

[0186] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). A bispecific antibody contains two antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. In some cases, the different antigen-binding domains are linked by peptide linkers.

[0187] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity against at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificity against different antigens (or epitopes), thereby enabling it to bind to at least two different binding sites and / or target molecules. In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0188] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap may be introduced in the first amino acid sequence or nucleic acid sequence to best align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of identity positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). In some embodiments, the two sequences are of the same length.

[0189] The determination of percentage identity between two sequences can also be achieved using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as an improvement upon that in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are integrated into the NBLAST and XBLAST procedures of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0190] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, variants have similar, identical, or improved functions to the polypeptide or peptide from which they are derived.

[0191] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0192] As used herein, the detectable markers described herein can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), and magnetic beads (e.g., ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0193] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0194] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0195] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0196] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0197] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0198] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0199] As used in this article, the term "subject" refers to mammals such as humans, cynomolgus monkeys, and mice.

[0200] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for disease treatment means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0201] Beneficial effects of the invention

[0202] This application yields an antibody or antigen-binding fragment thereof capable of specifically binding to TIGIT. Furthermore, a bispecific antibody capable of specifically binding to CCR8 and TIGIT, as well as a defucosylated bispecific antibody, were prepared. Compared to the target antibody, the bispecific antibody or defucosylated bispecific antibody of this application can: specifically induce ADCC effects against CCR8 / TIGIT co-expressing cells; induce PBMC killing of human CCR8 / human TIGIT co-expressing cells; and inhibit tumor growth in vivo, with significantly superior tumor growth inhibitory activity compared to the control monoclonal antibody. Therefore, the antibody of this application has significant clinical value.

[0203] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0204] Figure 1 A schematic diagram of the structure of the anti-CCR8-anti-TIGIT bispecific antibody of the present invention is shown.

[0205] Figure 2A The diagram shows the binding curves of the anti-CCR8-anti-TIGIT bispecific antibody of the present invention with cells overexpressing human CCR8.

[0206] Figure 2B The diagram shows the binding curves of the anti-CCR8-anti-TIGIT bispecific antibody of the present invention with cells overexpressing human TIGIT.

[0207] Figure 2C The diagram shows the binding curves of the anti-CCR8-anti-TIGIT bispecific antibody of the present invention with cells that simultaneously overexpress human CCR8 and human TIGIT.

[0208] Figure 3A This invention demonstrates the ADCC effect induced by the defucosylated anti-CCR8-anti-TIGIT bispecific antibody of the present invention on cells overexpressing human CCR8 based on a luciferase reporter gene system.

[0209] Figure 3B This invention demonstrates the ADCC effect induced by the defucosylated anti-CCR8-anti-TIGIT bispecific antibody based on the luciferase reporter gene system on cells overexpressing human TIGIT.

[0210] Figure 3C This invention demonstrates the ADCC effect of the defucosylated anti-CCR8-anti-TIGIT bispecific antibody induced by the luciferase reporter gene system on cells simultaneously overexpressing human CCR8 and human TIGIT.

[0211] Figure 4A This invention demonstrates the cytotoxic activity of defucosylated anti-CCR8-anti-TIGIT bispecific antibody-induced PBMCs against human CCR8-overexpressing cells.

[0212] Figure 4B This invention demonstrates the cytotoxic activity of PBMCs induced by the defucosylated anti-CCR8-anti-TIGIT bispecific antibody against human TIGIT-overexpressing cells.

[0213] Figure 4C This invention demonstrates the cytotoxic activity of defucosylated anti-CCR8-anti-TIGIT bispecific antibody-induced PBMCs against cells simultaneously overexpressing human CCR8 and human TIGIT.

[0214] Figure 5The graph shows the inhibition curve of CT26 tumor tissue growth in human CCR8 knock-in Balb / c mice by the anti-CCR8-anti-TIGIT bispecific antibody of the present invention.

[0215] Sequence information

[0216] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0217] Table 1: Sequence Description

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Detailed Implementation

[0224] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0225] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CELLCULTURE (edited by R.R. Freshney (1987)).

[0226] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0227] Example 1. Design and preparation of anti-CCR8-anti-TIGIT bispecific antibody

[0228] Design and preparation of anti-CCR8-anti-TIGIT bispecific antibodies

[0229] Nucleotide sequences encoding the variable region of the heavy chain of the anti-CCR8 antibody ADI-68741 (amino acid sequence as shown in SEQ NO: 16) and the variable region of the heavy chain of ADI-68741-R2-21-DA (amino acid sequence as shown in SEQ NO: 12) were synthesized separately. These sequences were then ligated with nucleotide sequences encoding the hIgG1 heavy chain constant region sequence 1 (amino acid sequence as shown in SEQ NO: 29) and the mIgG2a heavy chain constant region sequence 2 (amino acid sequence as shown in SEQ NO: 30), which can spontaneously form heterodimers, respectively. NheI and XbaI restriction enzyme sites were added to both ends, and the sequences were cloned into the vector pCDNA3.1 to construct plasmids 68741-hIgG1-FL (amino acid sequence as shown in SEQ NO: 37), 68741R2-21-DA-hIgG1-FL (amino acid sequence as shown in SEQ NO: 38), and 68741-mIgG2a-FL (amino acid sequence as shown in SEQ NO: 38). (as shown in SEQ NO: 39) and 68741R2-21-DA-mIgG2a-FL (amino acid sequence as shown in SEQ NO: 40).

[0230] The nucleotide sequence encoding the variable region of the heavy chain of the anti-TIGIT antibody ADI-50377 (amino acid sequence as shown in SEQ NO: 4) was synthesized and linked with the nucleotide sequences encoding the constant region sequence 3 of the hIgG1 heavy chain (amino acid sequence as shown in SEQ NO: 31) and the constant region sequence 4 of the mIgG2a heavy chain (amino acid sequence as shown in SEQ NO: 32), which can spontaneously form heterodimers. NheI and XbaI restriction enzyme sites were added to both ends, and the cloned into the vector pCDNA3.1 to construct plasmids 50377-hIgG1-KR (amino acid sequence as shown in SEQ NO: 41) and 50377-mIgG2a-KR (amino acid sequence as shown in SEQ NO: 42).

[0231] The nucleotide sequences encoding the light chain variable region of the anti-CCR8 antibody ADI-68741 (amino acid sequence identical to ADI-68741-R2-21-DA, as shown in SEQ NO: 20) and the light chain variable region of the anti-TIGIT antibody ADI-50377 (amino acid sequence as shown in SEQ NO: 8) were synthesized and ligated to the nucleotide sequence encoding the human Kappa light chain constant region (amino acid sequence as shown in SEQ NO: 35), respectively. NheI and BamHI restriction enzyme sites were added to both ends, and the sequences were cloned into the vector pCDNA3.1 to construct plasmids 68741LC (amino acid sequence as shown in SEQ NO: 43) and 50377LC (amino acid sequence as shown in SEQ NO: 44).

[0232] The nucleotide sequences encoding the light chain variable region of the anti-CCR8 antibody ADI-68741 (amino acid sequence identical to ADI-68741-R2-21-DA, as shown in SEQ NO: 20) and the light chain variable region of the anti-TIGIT antibody ADI-50377 (amino acid sequence as shown in SEQ NO: 8) were synthesized and ligated to the nucleotide sequence encoding the mouse kappa light chain constant region (amino acid sequence as shown in SEQ NO: 36), respectively. NheI and KpnI restriction enzyme sites were added to both ends, and the sequences were cloned into the vector pCDNA3.1 to construct plasmids 68741-muLC (amino acid sequence as shown in SEQ NO: 45) and 50377-muLC (amino acid sequence as shown in SEQ NO: 46).

[0233] Homologous recombination products were transfected into Top10 competent cells, plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked for sequencing, and plasmids were extracted. Normal antibodies and defucosylated antibodies were transfected and expressed using normal host cell lines (ExpiCHO-S cells F1) (Yida) and defucosylated gene knockout cell lines (CHOS-ADP Fut8 KO) (a self-built line by Promis, obtained by knocking out the FUT8 allele in CHO cells). Specific methods were followed according to the product instructions. The supernatant was collected after 7 days of cell culture.

[0234] Using Protein A affinity chromatography column (MabSelect) TM PrismA (GE Healthcare) purified the target protein. The purification column was equilibrated with 5–10 column volumes of equilibration buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) until the conductivity and pH of the eluent remained constant before loading the sample. After loading, the column was continuously washed with equilibration buffer until the UV value of the eluent no longer decreased. The sample was eluted with elution buffer (20 mM glycine-HCl, pH 2.7), and the eluent was collected. The eluent was neutralized with alkaline buffer (1 M Tris-HCl, pH 8.0). After obtaining the monoclonal antibody, it was concentrated and the buffer was changed to PBS buffer to achieve a final protein concentration of 5–10 mg / mL. Anti-CCR8 antibody and anti-TIGIT antibody were mixed at a 1:1 molar ratio, and DTT was added for reduction at 4°C for 4 hours. The buffer was then changed to 20 mM sodium phosphate, pH 6.0 buffer to remove DTT. Fragments and aggregates were removed using cation exchange. The highly purified components were collected and combined, and the protein was allowed to fully oxidize in air. The antibody Fc fragment formed a heterodimer, yielding an anti-CCR8-anti-TIGIT bispecific antibody. Figure 1(Table 2) The bispecific antibodies against hIgG1 obtained from gene knockout defucosylated cell lines were named 68741-50377-hIgG1(afu) and 68741R2-21-DA-50377-hIgG1(afu), respectively. The bispecific antibodies against mIgG2a obtained from normal host cells were named 68741-50377-mG2a and 68741R2-21-DA-50377-mG2a, respectively.

[0235] Table 2. Sequences of bispecific antibodies

[0236]

[0237] Preparation of control antibodies

[0238] Nucleotide sequences encoding the heavy chain variable regions of anti-CCR8 antibody ADI-68741 (amino acid sequence as shown in SEQ NO: 16), ADI-68741-R1-21-DA heavy chain variable regions (amino acid sequence as shown in SEQ NO: 12), and the heavy chain variable regions of anti-TIGIT antibody ADI-55796 (amino acid sequence as shown in SEQ NO: 24), ADI-50377 (amino acid sequence as shown in SEQ NO: 4) were synthesized and ligated with the nucleotide sequence encoding the wild-type hIgG1 heavy chain constant region sequence 5 (amino acid sequence as shown in SEQ NO: 33). NheI and KpnI restriction enzyme sites were added to both ends, and the sequences were cloned into the vector pCDNA3.1 to construct plasmids 68741HC (amino acid sequence as shown in SEQ NO: 47), 68741R2-21-DA HC (amino acid sequence as shown in SEQ NO: 48), and 55796HC (amino acid sequence as shown in SEQ NO: 48). (as shown in SEQ NO: 49), 50377HC (amino acid sequence as shown in SEQ NO: 50);

[0239] The nucleotide sequences encoding the heavy chain variable region of anti-CCR8 antibody ADI-68741 (amino acid sequence as shown in SEQ NO: 16) and the heavy chain variable region of anti-TIGIT antibody ADI-50377 (amino acid sequence as shown in SEQ NO: 4) were synthesized and ligated with the nucleotide sequence encoding the mIgG2a heavy chain constant region sequence 6 (amino acid sequence as shown in SEQ NO: 34), respectively. NheI and KpnI restriction enzyme sites were added to both ends, and the sequences were cloned into the vector pCDNA3.1 to construct plasmids 68741mIgG2a HC (amino acid sequence as shown in SEQ NO: 52) and 50377mIgG2a HC (amino acid sequence as shown in SEQ NO: 53).

[0240] The gene encoding the variable region of the light chain of the anti-TIGIT antibody ADI-55796 (amino acid sequence as shown in SEQ NO: 28) was synthesized and linked with the nucleotide sequence encoding the constant region of the human Kappa light chain (amino acid sequence as shown in SEQ NO: 35). NheI and BamHI restriction enzyme sites were added to both ends, and the gene was cloned into the vector pCDNA3.1 to construct plasmid 55796LC (amino acid sequence as shown in SEQ NO: 51).

[0241] Homologous recombination products were transfected into Top10 competent cells, plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked for sequencing, and plasmids were extracted. Normal antibodies and defucosylated antibodies were transfected and expressed using normal host cell lines (ExpiCHO-S cells F1) (Yida) and defucosylated gene knockout cell lines (CHOS-ADP Fut8 KO) (built by Pumis, by knocking out the FUT8 allele in CHO cells to obtain Fut8 knockout CHOS cells). The specific methods are as follows: The antibody heavy chain and corresponding light chain plasmid were linearized by enzyme digestion and co-transfected into Expi-CHO cells or CHOS-ADP Fut8 KO cells to form a normal antibody structure. Transfection was performed according to the product instructions. After 5 days of cell culture, the supernatant was collected and the target protein was purified using protein A magnetic beads (purchased from GenScript). Resuspend the magnetic beads in an appropriate volume of binding buffer (PBS + 0.1% Tween, pH 7.4) (1-4 times the volume of the magnetic beads) and add it to the sample to be purified. Incubate at room temperature for 1 hour with gentle shaking. Place the sample on a magnetic rack (purchased from Beaver), discard the supernatant, and wash the magnetic beads 3 times with binding buffer. Add elution buffer (0.1M sodium citrate, pH 3.2) at 3-5 times the volume of the magnetic beads, shake at room temperature for 5-10 minutes, return to the magnetic rack, collect the elution buffer, and transfer it to a collection tube containing neutralization buffer (1M Tris, pH 8.54) and mix well. Name the mIgG2a antibodies obtained from normal host cells as 68741-mG2a and 50377-mG2a, respectively. The hIgG1 antibodies obtained from the defucosylation gene knockout cell line were named 68741-hIgG1(afu), 68741R2-21-DA-hIgG1(afu), 55796-hIgG1(afu), and 50377-hIgG1(afu), respectively.

[0242] Example 2. Anti-CCR8-anti-TIGIT bispecific antibody with overexpression of human CCR8, overexpression of human TIGIT, and other similar antibodies. Overexpression of human CCR8 and human TIGIT cell binding

[0243] CHO cells were transfected with pCHO1.0 vector (purchased from Invitrogen) containing human CCR8 (amino acid sequence as shown in SEQ NO: 54) and human TIGIT (amino acid sequence as shown in SEQ NO: 55) cDNA cloned into MCS, respectively. After pressure selection, CHO cells overexpressing human CCR8 (CHO-huCCR8 cells) and CHO cells overexpressing human TIGIT (CHO-huTIGIT cells) were obtained. A human TIGIT overexpression lentiviral vector was constructed, and lentivirus was packaged and used to infect CHO-huCCR8 cells. Further pressure selection yielded CHO cells simultaneously overexpressing human CCR8 and human TIGIT (CHO-huCCR8 / huTIGIT cells). The expanded culture of overexpressing cells was resuspended in 1×PBS and the cell density was adjusted to 2×10⁶ cells / year. 6 Cells were added at 100 μL / well to a 96-well flow cytometry plate. After centrifugation, the antibody sample was serially diluted with 1×PBS and incubated at 4°C for 30 minutes. Cells were washed twice with 1×PBS, and then Goat F(ab')2 Anti-Human IgG-Fc(PE) fluorescent secondary antibody diluted 200-fold with 1×PBS was added and incubated at 4°C for 30 minutes. After washing twice with 1×PBS, the cells were resuspended in 1×PBS, and flow cytometry was performed on a CytoFlex flow cytometer to detect the fluorescence intensity and calculate the median fluorescence intensity.

[0244] The results are as follows Figure 2A As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =42.59nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =57.99nM) The binding activity of this antibody to CHO-huCCR8 cells was significantly weaker than that of its corresponding anti-CCR8 IgG monoclonal antibody 68741-hIgG1(afu)(EC) 50 =2.42nM). The result is as follows: Figure 2B As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =105.8nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =60.27nM) The cell binding activity of CHO-huTIGIT was significantly weaker than that of its corresponding anti-TIGIT IgG monoclonal antibody 55796-hIgG1(afu)(EC) 50 =1.14nM). The result is as follows: Figure 2CAs shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =13.22nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =11.6nM) The binding activity of CHO-huCCR8 / huTIGIT co-expressed cells with human CCR8 / human TIGIT was slightly weaker than that of its corresponding anti-CCR8 IgG monoclonal antibody 68741-hIgG1(afu)(EC) 50 =3.63nM), but its binding ability with CHO-huCCR8 / huTIGIT cells is still stronger than that with CHO-huCCR8 and CHO-huTIGIT cells.

[0245] The above results indicate that the anti-CCR8-anti-TIGIT bispecific antibody of the present invention can bind better to human CCR8 / human TIGIT co-expressing cells, while binding weaker to human CCR8 or human TIGIT single-expressing cells.

[0246] Example 3. ADCC activity of anti-CCR8-anti-TIGIT bispecific antibody based on luciferase reporter gene system

[0247] Expanded culture of CD16a-NFAT-Luc Jurkat effector cells (prepared by Promega, using hCD16a lentiviral vector and luc2P NFAT-RE Hygro (Promega, pGL4.30) co-transfected Jurkat cells, followed by antibiotic resistance selection to obtain CD16a-NFAT-Luc Jurkat reporter gene cells), resuspended cells in 1640 medium containing 10% low IgG FBS to 4 × 10⁻⁶ cells / year. 6 Cells / mL. CHO-huCCR8 cells, CHO-huTIGIT cells, and CHO-huCCR8 / huTIGIT cells were resuspended in 1640 medium containing 10% low IgG FBS and diluted to 1.6 × 10⁻⁶ cells / mL. 6 Cells / mL. The three cell suspensions were mixed 1:1 with CD16a-NFAT-Luc Jurkat cells, and 50 μL / well was added to sterile 96-well white plates. Finally, serially diluted antibody samples containing 10% low IgG FBS were added to 1640 medium. The cells were incubated at 37°C, 5% CO2, in the dark for 6 hours. After incubation, the cells were removed, equilibrated at room temperature for 5 minutes, and 100 μL / well was added to Bio-turbo reagent (purchased from Ruian). Fluorescence signal values ​​were read using a SpectraMAX multi-mode microplate reader.

[0248] The results are as follows Figure 3AAs shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =1.38nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =1.46nM) mediated ADCC activity in CHO-huCCR8 cells was significantly weaker than that mediated by its corresponding anti-CCR8 IgG monoclonal antibody 68741-hIgG1(afu)(EC) 50 =0.014nM). The result is as follows: Figure 3B As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =1.57nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =1.75nM) mediated ADCC activity in CHO-huTIGIT cells was significantly weaker than that mediated by its corresponding anti-TIGIT IgG monoclonal antibody 55796-hIgG1(afu)(EC) 50 =0.04nM). The result is as follows: Figure 3C As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =0.026nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =0.026nM) mediated ADCC activity in CHO-huCCR8 / huTIGIT cells compared to its corresponding anti-TIGIT IgG monoclonal antibody 55796-hIgG1(afu)(EC) 50 =0.066nM) is stronger.

[0249] The above results indicate that the anti-CCR8-anti-TIGIT bispecific antibody of the present invention can better induce ADCC effect on human CCR8 / human TIGIT co-expressing cells, while the ADCC effect on human CCR8 or human TIGIT single-expressing cells is weaker.

[0250] Example 4. PBMC Killing Activity

[0251] Target cells CHO-huCCR8, CHO-huTIGIT, and CHO-huCCR8 / CHO-huTIGIT were stained using the CellTraceViolet kit, with 1 × 10⁶ cells per well. 4 Seed 1 x 10⁶ cells / well into 96-well clear-bottomed, black-edged cell culture plates. Collect PBMCs that have been revived one day in advance and seed them at a rate of 1 x 10⁶ cells / well. 5DAPI cells were added to each well of the target cells, and then serially diluted test antibody was added to the wells for incubation for 48 hours. After 48 hours, DAPI fluorescence signals were collected using Cytation 5 and the corresponding killing intensity was calculated.

[0252] The results are as follows Figure 4A As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =0.017nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =0.019nM) The killing activity of PBMCs against CHO-huCCR8 cells induced by phosphodiesterase inhibitors (PFI) was significantly weaker than that induced by defucosylated anti-CCR8 IgG monoclonal antibody 68741-hIgG1(afu)(EC) 50 =0.001nM). The result is as follows: Figure 4B As shown, defucosylated 68741-50377-hIgG1(afu)(EC) 50 =0.011nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =0.007nM) induces PBMCs with significantly weaker killing activity against CHO-huTIGIT cells than defucosylated anti-TIGIT IgG monoclonal antibody 55796-hIgG1(afu)(EC) 50 =0.0008nM). The result is as follows: Figure 4C As shown, the defucosylated anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-hIgG1(afu)(EC) 50 =1.128nM), 68741-R2-21-DA-50377-hIgG1(afu)(EC 50 =1.233nM) induces the killing activity of PBMCs against human CCR8 / human TIGIT co-expressing CHO cells and defucosylated anti-CCR8 monoclonal antibody 68741-hIgG1(afu)(EC 50 =1.175nM) and anti-TIGIT monoclonal antibody 55796-hIgG1(afu) (EC 50 =1.31nM) is equivalent.

[0253] The above results indicate that the anti-CCR8-anti-TIGIT bispecific antibody of the present invention can better induce PBMCs to kill human CCR8 / human TIGIT co-expressing cells, while weakening the killing effect on human CCR8 or human TIGIT single-expressing cells.

[0254] Example 5. Anti-CCR8-anti-TIGIT bispecific antibody in human CCR8 knock-in mice inoculated with CT26 tumor model In vivo pharmacodynamic studies in the form of [type]

[0255] This experiment established a tumor-bearing model by inoculating human CCR8 knock-in Balb / c mice with CT-26 colon cancer cells (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to determine the anti-tumor effect of the anti-CCR8-anti-TIGIT bispecific antibody of this invention.

[0256] Specifically, CT26 cells were cultured and expanded in vitro. After trypsin digestion, the cells were collected, washed three times with PBS, counted, and subcutaneously inoculated into the right abdomen of human CCR8 knock-in Balb / c mice. The cells were cultured until the average tumor volume reached 100 mm². 3 Mice were divided into groups of 6. Different antibodies were administered via tail vein injection with the same volume and molar dose. Tumor volume and body weight changes in each group were monitored every 2-3 days for 2 to 3 weeks. The dosage and administration method are shown in Table 3.

[0257] Table 3: Assay protocol for anti-CCR8-anti-TIGIT dual antitumor inhibitory activity

[0258]

[0259]

[0260] The results are as follows Figure 5 As shown, compared with the PBS group, the anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-mG2a almost completely inhibited the increase in tumor volume. Specifically, the tumor growth inhibition index (TGI) on day 24 for anti-CCR8 monoclonal antibody 68741-mG2a, anti-TIGIT monoclonal antibody 50377-mG2a, monoclonal antibody combined with 68741-mG2a+50377-mG2a, and anti-CCR8-anti-TIGIT bispecific antibody 68741-50377-mG2a were 34%, 46%, 93%, and 94%, respectively. Furthermore, the anti-CCR8-anti-TIGIT bispecific antibody group exhibited the most significant tumor growth inhibition activity, significantly stronger than the anti-CCR8 monoclonal antibody group and the anti-TIGIT monoclonal antibody group, even at a lower dosage than the monoclonal antibody combined with 68741-mG2a+50377-mG2a group.

[0261] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to TIGIT, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:1, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:2, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:3, or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; And / or, (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VLCDR3, which consists of the following sequence: SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, any one of (i)-(vi) is a conservative substitution; Preferably, the CDR described in any one of (i)-(vi) is defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system.

2. The antibody of claim 1 or its antigen-binding fragment, comprising: three heavy chain CDRs as shown in SEQ ID NO:1, VH CDR2 as shown in SEQ ID NO:2, and VH CDR3 as shown in SEQ ID NO:3; and / or three light chain CDRs as shown in SEQ ID NO:5, VL CDR1 as shown in SEQ ID NO:6, and VL CDR3 as shown in SEQ ID NO:7; Preferably, the antibody or its antigen-binding fragment further comprises the framework region of a human immunoglobulin.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO:4; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:4; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:4; and / or (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:8; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:8; or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:8; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: VH having the sequence shown in SEQ ID NO:4 and VL having the sequence shown in SEQ ID NO:

8.

4. An antibody or antigen-binding fragment thereof capable of specifically binding to CCR8, said antibody or antigen-binding fragment comprising: (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs): (i) VH CDR1, which consists of the sequence shown in SEQ ID NO:9 or 13, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (ii) VH CDR2, which consists of the sequence shown in SEQ ID NO:10 or 14, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (iii) VH CDR3, which consists of the sequence shown in SEQ ID NO:11 or 15, or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; And / or, (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:17, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it. (v)VL CDR2, which consists of the following sequence: SEQ ID NO:18, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to it, and (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it; Preferably, any one of (i)-(vi) is a conservative substitution; Preferably, the CDR described in any one of (i)-(vi) is defined according to the Kabat, IMGT or Chothia numbering system; Preferably, the CDR described in any one of (i)-(vi) is defined according to the IMGT numbering system.

5. The antibody of claim 4 or its antigen-binding fragment, comprising: (1) The following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:9, VHCDR2 as shown in SEQ ID NO:10, and VH CDR3 as shown in SEQ ID NO:11; and / or, the following three light chain CDRs: VLCDR1 as shown in SEQ ID NO:17, VL CDR2 as shown in SEQ ID NO:18, and VL CDR3 as shown in SEQ ID NO:19; or, (2) The following three heavy chain CDRs: VH CDR1 as shown in SEQ ID NO:13, VHCDR2 as shown in SEQ ID NO:14, and VH CDR3 as shown in SEQ ID NO:15; and / or the following three light chain CDRs: VLCDR1 as shown in SEQ ID NO:17, VL CDR2 as shown in SEQ ID NO:18, and VL CDR3 as shown in SEQ ID NO:19; Preferably, the antibody or its antigen-binding fragment further comprises the framework region of a human immunoglobulin.

6. The antibody or antigen-binding fragment thereof of claim 4 or 5, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following: (i) The sequence shown in SEQ ID NO: 12 or 16; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 12 or 16; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 12 or 16; and / or (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following: (iv) The sequence shown in SEQ ID NO:20; (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:20; or (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:20; Preferably, the substitution described in (ii) or (v) is a conservative substitution; Preferably, the antibody or its antigen-binding fragment comprises: (1) VH having the sequence shown in SEQ ID NO:12 and VL having the sequence shown in SEQ ID NO:20; or (2) VH having the sequence shown in SEQ ID NO:16 and VL having the sequence shown in SEQ ID NO:

20.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, It further includes constant regions derived from mammalian (e.g., human, mouse) immunoglobulins; Preferably, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from human or mouse; for example, a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; for example, a mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3 heavy chain constant region. Preferably, the heavy chain constant region has an LALA mutation and / or a knock-into-hole modification; Preferably, the heavy chain constant region has a sequence as shown in any one of SEQ ID NO:29-34; Preferably, the light chain of the antibody or its antigen-binding fragment contains a light chain constant region derived from mammalian (e.g., human, mouse) immunoglobulins (e.g., κ or λ). Preferably, the light chain constant region has a sequence as shown in SEQ ID NO:35 or 36; Preferably, the antibody or its antigen-binding fragment is low-fucosylated or non-fucosylated.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein, The antigen-binding fragment is selected from Fab, Fab', and (Fab'). 2 Fv, disulfide-linked Fv, scFv, diabody, and sdAb; and / or, the antibody is a murine antibody, chimeric antibody, humanized antibody, or multispecific antibody.

9. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9; preferably, the vector is a cloning vector or an expression vector.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10; Preferably, the host cell is a mammalian cell; Preferably, the host cell has low or no fucosylation activity, for example, selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosylation enzymes.

12. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, comprising culturing the host cell of claim 11 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture; Preferably, the host cell has low or no fucosylation activity, for example, selected from mammalian cells (e.g., CHO cells) that lack expression of genes encoding fucosylation enzymes.

13. A multispecific antibody (e.g., a bispecific antibody) comprising the antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

14. Multispecific antibodies (e.g., bispecific antibodies) that specifically bind to CCR8 and TIGIT, comprising a first antigen-binding domain specific to CCR8 and a second antigen-binding domain specific to TIGIT.

15. The multispecific antibody of claim 14, wherein, The second antigen-binding domain comprises: the antibody or its antigen-binding fragment as described in any one of claims 1-3.

16. The multispecific antibody according to claim 14 or 15, wherein, The first antigen-binding domain comprises: the antibody or its antigen-binding fragment as described in any one of claims 4-6.

17. The multispecific antibody according to any one of claims 14-16, wherein, The first antigen-binding domain is a Fab fragment or scFv, and the second antigen-binding domain is a Fab fragment or scFv; Preferably, the first antigen-binding domain is a Fab fragment, and the second antigen-binding domain is a Fab fragment or scFv.

18. The multispecific antibody of any one of claims 14-17, further comprising an Fc domain, said Fc domain comprising a first monomer and a second monomer; Preferably, the first antigen-binding domain and the second antigen-binding domain are optionally connected to the N-terminus of the first monomer and the second monomer of the Fc domain via connectors, respectively.

19. The multispecific antibody of claim 18, wherein, The Fc domain contains modifications to promote the dimerization of the first and second monomers; Preferably, the modification includes an amino acid substitution in the CH3 domain of the Fc domain.

20. The multispecific antibody of claim 18 or 19, wherein, The Fc domain contains modifications to facilitate cFAE (Controlled Fab-arm Exchange); Preferably, the modification includes an amino acid substitution in the CH3 domain of the Fc domain.

21. The multispecific antibody according to any one of claims 18-20, wherein, The modification includes a "knob" modification in one of the two monomers in the Fc domain and a "knob-into-hole" modification in the other of the two monomers in the Fc domain, to form a "knob-into-hole" modification; Preferably, the modification comprises a substitution selected from F405L and / or R411T in one of the two monomers in the Fc domain and a substitution selected from T370K and / or K409R in the other of the two monomers in the Fc domain, to form a modification that promotes cFAE. Preferably, the two monomers of the Fc domain respectively contain amino acid sequences as shown in SEQ ID NO:29 and 30, or amino acid sequences as shown in SEQ ID NO:31 and 32, or amino acid sequences as shown in SEQ ID NO:33 and 34.

22. The multispecific antibody of any one of claims 14-21, wherein it is low-fucosylated or non-fucosylated.

23. The multispecific antibody according to any one of claims 14-22, wherein, The first antigen-binding domain is a Fab fragment, the second antigen-binding domain is a Fab fragment, and the multispecific antibody comprises: (i) A first peptide chain comprising the VL of the first antigen-binding domain and a first light chain constant region (CL); preferably, the first CL is kappa; (ii) A second peptide chain comprising the VH of the first antigen-binding domain and a first heavy chain constant region (CH); preferably, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; (iii) A third peptide chain comprising the VH of the second antigen-binding domain and the second heavy chain constant region (CH); preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; (iv) A fourth peptide chain comprising the VL of the second antigen-binding domain and the second light chain constant region (CL); preferably, the second CL is kappa; Preferably, the Fc domain monomers of the first CH and the second CH form a dimer; preferably, the Fc domain includes the modifications defined in claim 21; Preferably, the first CH and the first CL, and the second CH and the second CL can each form a dimer.

24. The multispecific antibody of claim 23, wherein, The multispecific antibody comprises: (1) A first peptide chain containing the sequence shown in SEQ ID NO:43, a second peptide chain containing the sequence shown in SEQ ID NO:37, a third peptide chain containing the sequence shown in SEQ ID NO:41, and a fourth peptide chain containing the sequence shown in SEQ ID NO:44; (2) A first peptide chain containing the sequence shown in SEQ ID NO:43, a second peptide chain containing the sequence shown in SEQ ID NO:38, a third peptide chain containing the sequence shown in SEQ ID NO:41, and a fourth peptide chain containing the sequence shown in SEQ ID NO:44; (3) A first peptide chain comprising the sequence shown in SEQ ID NO:45, a second peptide chain comprising the sequence shown in SEQ ID NO:39, a third peptide chain comprising the sequence shown in SEQ ID NO:42, and a fourth peptide chain comprising the sequence shown in SEQ ID NO:46; or (4) A first peptide chain containing the sequence shown in SEQ ID NO:45, a second peptide chain containing the sequence shown in SEQ ID NO:40, a third peptide chain containing the sequence shown in SEQ ID NO:42, and a fourth peptide chain containing the sequence shown in SEQ ID NO:

46.

25. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a multispecific antibody as described in any one of claims 13-24 or at least one peptide chain thereof.

26. A vector comprising the isolated nucleic acid molecule of claim 25; Preferably, the vector contains nucleotide sequences encoding each peptide chain of the multispecific antibody, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

27. A host cell comprising the isolated nucleic acid molecule of claim 25 or the vector of claim 26; Preferably, the host cell has low or no fucosylation activity, for example, it may be a mammalian cell lacking the expression of a gene encoding a fucosylation enzyme (e.g., FUT8), such as a CHO cell.

28. A method for preparing the multispecific antibody according to any one of claims 13-24, comprising culturing the host cell according to claim 27 under conditions that allow protein expression, and recovering the multispecific antibody from the cultured host cell culture.

29. The method of claim 28, wherein, The host cell has low or no fucosylation activity, thereby producing low or no fucosylated antibodies; preferably, the host cell can be a mammalian cell lacking the expression of a gene encoding a fucosylation enzyme (e.g., FUT8), such as a CHO cell.

30. An immunoconjugate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-8 or a multispecific antibody as described in any one of claims 13-24, and a therapeutic agent attached to said antibody or antigen-binding fragment thereof or multispecific antibody; Preferably, the therapeutic agent is selected from cytotoxic agents; Preferably, the therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive nuclides, and any combination thereof; Preferably, the immunoconjugate is an antibody-drug conjugate (ADC).

31. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, or a multispecific antibody as described in any one of claims 13-24, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents; Preferably, the additional pharmaceutically active agent is a drug with antitumor activity, such as an alkylating agent, mitotic inhibitor, antitumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, radiosensitizer, antiangiogenic agent, cytokine, molecularly targeted drug, immune checkpoint inhibitor, or oncolytic virus.

32. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 or the multispecific antibody as described in any one of claims 13-24; Preferably, the antibody or its antigen-binding fragment carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. Preferably, the multispecific antibody carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

33. A method for inhibiting the growth of tumor cells expressing CCR8 and / or TIGIT and / or killing said tumor cells, comprising contacting said tumor cells with an effective amount of an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, or a multispecific antibody as claimed in any one of claims 13-24, or an immunoconjugate as claimed in claim 30, or a pharmaceutical composition as claimed in claim 31.

34. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the multispecific antibody according to any one of claims 13-24, or the immunoconjugate according to claim 30, or the pharmaceutical composition according to claim 31, in the preparation of a medicament, wherein the medicament is used for: (1) Enhance the activity of immune cells in vitro or in subjects; (2) Enhance the immune response in subjects; (3) Prevention and / or treatment of tumors in subjects; or (4) Prevention and / or treatment of infection in subjects; Preferably, the tumor expresses CCR8 and / or TIGIT; Preferably, CCR8 and / or TIGIT are expressed on the surface of the tumor cells; Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematologic malignancies such as classical Hodgkin lymphoma. (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors, such as primary CNS lymphoma, spinal axis tumors, brainstem glioma; Preferably, the infection is selected from viral infection, bacterial infection, fungal infection, and parasitic infection; Preferably, the subject is a mammal, such as a human, a cynomolgus monkey, or a mouse.

35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8 or the multispecific antibody according to any one of claims 13-24 in the preparation of a kit for detecting whether a tumor can be treated by antitumor therapy targeting CCR8 and / or TIGIT; (1) Contact the sample containing the tumor cells with the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 or the multispecific antibody as described in any one of claims 13-24; (2) Detect the formation of the antibody or its antigen-binding fragment or the complex between the multispecific antibody and CCR8 and / or TIGIT; Preferably, the antibody or its antigen-binding fragment, or the multispecific antibody, carries a detectable label; Preferably, the CCR8 and / or TIGIT are CCR8 and / or TIGIT of mammals (e.g., humans, monkeys); Preferably, the tumor is selected from non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and other hematologic malignancies such as classical Hodgkin lymphoma. (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, Hodgkin lymphoma, central nervous system (CNS) tumors such as primary CNS lymphoma, spinal axis tumors, brainstem glioma.

Images