Antibodies targeting CCR8 and uses thereof

Abstract

The invention belongs to the field of biological medicine. Specifically, the invention provides an antibody targeting CCR8 and application of the antibody. More specifically, the invention provides an antibody targeting CCR8, STAR and CAR derived from the antibody, a therapeutic immune cell comprising the STAR or CAR, and applications thereof in disease treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the field of cell therapy, and specifically to a synthetic T-cell receptor and antigen receptor (STAR) targeting receptors in immunosuppressive active cells and tumor antigens, and to a vector comprising the receptor, a T cell comprising the receptor and a preparation method thereof. BACKGROUND

[0002] Chimeric antigen receptor T cell (CAR-T) therapy is an anti-cancer immunotherapy that has achieved good efficacy in recent years. Unlike the way in which natural T cells recognize tumor cells, the recognition of tumor cells by CAR-T cells does not depend on MHC molecules (such as HLA). The CAR molecule mainly consists of an extracellular region responsible for recognizing target antigens, a transmembrane region, and an intracellular region responsible for transmitting T cell activation signals after receiving stimulation. When the CAR molecule is transfected into the T cells of a patient by transfection technology, the patient's T cells express tumor antigen receptors, and the purified and expanded CAR-T cells have been used to treat B-cell lymphoma and hematological tumors. However, CAR-T therapy has encountered difficulties in treating solid tumors. There are multiple factors for the failure of CAR-T cell therapy to achieve good efficacy in the treatment of solid tumors, one of which is that CAR-T cells are inhibited in the tumor microenvironment and T cells are prone to exhaustion and apoptosis. Recent studies have shown that the presence of suppressive immune cells (MDSC and Treg cells, etc.) in the tumor microenvironment has a strong inhibitory effect on various tumor immunotherapies, including cell therapy, greatly limiting its therapeutic potential.

[0003] The T cell receptor (TCR) complex molecule contains multiple chains, and the TCR alpha chain and the TCR beta chain are responsible for recognizing MHC-polypeptide molecules, and the other six CD3 subunits combine with the TCR alpha / beta chain to function as a signal transduction. The natural TCR complex contains a total of 10 ITAM signal sequences, which can theoretically transmit stronger signals than CAR. Previous studies have shown that although the signal of TCR is slower than that of CAR, the signal of TCR is more persistent. Therefore, the use of the signal transduction function of natural TCR can alleviate T cell dysfunction and enable it to better play a role in anti-solid tumor. However, the recognition of tumor cells by TCR needs to rely on MHC molecules, and the affinity is lower than that of CAR.

[0004] Based on the high similarity between the extracellular region of TCR and the Fab domain of antibody, the variable region sequence of TCR is replaced by the variable region sequence of antibody (such as scFv), so as to obtain a synthetic T cell receptor and antigen receptor (STAR). STAR combines the advantages of TCR and CAR, has the specificity of antibody and the superior signal transmission function of natural TCR, can mediate complete T cell activation, and has great improvement in safety and effectiveness, so it has become a promising new type of cellular immunotherapy.

[0005] However, STAR derived from natural TCR still cannot overcome the shortcomings that CAR-T cells are inhibited in the tumor microenvironment and T cells are prone to exhaustion and apoptosis. Regulatory T (Treg) cells expressing Foxp3 inhibit abnormal immune responses to self-antigens, and are also a key cell component that inhibits anti-tumor immune responses in the tumor microenvironment. A large number of Treg cells infiltrating into tumor tissues are often associated with poor prognosis. More and more evidence shows that removing Treg cells can enhance anti-tumor immune responses. On the other hand, systemic depletion of Treg cells can also cause harmful autoimmune responses to the body (such as anti-CTLA-4 mAb, IL2-Fc and anti-CCR4 mAb, etc.). Therefore, it is a more ideal tumor immunization strategy to specifically target tumor-infiltrating Treg cells with immunosuppressive activity, without targeting inflammatory Treg cells and non-Treg type T cells.

[0006] CCR8 is a member of the chemokine receptor subfamily, which is a seven-transmembrane G protein-coupled receptor. CCR8 is specifically expressed on tumor-infiltrating regulatory T cells (Treg), but is not expressed on peripheral blood Treg or normal tissues. It is the receptor of chemokine CCL1 and is involved in the recruitment of Tregs and Th2 cells to inflammatory and tumor sites. CCR8 is generally up-regulated in tumor tissues and has a high correlation with Foxp3. The expression of CCR8 and Foxp3 is related to survival rate and disease stage in various cancers. Antibodies targeting CCR8 can specifically eliminate tumor-infiltrating Tregs with immunosuppressive activity, inhibit tumor growth, and have no function of targeting inflammatory Treg cells, peripheral Treg cells and Teff cells in tumor tissues.

[0007] Based on the advantages of STAR and the specific expression of CCR8 on the surface of tumor-infiltrating Treg cells, the present application replaces the variable region of one chain of TCR with a CCR8-targeting antibody or ligand that binds to CCR8 on the surface of Treg cells, and replaces the variable region of the other chain of TCR with an antibody sequence (such as scFv or VHH, etc.) targeting a tumor antigen, thereby constructing and expressing a novel STAR that can target both CCR8 and tumor antigens. Since Tregs with immunosuppressive activity are removed, the targeting killing of immune cells (such as T cells, NK cells, and macrophages, etc.) on tumors can be enhanced, thereby further improving the effect of cell therapy. SUMMARY

[0008] The present application relates to a single-domain antibody that specifically binds to CCR8 and its application in synthetic T cell receptor antigen receptor (STAR) and chimeric antigen receptor (CAR). The single-domain antibody comprises specific CDR1, CDR2 and CDR3 sequences, and can specifically bind to CCR8 with high efficiency. Based on this single-domain antibody, the present application further develops various STAR and CAR structures targeting CCR8, which can be used to treat various CCR8-related diseases.

[0009] The core content of the present application includes the following aspects:

[0010] Development of single-domain antibody: a single-domain antibody that specifically binds to CCR8 is provided, which comprises specific CDR1, CDR2 and CDR3 sequences (as shown in SEQ ID NO: 8). The antibody can be a murinized antibody or a humanized antibody, which has high specificity and high affinity.

[0011] Synthetic T cell receptor antigen receptor (STAR): a STAR targeting CCR8 is developed, which comprises the single-domain antibody in the antigen binding region. The STAR structure can comprise a single or multiple antigen binding regions, and can be combined with an antibody or its antigen binding fragment that specifically binds to other antigens (such as MSLN or Claudin18.2) to form a dual-targeting structure.

[0012] Chimeric antigen receptor (CAR): a CAR targeting CCR8 is developed, which comprises the single-domain antibody in the extracellular antigen binding region. The CAR structure can also comprise an antigen binding region that specifically binds to other antigens to achieve multi-targeting therapy.

[0013] Nucleic acid molecules and expression vectors: nucleic acid molecules encoding the single-domain antibody, STAR and CAR are provided, as well as expression vectors comprising these nucleic acid molecules. These expression vectors can be used to express the corresponding protein structures in host cells.

[0014] Host cells and therapeutic immune cells: Cells expressing the single domain antibody, STAR or CAR are obtained by transforming host cells. These cells can be further used to prepare therapeutic immune cells, such as T cells or NK cells.

[0015] Pharmaceutical compositions and therapeutic uses: Pharmaceutical compositions comprising the single domain antibody, STAR, CAR or therapeutic immune cells are developed for treating a variety of CCR8-related diseases, particularly hematological tumors and solid tumors.

[0016] The single domain antibody, STAR and CAR structure of the present application has high specificity and high affinity, which can effectively target CCR8, and the design of combining other antigen binding regions can further improve the therapeutic effect. Through the development of nucleic acid molecules and expression vectors, the present application provides a basis for large-scale production and application. In addition, the present application also covers the application of these structures in pharmaceutical compositions, which provides a new strategy for treating a variety of diseases.

[0017] In a first aspect, the present application provides a single domain antibody specifically binding to CCR8, comprising a CDR1, a CDR2 and a CDR3 selected from the sequence of SEQ ID NO: 8.

[0018] In some aspects, the single domain antibody as previously described comprises a CDR1 as shown in SEQ ID NO: 5, a CDR2 as shown in SEQ ID NO: 6, and a CDR3 as shown in SEQ ID NO: 7.

[0019] In some aspects, the single domain antibody as previously described comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 8, preferably it comprises the amino acid sequence shown in SEQ ID NO: 8.

[0020] In some aspects, the single domain antibody as previously described is a murinized antibody or a humanized antibody.

[0021] In a second aspect, the present application provides a synthetic T cell receptor antigen receptor (STAR) targeting CCR8, comprising a single domain antibody specifically binding to CCR8 as previously described.

[0022] In some aspects, the STAR as previously described comprises a first peptide chain and a second peptide chain:

[0023] i) the first peptide chain comprises a first target binding region and a first constant region, and the second peptide chain comprises a second target binding region and a second constant region; or,

[0024] ii) the first peptide chain comprises a first constant region, the second peptide chain comprises a second constant region, the first peptide chain or the second peptide chain comprises a first target binding region;

[0025] wherein the first target binding region and / or the second target binding region comprises one or more antigen binding regions, which are the same or different;

[0026] The antigen binding region in the first target binding region and / or the second target binding region comprises a CCR8 single domain antibody as described in any of the preceding.

[0027] In some aspects, the STAR as described in any of the preceding, the antigen binding region in the first target binding region and / or the second target binding region further comprises an antibody or antigen binding fragment thereof that specifically binds to MSLN or Claudin 18.2.

[0028] Preferably, the antibody or antigen binding fragment thereof is selected from a scFv, a nanobody or a single domain antibody.

[0029] In some aspects, the STAR as described in any of the preceding, the STAR comprises any one of the following groups:

[0030] a) the first peptide chain comprises a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, at least one CCR8 single domain antibody as described in any of the preceding, a second constant region;

[0031] b) the first peptide chain comprises, from N-terminus to C-terminus, a CCR8 single domain antibody as described in any of the preceding, a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, an antibody or antigen binding fragment thereof that specifically binds to MSLN or Claudin 18.2, a second constant region;

[0032] c) the first peptide chain comprises a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, at least one CCR8 single domain antibody as described in any of the preceding, an antibody or antigen binding fragment thereof that specifically binds to MSLN or Claudin 18.2, a second constant region;

[0033] The first constant region of the first peptide chain and the second constant region of the second peptide chain in each of the above groups a)-c) are a constant region of a TCR a chain or a constant region of a TCR β chain; the constant regions of the first peptide chain and the second peptide chain are not simultaneously a constant region of a TCR a chain or not simultaneously a constant region of a TCR β chain.

[0034] In some aspects, the STAR as previously described, wherein the antibody or antigen binding fragment thereof that specifically binds MSLN comprises a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33, preferably the antibody or antigen binding fragment thereof that specifically binds MSLN comprises a single domain antibody as set forth in the amino acid sequence of SEQ ID NO: 34; and

[0035] the antibody or antigen binding fragment thereof that specifically binds Claudin 18.2 comprises a CDR1 as set forth in SEQ ID NO: 41, a CDR2 as set forth in SEQ ID NO: 42, and a CDR3 as set forth in SEQ ID NO: 43, preferably the antibody or antigen binding fragment thereof that specifically binds Claudin 18.2 comprises a single domain antibody as set forth in the amino acid sequence of SEQ ID NO: 44.

[0036] In some aspects, the STAR as previously described,

[0037] the first constant region is a TCR a chain constant region or a TCR β chain constant region, preferably a modified TCR a chain constant region or a TCR β chain constant region;

[0038] the second constant region is a TCR a chain constant region or a TCR β chain constant region, preferably a modified TCR a chain constant region or a TCR β chain constant region;

[0039] Preferably, the TCR a chain constant region is selected from the group consisting of a wild type human or wild type mouse TCR a chain constant region;

[0040] Preferably, the TCR β chain constant region is selected from the group consisting of a wild type human or wild type mouse TCR β chain constant region.

[0041] In some aspects, the STAR, wherein the modified TCR a chain constant region is derived from a mouse TCR a chain constant region that comprises one or more modifications, which are amino acid substitutions or deletions, at positions 6, 13, 15-18, 48, 112, 114, 115, 122, 136, 137, relative to a wild type mouse TCR a chain constant region;

[0042] the modified TCR β chain constant region is derived from a mouse TCR β chain constant region that comprises one or more modifications, which are amino acid substitutions or deletions, at positions 3, 6, 9, 11, 12, 17, 21-25, 56, 150, 162-172, 168, or 170, relative to a wild type mouse TCR β chain constant region; or

[0043] the modified TCR a chain constant region is derived from a human TCR a chain constant region which comprises one or more modifications, which are amino acid substitutions or deletions, at positions 47, 90-93, 115, and 118, relative to a human wild-type TCR a chain constant region; and / or

[0044] the modified TCR β chain constant region is derived from a human TCR β chain constant region which comprises one or more modifications, which are amino acid substitutions or deletions, at positions 17, 21, 56, 132, 135, and 138, relative to a human wild-type TCR β chain constant region.

[0045] In some aspects, a STAR as previously described, characterized in that the modified TCR a chain constant region is derived from a mouse wild-type TCR a chain constant region which comprises one or more amino acid mutations or combinations thereof selected from the group consisting of:

[0046] i) a T48C amino acid substitution;

[0047] ii) S112L, M114I, and / or G115V amino acid substitutions;

[0048] iii) E6D, K13R amino acid substitutions and deletion of amino acids at positions 15-18;

[0049] iv) a K122R amino acid substitution;

[0050] v) deletion of the constant region intracellular domain, deletion of amino acids at positions 136-137;

[0051] and / or the modified TCR β chain constant region is derived from a mouse wild-type TCR β chain constant region which comprises one or more amino acid mutations or combinations thereof selected from the group consisting of:

[0052] i) a S56C amino acid substitution;

[0053] ii) substitution of amino acids at positions 150, 168, 170 to R;

[0054] iii) R3K, T6F, K9E, S11A, L12V amino acid substitutions, and / or deletion of amino acids at positions 17, 21-25;

[0055] iv) deletion of the constant region intracellular domain, deletion of amino acids at positions 167-172.

[0056]

[0057] ​In some aspects, a STAR as previously described, characterized in that the modified TCR a chain constant region is derived from a human wild-type TCR a chain constant region comprising an amino acid mutation or a combination thereof selected from the group consisting of:

[0058] i) a T47C amino acid substitution;

[0059] ii) a P90S, E91D, S92V, S93P amino acid substitution;

[0060] iii) a S115L, G118V amino acid substitution.

[0061] and / or the modified TCR β chain constant region is derived from a human wild-type TCR β chain constant region comprising an amino acid mutation or a combination thereof selected from the group consisting of:

[0062] i) a S56C amino acid substitution;

[0063] ii) a E17K, S21A, F132I, E135A and / or Q138H amino acid substitution.

[0064] In some aspects, a STAR as previously described, wherein the amino acid sequence of the constant region of the modified TCR a chain is selected from the sequence as set forth in any one of SEQ ID NOs: 11-15, and / or the amino acid sequence of the constant region of the modified TCR β chain is selected from the sequence as set forth in any one of SEQ ID NOs: 18-22.

[0065] In some aspects, a STAR as previously described, wherein the first peptide chain and / or the second peptide chain has at least one exogenous intracellular functional domain, such as an intracellular domain of a costimulatory molecule, preferably an intracellular domain of OX40, more preferably the intracellular domain of OX40 comprises an amino acid sequence as set forth in SEQ ID NO: 23, linked at its C-terminus.

[0066] In some aspects, a STAR as previously described, wherein the exogenous intracellular functional domain is linked directly or via a linker to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain,

[0067] preferably the exogenous intracellular functional domain is linked via a linker to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain which is deleted of the intracellular region,

[0068] preferably the linker is a (G4S)nlinker or a (EAAAK)nlinker, wherein n represents an integer from 1 to 10, preferably n is 3 or 4.

[0069] In some aspects, a STAR as previously described, wherein the STAR is co-expressed with a membrane-bound IL-15 protein (mbIL-15).

[0070] In some aspects, a STAR as previously described, wherein

[0071] a) the STAR comprises a first peptide chain set forth in SEQ ID NO: 48 and a second peptide chain set forth in SEQ ID NO: 47;

[0072] b) the STAR comprises a first peptide chain set forth in SEQ ID NO: 50 and a second peptide chain set forth in SEQ ID NO: 49;

[0073] c) the STAR comprises a first peptide chain set forth in SEQ ID NO: 52 and a second peptide chain set forth in SEQ ID NO: 51;

[0074] d) the STAR comprises a first peptide chain set forth in SEQ ID NO: 54 and a second peptide chain set forth in SEQ ID NO: 53;

[0075] e) the STAR comprises a first peptide chain set forth in SEQ ID NO: 56 and a second peptide chain set forth in SEQ ID NO: 55; or

[0076] f) the STAR comprises a first peptide chain set forth in SEQ ID NO: 58 and a second peptide chain set forth in SEQ ID NO: 57.

[0077] In some aspects, a STAR as previously described, wherein

[0078] i) the IL-15 amino acid sequence is set forth in SEQ ID NO: 27;

[0079] ii) the IL-15Ra extracellular domain amino acid sequence is set forth in SEQ ID NO: 28;

[0080] iii) the linker amino acid sequence linking the IL-15Ra extracellular domain to IL-15 is set forth in SEQ ID NOs: 24-25, 29; and / or

[0081] iv) the mbIL-15 amino acid sequence is set forth in SEQ ID NO: 30.

[0082] In a third aspect, the present application provides a chimeric antigen receptor (CAR) targeting CCR8, comprising an extracellular antigen binding region, wherein the extracellular antigen binding region comprises the CCR8 single domain antibody of any one of the previous aspects, and the CAR comprises, from N-terminus to C-terminus, the extracellular antigen binding region, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0083] In some aspects, the CAR as described, wherein the extracellular antigen binding region further comprises an antigen binding region that specifically binds to another antigen, preferably the antigen binding region that specifically binds to another antigen comprises a single chain antibody (scFv) or a single domain antibody that specifically binds to the another antigen.

[0084] In some aspects, the CAR as previously described, wherein the another antigen is Claudin 18.2, preferably the antigen binding region that specifically binds to Claudin 18.2 comprises a CDR1 as set forth in SEQ ID NO: 41, a CDR2 as set forth in SEQ ID NO: 42, and a CDR3 as set forth in SEQ ID NO: 43, preferably the antigen binding region that specifically binds to Claudin 18.2 comprises an amino acid sequence as set forth in SEQ ID NO: 44.

[0085] Alternatively, the another antigen is MSLN, preferably the antigen binding region that specifically binds to MSLN comprises a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33, preferably the antigen binding region that specifically binds to MSLN comprises an amino acid sequence as set forth in SEQ ID NO: 34.

[0086] In some aspects, the CAR as previously described, wherein the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 59 to 64.

[0087] In a fourth aspect, the present application provides an isolated nucleic acid molecule encoding the single domain antibody as described in any one of the preceding aspects, or the STAR as described in any one of the preceding aspects, or the CAR as described in any one of the preceding aspects.

[0088] In a fifth aspect, the present application provides an expression vector comprising the nucleic acid molecule as described in the preceding aspect, operably linked to an expression control element such as a promoter.

[0089] In some aspects, the expression vector as described, wherein comprises:

[0090] 1) a nucleotide sequence encoding a fusion polypeptide of the first peptide chain of the STAR and the second peptide chain of the STAR linked by a self-cleaving peptide; or

[0091] 2) a nucleotide sequence encoding a fusion polypeptide of the first peptide chain of the STAR, the second peptide chain of the STAR, and the mbIL-15 linked by a self-cleaving peptide.

[0092] In some aspects, the expression vector, wherein the self-cleaving peptide is a 2A polypeptide, preferably the self-cleaving peptide is a Furin-2A polypeptide, such as the Furin-P2A polypeptide of SEQ ID NO: 26.

[0093] In a sixth aspect, the present application provides a host cell obtained by transforming a cell with the nucleic acid molecule as described in the preceding or the expression vector of any one of the preceding.

[0094] In a seventh aspect, the present application provides an isolated therapeutic immune cell comprising the STAR of any one of the preceding or the CAR of any one of the preceding.

[0095] In some aspects, the therapeutic immune cell, wherein the immune cell is a T cell or a NK cell, preferably a T cell.

[0096] In an eighth aspect, the present application provides a method of preparing a therapeutic immune cell as described in the preceding, comprising

[0097] Step 1) providing a starting immune cell;

[0098] Step 2) introducing into the starting immune cell the expression vector of any one of the preceding; and

[0099] Step 3) harvesting the immune cell obtained in step 2).

[0100] In a ninth aspect, the present application provides a method of producing a single-domain antibody that specifically binds to CCR8, comprising:

[0101] (i) culturing the host cell as described in the preceding under conditions suitable for expression of the nucleic acid molecule or the expression vector, and

[0102] (ii) isolating and purifying the single-domain antibody that specifically binds to CCR8 expressed by the host cell.

[0103] In a tenth aspect, the present application provides a pharmaceutical composition comprising the single-domain antibody of the preceding, the STAR of the preceding, the CAR of the preceding, the therapeutic immune cell of the preceding, and / or the expression vector of the preceding, and a pharmaceutically acceptable carrier, preferably for use in treating a disease in a subject.

[0104] In an eleventh aspect, the present application provides the use of the single-domain antibody of the preceding, the STAR of the preceding, the CAR of the preceding, the therapeutic immune cell of the preceding, and / or the expression vector of the preceding and / or the pharmaceutical composition of the preceding for the manufacture of a medicament for treating a disease in a subject.

[0105] In some aspects, the use, wherein the disease is a CCR8-related disease, such as a CCR8- related autoimmune disease, a hematological tumor or a solid tumor;

[0106] Preferably the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), polymyositis and dermatomyositis, systemic scleroderma, Sjogren’s syndrome, autoimmune hemolytic anemia, rheumatoid arthritis;

[0107] Preferably the hematological tumor or solid tumor is selected from the group consisting of epithelial cell carcinoma, glioblastoma, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, cancer of the digestive tract, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma;

[0108] More preferably the hematological tumor or solid tumor is selected from the group consisting of epithelial cell carcinoma, glioblastoma, rectal cancer, breast cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, cancer of the digestive tract, mesothelioma, pancreatic cancer, ovarian cancer and B-cell malignancies.

[0109] In a twelfth aspect of the present application, a method of treating a disease in a subject is provided, comprising administering to said subject a therapeutically effective amount of a single-domain antibody according to any one of the preceding aspects, a STAR according to any one of the preceding aspects, a CAR according to the preceding aspect, a therapeutic immune cell according to the preceding aspect, and / or an expression vector according to any one of the preceding aspects and / or a pharmaceutical composition according to the preceding aspect, preferably the disease is a CCR8-related disease, such as a CCR8-related autoimmune disease, a hematological tumor or a solid tumor;

[0110] Preferably the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), polymyositis and dermatomyositis, systemic scleroderma, Sjogren’s syndrome, autoimmune hemolytic anemia, rheumatoid arthritis;

[0111] Preferably the hematological tumor or solid tumor is selected from the group consisting of epithelial cell carcinoma, glioblastoma, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, cancer of the digestive tract, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma;

[0112] More preferably, the blood tumor or solid tumor is selected from the group consisting of epithelial cell cancer, glioblastoma, colorectal cancer, breast cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, digestive tract cancer, mesothelioma, pancreatic cancer, ovarian cancer and B-cell malignancies. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 CCR8 nanobody EC50 affinity test results

[0114] Figure 2 CCR8 nanobody MPA specific detection diagram

[0115] Figure 3 Structural diagrams of various STARs targeting CCR8

[0116] Figure 4 Comparison of CCR8 STAR killing efficiency in vitro

[0117] Figure 5 Fluorescence images of mouse tumors for in vivo functional verification of CCR8 STAR

[0118] Figure 6 Fluorescence counting diagram of CCR8 STAR in vivo functional verification

[0119] Figure 7 CCR8 STAR in vivo functional verification of tumor volume changes

[0120] Figure 8. In vitro killing effect of dual-target CCR8-MSLN STAR-T on different cells

[0121] Figure 9 Fluorescence images of mouse tumors for in vivo functional verification of CCR8-MSLN STAR

[0122] Figure 10 Fluorescence counting diagram of CCR8-MSLN STAR in vivo functional verification

[0123] Figure 11 Figure 3. Body weight changes of mice verified by CCR8-MSLN STAR in vivo.

[0124] Figure 12 In vivo functional verification of CCR8-MSLN STAR and tumor volume changes

[0125] Figure 13 The results show that the infection efficiency of STAR viruses with different structures is similar.

[0126] Figure 14Results of CCR8-Claudin18.2 STAR co-expressing mbIL-15 killing Claudin18.2 positive cells in vitro

[0127] Results of CCR8-Claudin18.2 STAR co-expressing mbIL-15 killing CCR8 positive cells in vitro

[0128] Results of CCR8-Claudin18.2 STAR co-expressing mbIL-15 secreting cytokines

[0129] Figure 17 In vivo functional verification of CCR8-Claudin18.2 STAR co-expressing mbIL-15 mouse tumor fluorescence photos

[0130] Figure 18 In vivo functional verification of CCR8-Claudin18.2 STAR co-expressing mbIL-15 mouse weight change results

[0131] Figure 19 In vivo functional verification of CCR8-Claudin18.2 STAR co-expressing mbIL-15 tumor fluorescence value change curve

[0132] Figure 20 In vivo functional verification of CCR8-Claudin18.2 STAR co-expressing mbIL-15 tumor volume change curve

[0133] Figure 21 In vivo functional verification of CCR8-Claudin18.2 STAR co-expressing mbIL-15 tumor volume inhibition rate change curve

[0134] Figure 22 Structure diagram of CCR8 and double-target CCR8-MSLN CAR-T

[0135] Figure 23 Infection efficiency detection diagram of different target CARs

[0136] Figure 24C Results of CR8-MSLN CAR-T killing target cells in vitro

[0137] Figure 25 Structure diagram of CCR8 and double-target CCR8-Claudin18.2 CAR-T

[0138] Figure 26 Infection efficiency detection diagram of different target CARs

[0139] Figure 27CCR8-Claudin18.2 CAR-T in vitro killing target cell test results DETAILED DESCRIPTION

[0141] 1. Definitions

[0142] Unless otherwise indicated or defined, all terms used have the ordinary meaning that would be understood by one of skill in the art, which would be in line with the general art as referenced, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nded.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nded.), Gower Medical Publishing, London, New York (1989), and the general art referenced herein; and further, all methods, steps, techniques, and procedures that are not specifically detailed are to be carried out in and with methods that are per se known to those skilled in the art, which would be in line with the general art, unless otherwise indicated. Reference is also made, for example, to standard manuals, the general art described above, and other references cited therein.

[0143] As used herein, the term "and / or" encompasses all combinations of the items linked by the term "and / or". For example, "A and / or B" covers "A", "B", and "A and B". For example, "A, B, and / or C" covers "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0144] The word "comprise" when used in this document to describe a protein or nucleic acid sequence, the protein or nucleic acid can be composed of the sequence, or can have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present application. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide will be retained in some practical cases (for example when expressed in a particular expression system), but does not materially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of the present application, although it can not contain the methionine encoded by the start codon at the N-terminus, at this time the sequence containing the methionine is also covered, and accordingly, the encoding nucleotide sequence can also contain the start codon; vice versa.

[0145] The term "isolated" with respect to a polypeptide or nucleic acid molecule is considered to be "isolated" when it has been separated from at least one other component (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component) with which it is ordinarily associated in the source or medium from which it is obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0146] As used herein, Synthetic T-Cell Receptor and Antibody Receptor (STAR) refers to a modified TCR in which the TCR variable region sequence is replaced with an antibody variable region sequence or other receptor sequence, and the constant region sequence can also be modified.

[0147] As used herein, "antigen binding region" (e.g., in a STAR) means that it, by itself or in combination with another antigen binding region, can specifically bind to a target antigen. The antigen binding region can be derived from an antibody that specifically binds to a target antigen, including any commercially available antibody. The antigen binding region can also be derived from a receptor that binds to a particular target protein.

[0148] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or produced in whole or in part synthetically (e.g., recombinantly), including any fragment thereof that retains the ability of a full-length immunoglobulin to bind with specificity to an immunoglobulin antigen. Thus, antibodies include any protein having a binding domain homologous or substantially homologous to an immunoglobulin antigen binding region (antibody binding site). Antibodies include antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, camelid antibodies, single domain antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen binding fragments thereof of any of the above. Antibodies described herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass (e.g., IgG2a and IgG2b) of either.

[0149] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain that comprises an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region, VL (also denoted V L ) and VH (or also denoted V L ), respectively. The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region comprises CDRs and framework regions (FRs), the CDRs being the part of the antigen binding site.

[0150] As used herein, "hypervariable region," "HV," "complementarity determining region," and "CDR," and "antibody CDR," are used interchangeably to refer to one of a number of portions within each variable region that together form the antigen binding site of an antibody. Each variable region comprises 3 CDRs, designated CDR1, CDR2, and CDR3. For example, for a conventional 4-chain antibody, the light chain variable region comprises 3 CDRs, designated VL CDR1, VL CDR2, and VL CDR3 (or LCDR1, LCDR2, and LCDR3); the heavy chain variable region domain comprises 3 CDRs, designated VH CDR1, VH CDR2, and VH CDR3 (or HCDR1, HCDR2, and HCDR3). For a camelid antibody or single domain antibody, since it has only one variable region, it contains only 3 CDRs, designated CDR1, CDR2, and CDR3.

[0151] In the context of the present application, the terms "single domain antibody", "nanobody", "heavy chain single domain antibody", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0152] A "single domain antibody" is the variable domain of an antigen binding immunoglobulin termed "heavy chain antibody" (i.e. "antibody devoid of light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH" is used to distinguish said variable region of a heavy chain antibody from the variable region of a heavy chain present in a conventional 4-chain antibody (which is referred to herein as "VH") as well as from the variable region of a light chain present in a conventional 4-chain antibody (which is referred to herein as "VL"). A VHH specifically binds an epitope without the need for other antigen binding regions (as opposed to VH or VL in a conventional 4-chain antibody, in which case the epitope is recognized by VL together with VH). A VHH is a small, stable and highly efficient antigen recognition unit formed by a single domain.

[0153] e.g. Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999) Figure 2As shown in the numbering scheme for amino acid residues applied to VHH domains of Camelidae, numbering can be according to the general numbering scheme for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). However, alternative methods of numbering amino acid residues of VH domains are known in the art and can similarly be applied to VHH domains. For example, Chothia CDRs refer to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and Chothia structural loops, and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on an analysis of available complex crystal structures. The CDRs of the single domain antibodies of the application are determined according to the Kabat database.

[0154] A VHH domain derived from Camelidae can be "humanized" (also referred to herein as "sequence optimized", except that "sequence optimized" can also encompass other modifications to the sequence by providing one or more mutations that improve the properties of the VHH, such as removal of potential post-translational modification sites, in addition to humanization) by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues that occur at the corresponding position in a human canonical 4-chain antibody VH domain. A humanized VHH domain can contain one or more fully human framework region sequences. Humanization can be accomplished using methods of protein surface amino acid resurfacing and / or humanization by CDR grafting to a universal framework.

[0155] In general, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (e.g., an antibody of the application) can bind. Specificity can be determined based on the affinity and / or avidity of the antigen-binding protein. Affinity, as represented by the dissociation equilibrium constant (KD) of an antigen for an antigen-binding protein, is a measure of the strength of binding between an epitope and an antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be determined in known ways, depending on the particular antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an antibody) and a cognate antigen. Affinity is related to both the affinity between the antigen and the antigen-binding site on its antigen-binding protein, and the number of relevant binding sites present on the antigen-binding protein.

[0156] As used herein, "amino acid numbering with reference to SEQ ID NO:x" (where SEQ ID NO:x is a particular sequence set forth herein) means that the position number of the particular amino acid described is the position number of the corresponding amino acid on SEQ ID NO:x. Correspondence of amino acids in different sequences can be determined according to methods of sequence alignment known in the art. For example, amino acid correspondence can be determined by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ), in which two sequences can be aligned using the Needleman-Wunsch algorithm, using default parameters. For example, an alanine at position 46 from the N-terminus of a polypeptide that aligns with the amino acid at position 48 of SEQ ID NO:x in a sequence alignment, that amino acid in the polypeptide can also be described herein as "an alanine at position 48 of the polypeptide, the amino acid position being with reference to SEQ ID NO:x".

[0157] The proteins / polypeptides referred to in the present application can comprise a signal peptide (or leader sequence) at the N-terminus. Those skilled in the art will appreciate that a signal peptide sequence can direct a protein / polypeptide to a particular location in a cell, e.g., the cell membrane, which can itself be cleaved off and not included in the final product. Exemplary signal peptides include, but are not limited to, an IgE signal peptide, a GM-CSF signal peptide, a bovine prolactin pre-signal peptide, and the like. These signal peptide sequences are known in the art or can be readily identified by those skilled in the art according to the knowledge in the art.

[0158] An "expression vector" of the application can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or can be a translatable RNA (such as an mRNA). In some preferred embodiments, the expression vector is a viral vector, e.g., a lentiviral vector.

[0159] As used herein, the term "operably linked" refers to the connection of an expression control element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) so that the transcription of the nucleotide sequence is controlled and regulated by the transcription control element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art. "Regulatory sequence" and "regulatory element" are used interchangeably and refer to a nucleotide sequence that is located upstream (5' non-coding sequence), in the middle, or downstream (3' non-coding sequence) of a coding sequence and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. An expression control element refers to a nucleotide sequence that is capable of controlling the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, enhancers, and polyadenylation recognition sequences. Suitable promoters include, but are not limited to, the PGK promoter, the hEF1a promoter (as shown in SEQ ID NO: 36), and the MND promoter (as shown in SEQ ID NO: 35).

[0160] As used herein, "subject" refers to an organism that has or is susceptible to a disease (e.g., cancer) that can be treated by the antibodies, cells, methods, or pharmaceutical compositions of the invention. Non-limiting examples include humans, cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals. In a preferred embodiment, the subject is a human.

[0161] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0162] As used herein, "therapeutically effective amount" or "therapeutically effective dose" or "effective amount" refers to the amount of a substance, compound, material, or cells that, when administered to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is the amount necessary to prevent, cure, ameliorate, retard or partially retard symptoms of a disease or disorder. For example, an "effective amount" of the cells or pharmaceutical compositions of the application preferably results in a decrease in severity of the symptoms of the disease, an increase in frequency and duration of symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of a tumor, an "effective amount" of the antibodies, cells, expression vectors or pharmaceutical compositions of the application preferably inhibits tumor cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received the treatment. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in humans. Alternatively, the ability to inhibit tumor cell growth can also be evaluated by examining the ability to inhibit tumor cell growth, which can be determined in vitro by assays known to one of skill in the art.

[0163] Single-domain antibodies that specifically bind CCR8

[0164] In one aspect, the application provides a single-domain antibody that specifically binds CCR8, comprising CDR1, CDR2, and CDR3 selected from the sequences of any one of SEQ ID NOs: 4 and 8. The CDRs can be Kabat CDRs, AbM CDRs, Chothia CDRs, or Contact CDRs. In some embodiments, the CDRs are Kabat CDRs.

[0165] In some embodiments, the single-domain antibody that specifically binds CCR8 can further comprise one or more additional tag sequences for ease of purification and / or labeling. For example, the additional tag can be a His tag (such as a 6xHis tag) or an Fc tag, which facilitates isolation and purification of the polypeptide, or which facilitates prolongation of its half-life in vivo. One of skill in the art will appreciate that these additional tags do not substantially affect the binding ability of the antibody.

[0166] The single-domain antibody that specifically binds CCR8 of the application binds to the K D value can be less than about 1 x 10 -7 M, preferably less than about 1 x 10 -8 M, more preferably less than about 1 x 10 -9 M, more preferably less than about 1 x 10 -10 M.

[0167] Expression vectors and methods for making single-domain antibodies

[0168] In another aspect, the present application provides an isolated nucleic acid molecule encoding a single-domain antibody of the present application that specifically binds to CCR8. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized for the host cell used for expression. In some embodiments, the nucleic acid molecule of the present application is operably linked to an expression control element such as a promoter.

[0169] The present application also provides an expression vector for expressing a single-domain antibody of the present application comprising the nucleic acid molecule encoding a single-domain antibody of the present application that specifically binds to CCR8.

[0170] The present application also provides a host cell for producing a single-domain antibody of the present application, which is transformed by the nucleic acid molecule or the expression vector of the present application described above. As used herein, a “host cell” is a cell used to accept, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a nucleic acid or a vector. When a host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. A host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0171] In another aspect, the present application provides a method of producing a single-domain antibody of the present application that specifically binds to CCR8, comprising:

[0172] (i) culturing a host cell of the present application in a condition suitable for expression of the nucleic acid molecule or the expression vector, and

[0173] (ii) isolating and purifying a single-domain antibody of the present application that specifically binds to CCR8 expressed by the host cell.

[0174] Methods and reagents for the recombinant production of polypeptides, such as expression vectors specifically adapted, methods of transformation or transfection, selection markers, methods of inducing protein expression, culture conditions, etc. are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods of manufacturing a single-domain antibody of the present application that specifically binds to CCR8 are well known to those skilled in the art.

[0175] However, a single-domain antibody of the present application that specifically binds to CCR8 can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0176] STARs targeting CCR8

[0177] In some aspects, the present application provides a synthetic T cell receptor antigen receptor (STAR) targeting CCR8, comprising an antigen binding region that specifically binds to CCR8.

[0178] In some embodiments, the synthetic T cell receptor antigen receptor (STAR) targeting CCR8 comprises a first peptide chain comprising a first constant region and a second peptide chain comprising a second constant region, and wherein the first peptide chain and / or the second peptide chain further comprises an antigen binding region that specifically binds to CCR8, the first or second constant region is selected from a TCR a chain constant region or a TCR β chain constant region, respectively.

[0179] As used herein, “exogenous” means a protein or nucleic acid sequence from a foreign species, or if from the same species, a protein or nucleic acid sequence that has been significantly altered in composition and / or position from its natural form by deliberate, artificial intervention.

[0180] As used herein, an “exogenous intracellular functional domain” can be an intracellular domain of a costimulatory molecule such as the intracellular domain of CD40, OX40, ICOS, CD28, 4-1BB, CD27, CD137; can also be an intracellular domain of a co-inhibitory molecule, for example, the intracellular domain of TIM3, PD1, CTLA4, LAG3; can also be an intracellular domain of a cytokine receptor such as an interleukin receptor (e.g., IL-2 beta receptor, IL-7 alpha receptor, or IL-21 receptor), an interferon receptor, a tumor necrosis factor superfamily receptor, a colony stimulating factor receptor, a chemokine receptor, a growth factor receptor, or other membrane protein; or a domain of an intracellular protein such as NIK.

[0181] In some preferred embodiments, the exogenous intracellular functional domain is an intracellular domain of a costimulatory molecule, preferably the intracellular domain of OX40. In some embodiments, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO: 23.

[0182] Target binding region, refers to the region in an antigen receptor (e.g., STAR or CAR) that is used to specifically bind to the target antigen. The target binding region can comprise a single or multiple antigen binding regions that bind to a single protein region or epitope (antigenic determinant) of an antigen through antigen-antibody interactions, or receptor-ligand interactions, in some embodiments, the antigen binding region is an antigen binding fragment of an antibody, such as a single domain antibody, scFv, or nanobody, etc.

[0183] Chimeric antigen receptors (CARs) targeting CCR8

[0184] In some aspects, the present application provides a chimeric antigen receptor (CAR) targeting CCR8, comprising an extracellular antigen binding region (antigen binding domain), wherein the extracellular antigen binding domain comprises a CCR8 single domain antibody, the CAR comprising, in order from N-terminus to C-terminus, an extracellular antigen binding region, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0185] Hinge region, a region in a chimeric antigen receptor (CAR) connecting the extracellular antigen binding region and the transmembrane domain. The hinge region is usually composed of a flexible peptide chain, which provides spatial freedom and enhances the efficiency of antigen binding.

[0186] Transmembrane domain, a region in a chimeric antigen receptor (CAR) used to anchor the CAR in the cell membrane. The transmembrane domain is usually composed of hydrophobic amino acids, which can stably embed in the cell membrane.

[0187] Intracellular signaling domain, a region in a chimeric antigen receptor (CAR) used to transmit activation signals, usually containing an immunoreceptor tyrosine-based activation motif (ITAM) or other signaling motifs. These domains can activate the immune response of T cells.

[0188] In some embodiments, the CAR further comprises a transmembrane domain, for example a CD8a transmembrane domain or a CD28 transmembrane domain, preferably a CD8a transmembrane domain.

[0189] In some embodiments, the CAR further comprises a hinge region between the extracellular antigen binding region and the transmembrane domain, for example, the hinge region is a CD8a hinge region.

[0190] In some embodiments, the CAR further comprises a signal transduction domain, for example a signal transduction domain useful for T cell activation, for example a signal transduction domain selected from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3zeta, CD5, CD22, CD79a, CD79b, and CD66d. In some preferred embodiments, the CAR comprises a CD3zeta signal transduction domain.

[0191] In some embodiments, the CAR further comprises one or more costimulatory domains, for example a costimulatory domain selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, and 4-1BBL.

[0192] In some embodiments, the CAR comprises, in the N-terminal to C-terminal direction, the extracellular antigen binding region, the hinge region, the transmembrane domain, the costimulatory domain, and the signal transduction domain. In some embodiments, the hinge region is a CD8a hinge region, the transmembrane domain is a CD8a transmembrane domain, the signal transduction domain is a CD3 zeta signal transduction domain, and the costimulatory domain is a 4-1BB costimulatory domain.

[0193] In some embodiments, the extracellular antigen binding region further comprises an antigen binding region that specifically binds to another antigen. Thereby, the CAR can also target the other antigen. Preferably, the antigen binding region that specifically binds to another antigen comprises a single chain antibody (scFv) or a single domain antibody that specifically binds to the other antigen.

[0194] In some embodiments, the other antigen is MSLN, and the antigen binding region that specifically binds to MSLN comprises a CDR1 as set forth in SEQ ID NO: 31, a CDR2 as set forth in SEQ ID NO: 32, and a CDR3 as set forth in SEQ ID NO: 33. In some embodiments, the other antigen is MSLN, and the antigen binding region that specifically binds to MSLN comprises an amino acid sequence as set forth in SEQ ID NO: 34 (single domain antibody).

[0195] In some embodiments, the other antigen is Claudin 18.2, and the antigen binding region that specifically binds to Claudin 18.2 comprises a CDR1 as set forth in SEQ ID NO: 41, a CDR2 as set forth in SEQ ID NO: 42, and a CDR3 as set forth in SEQ ID NO: 43. In some embodiments, the other antigen is Claudin 18.2, and the antigen binding region that specifically binds to Claudin 18.2 comprises an amino acid sequence as set forth in SEQ ID NO: 44 (single domain antibody).

[0196] In some specific embodiments, the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 59 to 64.

[0197] Therapeutic immune cells

[0198] In another aspect, the present application provides an isolated therapeutic immune cell comprising a STAR of the present application.

[0199] In some embodiments, the immune cell is a T cell. In other embodiments, the immune cell is an NK cell.

[0200] In some embodiments, the therapeutic immune cells co-express a STAR of the application and a membrane-bound IL-15 protein (mbIL-15).

[0201] mbIL-15 refers to a fusion protein of IL-15 linked (e.g., via a linker) to an IL-15Ra extracellular domain. An exemplary amino acid sequence of IL-15 is set forth in SEQ ID NO: 27, but also encompasses amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity to SEQ ID NO: 27. An exemplary amino acid sequence of IL-15Ra extracellular domain is set forth in SEQ ID NO: 28, but also encompasses amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity to SEQ ID NO: 28. An exemplary amino acid sequence of a linker linking IL-15Ra extracellular domain to IL-15 is set forth in SEQ ID NO: 29. An exemplary amino acid sequence of mbIL-15 is set forth in SEQ ID NO: 30, but also encompasses amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% sequence identity to SEQ ID NO: 30.

[0202] The immune cells, such as T cells, of the application can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors, by various non-limiting methods. In some embodiments, the cells can be derived from a healthy donor or from a patient diagnosed with cancer. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. For example, immune cells, such as T cells, can be obtained by isolating peripheral blood mononuclear cells (PBMCs) and then activating and expanding with specific antibodies.

[0203] In some embodiments, the immune cells, such as T cells, of the application are isolated (ex vivo) immune cells, such as cells.

[0204] In some embodiments of each aspect of the application, the immune cells, such as T cells, are derived from autologous cells of a subject. As used herein, “autologous” refers to cells, cell lines, or cell populations used for therapy of a subject being derived from that subject. In some embodiments, the immune cells, such as T cells, are derived from allogeneic cells, for example, from a donor that is compatible with the human leukocyte antigens (HLAs) of the subject. Cells from a donor can be transformed into non-alloreactive cells using standard protocols and replicated as needed, thereby generating cells that can be administered to one or more patients.

[0205] In some embodiments, the therapeutic immune cells such as T cells are therapeutic immune cells such as T cells obtainable or obtained by the expression vector of the present application or the method of the present application as described below.

[0206] Expression vectors and methods for making therapeutic immune cells

[0207] In one aspect, the present application provides an expression vector comprising a coding sequence of the STAR of the present application.

[0208] The coding sequence in the expression vector of the present application can be operably linked to a regulatory element such as a promoter for expression in a cell.

[0209] In some embodiments, the expression vector further comprises a coding sequence of the membrane-bound IL-15 protein (mbIL-15) described in the present application.

[0210] The coding sequence in the expression vector of the present application can be operably linked to a regulatory element such as a promoter for expression in a cell.

[0211] In some embodiments, the mbIL-15 can be driven for expression by a separate promoter.

[0212] In some embodiments, the expression vector comprises

[0213] a) a coding nucleotide sequence of a fusion polypeptide comprising the first peptide chain of the STAR of the present application, the second peptide chain of the STAR of the present application linked by a self-cleaving peptide;

[0214] b) a coding nucleotide sequence of a fusion polypeptide comprising the first peptide chain of the STAR of the present application, the second peptide chain of the STAR of the present application and the mbIL-15 described in the present application linked by a self-cleaving peptide; or

[0215] c) a coding nucleotide sequence of a fusion polypeptide comprising the mbIL-15 described in the present application linked by a self-cleaving peptide.

[0216] In some embodiments, the expression vector comprises a coding nucleotide sequence of a fusion polypeptide comprising the first peptide chain of the STAR of the present application, the second peptide chain of the STAR of the present application linked by a self-cleaving peptide;

[0217] As used herein, "self-cleaving peptide" means a peptide that can achieve self-cleavage within a cell. For example, the self-cleaving peptide can comprise a protease recognition site, so as to be recognized and specifically cleaved by a protease within the cell. Alternatively, the self-cleaving peptide can be a 2A polypeptide. 2A polypeptides are a class of short peptides from viruses, whose self-cleavage occurs during translation. When two different proteins of interest are expressed in the same reading frame with a 2A polypeptide, the two proteins of interest are generated in almost 1 : 1 ratio. Commonly used 2A polypeptides can be P2A from porcine techovirus-1, T2A from Thosea asigna virus, E2A from equine rhinitis A virus, and F2A from foot-and-mouth disease virus. Among them, P2A has the highest cleavage efficiency and is therefore preferred. A variety of functional variants of these 2A polypeptides are also known in the art, which can also be used in the present application. 2A polypeptides can also be combined with Furin recognition sequences to remove additional introduced amino acid sequences.

[0218] In some embodiments, the self-cleaving peptide is a 2A polypeptide, e.g., a P2A polypeptide. In some embodiments, the self-cleaving peptide is a Furin-2A polypeptide, e.g., a Furin-P2A polypeptide as set forth in SEQ ID NO: 26.

[0219] In some embodiments, the different parts of the fusion polypeptide can be arranged in different ways, as long as they are separated by a self-cleaving peptide. For example, in some embodiments, the fusion polypeptide can comprise, from N- to C-terminal direction, the second peptide chain (the peptide chain comprising the constant region of the TCR β chain), a self-cleaving peptide such as Furin-P2A, the first peptide chain (the peptide chain comprising the constant region of the TCR α chain). When there are multiple self-cleaving peptides, the self-cleaving peptides can be the same or different.

[0220] In another aspect, the present application provides a method for preparing a therapeutic immune cell, comprising

[0221] Step 1) providing a starting immune cell;

[0222] Step 2) introducing into the starting immune cell an expression vector of the present application; and

[0223] Step 3) harvesting the immune cell obtained in step 2).

[0224] In some embodiments, the starting immune cell is a T cell. In other embodiments, the starting immune cell is an NK cell.

[0225] The starting immune cells, such as T cells, of the present application can be obtained from a number of non-limiting sources by various non-limiting methods, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the cells can be derived from a healthy donor or from a patient diagnosed with cancer. In some embodiments, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. For example, the starting immune cells, such as T cells, can be obtained by isolating peripheral blood mononuclear cells (PBMCs) and then activating, expanding with specific antibodies.

[0226] In some embodiments, the starting immune cells, such as T cells, of the present application are isolated (ex vivo) immune cells, such as T cells. As such, the therapeutic immune cells, such as T cells, obtained by the present application are isolated (ex vivo) therapeutic immune cells, such as T cells.

[0227] In some embodiments, the methods of the present application are in vitro methods.

[0228] In some embodiments of each aspect of the present application, the starting immune cells, such as T cells, are derived from autologous cells of a subject. As used herein, "autologous" refers to cells, cell lines, or cell populations used for therapy of a subject being derived from that subject. In some embodiments, the starting immune cells, such as T cells, are derived from allogeneic cells, for example from a donor that is human leukocyte antigen (HLA) compatible with the subject. Cells from a donor can be converted to non-alloreactive cells using standard protocols and replicated as needed to generate cells that can be administered to one or more patients.

[0229] Introduction of the expression vector into the immune cells, such as T cells, can be performed by methods known in the art, including but not limited to microinjection, electroporation, virus-mediated transfection, liposome-mediated transfection, and the like.

[0230] In some embodiments, the method further comprises between steps 2) and 3) a step x) expanding the immune cells, such as T cells, obtained in step 2). The immune cells, such as T cells, can be expanded using methods known in the art.

[0231] In some embodiments, the method further comprises a step y) screening the immune cells, such as T cells, expressing the STAR. In some embodiments, step y) can be performed after step 2). In some embodiments, step y) can be performed after step 2) and before step x). In some embodiments, step y) can be performed after step x). In some embodiments, the screening is performed by flow cytometry.

[0232] In another aspect, the present application provides a therapeutic immune cell, such as a T cell, obtainable or obtained by the expression vector of the present application or the method of the present application.

[0233] Pharmaceutical compositions and uses

[0234] In another aspect, the present application provides a pharmaceutical composition comprising the single domain antibody of the present application, the therapeutic immune cell of the present application and / or the expression vector of the present application, and a pharmaceutically acceptable carrier.

[0235] In another aspect, the present application provides the use of the single domain antibody of the present application, the therapeutic immune cell of the present application, the expression vector of the present application and / or the pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease in a subject.

[0236] In another aspect, the present application provides a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the single domain antibody of the present application, the therapeutic immune cell of the present application, the expression vector of the present application and / or the pharmaceutical composition of the present application.

[0237] In actual practice, the dosage level of the antibody, cell or expression vector of the pharmaceutical composition of the present application may be varied to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. Selection of the dose level will depend on a variety of factors including the activity of the particular composition of the present application employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0238] Administration of the antibody, expression vector, therapeutic immune cell or pharmaceutical composition or medicament according to the present application can be carried out in any convenient way, including by injection, infusion, implantation or transplantation. Administration of the antibody, expression vector, therapeutic immune cell or pharmaceutical composition described herein can be by intravenous, intralymphatic, intradermal, intratumoral, intramedullary, intramuscular or intraperitoneal administration. In one embodiment, the antibody, expression vector, therapeutic immune cell or pharmaceutical composition of the present application is preferably administered by intravenous injection.

[0239] In embodiments of each aspect of the present application, the disease is a CCR8- associated disease, e.g. a CCR8 expression abnormality-associated disease, e.g. is a CCR8- associated autoimmune disease, a hematological tumor or a solid tumor;

[0240] Preferably the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), myositis, scleroderma, Sjogren's syndrome, autoimmune hemolytic anemia, rheumatoid arthritis;

[0241] Preferably the tumor is selected from the group consisting of lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, gastrointestinal cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma;

[0242] More preferably the tumor is selected from the group consisting of rectal cancer, breast cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, gastrointestinal cancer, mesothelioma, pancreatic cancer, ovarian cancer and B-cell malignancies

[0243] The technical solution of the present application has the following benefits or technical advantages:

[0244] The present application is a cell therapy product that simultaneously targets tumor antigens and receptors in immunosuppressive active cells, which has the advantage of killing tumor cells while specifically targeting Treg cells infiltrated in tumor tissues, thus relieving the inhibition of Tregs, improving the tumor immunosuppressive microenvironment and the body's immunity, and enhancing the in vivo persistence and function of immune cell products, thereby improving the clinical efficacy of existing cell immunotherapy.

[0245] (1) The bifunctional STAR-T cell of the present application has the advantages of both CAR-T and TCR-T, with high affinity comparable to CAR-T cells, high signal transduction capacity and high sensitivity to antigens similar to TCR-T; at the same time, it avoids the defects of both CAR-T and TCR-T, avoiding the dependence of TCR-T on HLA and the severe self-activation of CAR-T, thus avoiding exhaustion;

[0246] (2) The bifunctional STAR-T cell of the present application fully utilizes its natural advantage of multiple chains, by connecting an antibody part against receptors in immunosuppressive active cells (such as CCR8) on one chain and an antibody part targeting tumors on the other chain, thus realizing the bifunctionality. Due to the structural advantage, the two antibodies do not interfere with each other and can function independently. Moreover, since the antibody part against receptors in immunosuppressive active cells can eliminate the immunosuppressive effect of Tregs and improve the tumor immunosuppressive microenvironment, it can improve the ability of the antibody targeting tumors to kill tumors;

[0247] (3) In animal models of epithelial cell carcinoma, brain glioblastoma and liver cancer, the bifunctional STAR-T cells of the present application exhibit superior anti-tumor effect to conventional CAR-T cells, and no obvious toxic side effects;

[0248] (4) The present application further optimizes the STAR structure by further coupling the costimulatory molecule OX40. Cell level results show that after coupling OX40 in the intracellular domain of the bifunctional STAR of the present application, the STAR-T cells can significantly increase IL-2 secretion and proliferation level under the stimulation of target cells; and

[0249] (5) In the prior art, in order to kill tumors while improving the tumor immunosuppressive microenvironment, the patient needs to be treated or medicated separately, which affects the tumor treatment effect, increases the burden of the patient, and also increases the risk of adverse reactions of the drug. The bifunctional STAR-T cells of the present application can achieve the effect of killing tumors while improving the tumor immunosuppressive microenvironment in the form of a single drug. DETAILED DESCRIPTION

[0250] The present application will be further described below in conjunction with specific examples, but these specific examples cannot be understood as limiting the protection scope of the present application. Those skilled in the art can make various changes or modifications to these specific examples without departing from the scope of the technical solutions of the present application, and the changed and modified embodiments still fall within the protection scope of the present application.

[0251] The lentiviral vectors and lentiviral packaging plasmids used in the embodiments of the present application are purchased from commercial companies or synthesized by commercial companies. The gene fragments used in the embodiments of the present application, including signal peptides, antibody binding regions, hinge regions, TCR constant regions, tag proteins, etc., are all synthesized by commercial companies. The embodiments of the present application are only for further description of the present application, and are not intended to limit the scope of the present application.

[0252] Optimization of STAR

[0253] The secreted antibody (Antibody, Ab) produced by B cells or B cell receptor (BCR) has great similarity with T cell receptor (TCR) in gene structure, protein structure and spatial conformation. Both antibody and TCR are composed of variable region and constant region, wherein the variable region plays a role in antigen recognition and binding, and the constant region domain plays a role in structural interaction and signal transduction. By replacing the variable regions of TCR alpha and beta chains (or TCR gamma and delta chains) with the heavy chain variable region (VH) and light chain variable region (VL) of antibody, VHH or scFv, an artificial chimeric molecule called synthetic T cell receptor and antibody receptor (Synthetic T-Cell Receptor and Antibody Receptor, STAR) can be constructed.

[0254] In some embodiments, the STAR molecule has two peptide chains, the first chain is a fusion of an antigen recognition sequence (such as the heavy chain variable region VH of an antibody) and the constant region of the T cell receptor alpha chain (Cα), and the second chain is a fusion of an antigen recognition sequence (such as the light chain variable region VL of an antibody) and the constant region of the T cell receptor beta chain (Cβ). The antigen recognition domain (such as VH, VL or scFv, etc.) and the constant region domain (the constant region of TCR alpha, beta, gamma and delta) in the construct can be arranged in combination to form various configurations of functionally similar constructs.

[0255] After the first and second chains of the STAR molecule are expressed in T cells, they will combine with the endogenous CD3εδ, CD3γε and CD3ζζ chains in the endoplasmic reticulum to form an 8-subunit complex, and are displayed on the cell membrane in the form of a complex. Immunoreceptor tyrosine-based activation motif (ITAM) is a motif in TCR molecules that plays a role in signal transduction, and its conserved sequence is YxxL / V. The intracellular region of CD3ε, δ, γ and ε chains contains one ITAM sequence, and the intracellular region of CD3ζ chain contains three ITAM sequences, so a complete STAR complex contains a total of 10 ITAM sequences. When the antigen recognition sequence of the STAR receptor binds to its specific antigen, the intracellular ITAM sequence is sequentially phosphorylated, thereby activating the downstream signaling pathway, activating transcription factors such as NF-κΒ, NFAT and AP-1, and triggering activated T cells to produce effector functions.

[0256] Because the constant region sequences of human, primate and mouse TCR alpha / beta chains (mouse TCR AC / mouse TCR BC) are highly functionally conserved and have the same key amino acid sequences, they can be replaced with each other. After interchanging, on the one hand, the efficiency of correct pairing of the STAR molecule is increased, and on the other hand, the possibility of mismatch to produce unknown specificity is reduced, and the safety is increased. The inventors have previously made cysteine substitutions, transmembrane domain hydrophobic amino acid modifications, etc. to the constant region of STAR to improve its performance.

[0257] 1) Modification of constant region derived from mouse

[0258] The constant region sequences of TCR alpha / beta chains derived from mice can be modified by humanization, cysteine substitution, and transmembrane domain hydrophobic amino acid substitution.

[0259] Introducing cysteine point mutations into disulfide bonds: The threonine T at position 48 in the constant region of the mouse TCRa chain was mutated to a cysteine C, and the serine S at position 56 in the constant region of the mouse TCRP chain was mutated to a cysteine C. These two newly added cysteines form disulfide bonds between the two chains of STAR, reducing the mispairing of the two chains of STAR with the endogenous TCR chains, and helping the STAR molecule to form a more stable complex. The obtained a chain constant region is named TRAC(Cys), and the obtained P chain constant region is named TRBC(Cys).

[0260] Design of STAR transmembrane region hydrophobic amino acid substitution: Three amino acid sites in the region from 111 to 119 amino acids in the transmembrane region of the TCRa chain constant region were mutated, with the serine S at position 112 changed to leucine L, the methionine M at position 114 changed to isoleucine I, and the glycine G at position 115 changed to valine V. The overall amino acid sequence of this region changed from LSVMGLRIL (SEQ ID NO: 65) to LLVIVLRIL (SEQ ID NO: 66). This design increases the hydrophobicity of the transmembrane region, offsetting the instability caused by the positive charge carried by the TCR transmembrane region, allowing the STAR molecule to exist more stably on the cell membrane, and thus obtaining better function. The a chain constant region obtained by combining the cysteine mutation and the hydrophobic region mutation is named TRAC(Cys-TM), and the corresponding P chain constant region is named TRBC(Cys-TM), wherein TRBC(Cys-TM) is the same as TRBC(Cys).

[0261] To further optimize the design of the STAR molecule, on the basis of the cysteine point mutation derived from the mouse constant region and the hydrophobic amino acid mutation of the a chain constant region, specific rearrangement was performed on the N terminus of the constant region of the STAR molecule to obtain a more optimal effect. Rearrangement means that part of the sequence is deleted, and part of the sequence is mutated to be humanized. The significance of humanization mutation is to minimize the non-human sequences in the STAR molecule as much as possible while ensuring the function of the STAR molecule, so as to maximize the possibility of avoiding rejection of STAR-T cells in clinical application. Therefore, the N terminus of the TCRa chain constant region was further modified, including substituting the amino acid such as E at position 6 with D, substituting K at position 13 with R, and deleting the amino acids at positions 15-18, and the obtained a chain constant region is named TRAC(Nrec-Cys-TM). The N terminus of the TCRP chain constant region was further modified, including substituting the amino acid such as R at position 3 with K, substituting the amino acid such as T at position 6 with F, substituting K at position 9 with E, substituting S at position 11 with A, substituting L at position 12 with V, and deleting the amino acids at positions 17, 21-25, and the obtained P chain constant region is named TRBC(Nrec-Cys-TM).

[0262] The modified TCR a chain constant region is derived from a rodent (preferably murine, more preferably mouse) TCR a chain constant region, which comprises substitution of the amino acid K at position 122 with R, relative to the wild-type rodent (preferably murine, more preferably mouse) TCR a chain constant region; and the modified TCR β chain constant region is derived from a rodent (preferably murine, more preferably mouse) TCR β chain constant region, which comprises substitution of the lysine at position 150, 168 or 170 with arginine.

[0263] In addition, a co-stimulatory molecule, such as OX40 cytoplasmic region, can be linked to the C-terminus of the a chain constant region and / or the β chain constant region to further enhance the function of STAR. The co-stimulatory molecule can be linked to the C-terminus of the a chain constant region and / or the β chain constant region via a linker, such as a (G4S)3 linker. The constant region linked to the co-stimulatory molecule can further lack the native intracellular region relative to the wild-type constant region, in addition to the above-mentioned modification, which further improves the function of STAR. For example, the a chain constant region can lack the amino acids at positions 136-137; and / or, the β chain constant region can lack the amino acids at positions 167-172.

[0264] 2) Modification of constant region derived from human

[0265] For the constant region sequence of TCR a / β chain derived from human, one or more than two of the following modifications can be made: minimal murinization, cysteine substitution, transmembrane domain hydrophobic amino acid substitution, or rearrangement, wherein:

[0266] STAR human constant region cysteine point mutation (hereinafter referred to as hcSTAR). The wild-type human TCR a chain constant region is mutated from threonine T at position 47 to cysteine C, and the wild-type human TCR β chain constant region is mutated from serine S at position 56 to cysteine C (this mutation is named Cys2). An additional disulfide bond is formed between the a chain constant region and the β chain constant region of the STAR molecule, which reduces the mispairing of the two chains of STAR with endogenous TCR chains, helps the STAR molecule to form a more stable complex, and thus obtains better function.

[0267] Minimal murinization of human constant region STAR (Minimal murinization, referred to as MM): the TCR a chain constant region is mutated from P at position 90 to S, from E at position 91 to D, from S at position 92 to V, and from S at position 93 to P; the TCR β chain constant region is mutated from E at position 17 to K, from serine S at position 21 to A, from F at position 132 to I, from E at position 135 to A, and from Q at position 138 to H.

[0268] STAR transmembrane hydrophobic mutation: mutations of two amino acid sites in the region from 110th to 118th amino acid in the transmembrane region of the constant region of TCR alpha chain, in which the 115th serine S is mutated to leucine L and the 118th glycine G is mutated to valine V. This design increases the hydrophobicity of the transmembrane region, offsets the instability caused by the positive charge carried by the TCR transmembrane region, so that the STAR molecule can more stably exist on the cell membrane, and thus obtain better function.

[0269] In addition, a costimulatory molecule such as OX40 cytoplasmic region can be connected to the C-terminus of the alpha chain constant region and / or the beta chain constant region to further enhance the function of STAR. The costimulatory molecule can be connected to the C-terminus of the alpha chain constant region and / or the beta chain constant region through a linker, such as a (G4S)3 linker. In addition to the above modifications, the constant region connected to the costimulatory molecule can also lack the natural intracellular region relative to the wild-type constant region, which further improves the function of STAR.

[0270] In the prior art, in order to kill tumors while improving the tumor immunosuppressive microenvironment, the patient needs to be treated or medicated separately, which affects the tumor treatment effect, increases the burden on the patient, and also increases the risk of adverse reactions of the drug. The bifunctional STAR-T cell of the present application makes full use of its natural advantage of multiple chains, by connecting an antibody part against a receptor (such as CCR8) in an immunosuppressive active cell on one chain and an antibody part targeting a tumor (such as MSLN or Claudin18.2) on the other chain, thereby realizing the dual function. Due to the advantage of structure, the two antibodies do not interfere with each other and can act independently. Not only that, but also because the antibody part against the receptor in the immunosuppressive active cell can eliminate the immunosuppressive effect of Treg, improve the tumor immunosuppressive microenvironment, and thus improve the ability of the antibody targeting the tumor to kill the tumor; in animal models of epithelial cell carcinoma, glioblastoma and liver cancer, the bifunctional STAR-T cell of the present application exhibits better anti-tumor effect than traditional CAR-T cells, and has no obvious toxic side effects.

[0271] The bifunctional STAR-T cell of the present application can achieve the effect of killing tumors while improving the tumor immunosuppressive microenvironment in the form of a single drug.

[0272] Example 1, screening of nanobodies targeting CCR8

[0273] 1.1. Immunization of alpaca with human CCR8 protein

[0274] Healthy alpacas were immunized with the commercially available extracellular domain of human CCR8 protein (100 μg) (company name ACRO Biosystems), and adjuvants included complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified extracellular domain of human CCR8 protein was diluted with PBS and then mixed with the corresponding adjuvant in a 5:1 ratio. The antigen and adjuvant were completely mixed to form a stable emulsion. The antigen mixture was extracted with a syringe and injected subcutaneously four times under the skin of the alpaca's neck (immunization injections were performed on days 1, 14, 28, and 42, respectively), with 100-200 μL injected at each point, and CCR8 antigen (100 μg) was injected each time.

[0275] On the 53rd day, alpaca blood samples were collected from the alpaca's ear vein, serum was extracted, and antibody titer was tested. The P / N value of the 200,000-fold diluted serum was greater than 2.

[0276] Days 54, 57, and 60: 30-40 mL of alpaca blood samples were collected from the alpaca hind leg vein for PBMC isolation.

[0277] PBMC isolation, RNA extraction, reverse transcription, and phage library construction

[0278] PBMCs were separated from the alpaca blood sample obtained in the previous step using Ficoll separation solution (Cytiva, product number 17544202) according to conventional procedures; RNA was isolated from PBMCs using conventional methods, and cDNA was synthesized using an Invitrogen reverse transcription kit (ThermoScientific, product number #K1622).

[0279] The VHH sequence was obtained through two rounds of PCR, and homology arms of the phagemid vector were added to both ends of the sequence. The VHH fragment was ligated to the phagemid vector, and the ligation product was subsequently concentrated and purified (gel recovery kit, company name TIANGEN, product number #19-03). The purified ligation product was electroporated into competent E. coli cells. Helper phage was added for infection and further cultured. The phage was harvested and concentrated, and the titer was determined.

[0280] 1.3. Phage library antibody screening

[0281] The phage library obtained in the above steps was subjected to three rounds of antibody screening, each including a positive selection and a negative selection. The phage were first incubated with the antigenic peptide, and those that did not bind were discarded, retaining those that bound to the antigenic peptide. The phage were then incubated with BSA for negative selection, retaining those that did not bind to BSA.

[0282] The phages obtained by the three rounds of screening were used to co-infect TG1 cells with M13KO7 helper phage, and the cells were plated on 2YT-AK plates. Single colonies were picked and used for phage expansion. The phages were collected and used for binding detection to determine the available phage / antibody. Two obtained antibodies were named NCR801 and NCR802, respectively.

[0283] Example 2, performance detection of CCR8 nanobody

[0284] 2.1. EC50 affinity of CCR8 nanobody

[0285] The affinity E50 of CCR8 nanobody was determined. When the cell density of 293T-hCCR8, 293T-hCCR4, and 293T cells reached 80%, the cells were added to a 96-well plate at a volume of 100 μl per well at a concentration of 1E6 per well for flow cytometry staining. The antibody was gradiently diluted at an initial concentration of 250 nM, 3-fold dilution, and 11 samples for each antibody. The prepared antibody was added to the cells in the 96-well plate, and the cells were stained at 4°C for 30 min. After the antibody was washed away with PBS, a secondary antibody was added for staining: APC anti-human IgG Fc Antibody (Cat: 410711, Lot: B343074), diluted at 1:200. After staining, the APC fluorescence value was detected by flow cytometry, and the EC50 value was calculated according to the experimental results.

[0286] The results are shown in Table 1. Figure 1 The affinity EC50 value of NCR802 to CCR8 protein was 0.19 nM, and the affinity detection result of NCR801 showed that it had non-specific binding. The sequences of these antibodies are shown in Table 1.

[0287] Table 1. CCR8 nanobody sequences

[0288]

[0289]

[0290] 2.2. MPA specificity detection of CCR8 nanobody

[0291] The MPA developed and used by Integral Molecular is an array composed of more than 5220 human membrane proteins (covering 94% of human membrane proteins). Each human membrane protein in the MPA has a complete structure and can be expressed in its native conformation in living cells. MPA is an in vitro tool that can quickly and comprehensively screen the specificity of candidate therapeutic drugs. As shown in Figure 2As shown, the present study successfully completed the MPA detection of NCR802-Fc antibody, no off-target was detected, and the results showed that NCR802 specifically binds to CCR8.

[0292] Example 3 CCR8 STAR

[0293] 3.1. CCR8 STAR vector construction

[0294] The STAR structure targeting CCR8 is shown in Figure 3 A1-A2. The CCR8 nanobody NCR802 sequence was assembled with the constant region of the STAR molecule (the constant region of the alpha and beta chains of the STAR is selected from any one of SEQ ID NO: 9-15 and any one of SEQ ID NO: 16-22, the alpha chain is preferably SEQ ID NO: 11 or SEQ ID NO: 14, and the beta chain is preferably SEQ ID NO: 18, and the C-terminus of the constant region of the alpha and / or beta chain of the STAR is directly connected to the OX40 costimulatory domain), and inserted into a lentivirus vector using a homologous recombination method to construct a complete CCR8-STAR plasmid.

[0295] Lentix-293T cells were seeded at 5x10 5 / mL into a 10 cm dish and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 80% (observed under a microscope), transfection was performed. Four plasmids were mixed with 500 μL of serum-free DMEM at a ratio of PMD2.G:PRSV-Rev:PMDlg:transfer plamid = 1:1:2:4. 54 μL of PEI-max was mixed with 500 μL of serum-free DMEM and incubated at room temperature for 5 min (the volume-to-mass ratio of PEI-Max and plasmid was 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently blown and mixed, and incubated at room temperature for 15 min. The final mixture was slowly added to the culture medium, mixed thoroughly, and then returned to the incubator for 12-16 h. The medium was replaced with 6% FBS DMEM and the virus solution was collected at 48 h and 72 h.

[0296] Jurkat-C4 cells with knocked-out TCR were seeded at 1.5x10 5The cells were inoculated into a flat-bottom 96-well plate at 100 μL / mL, and 100 μL of 1640 medium containing 10% FBS and 0.2 μL 1000×polybrene was added to each well. When diluting the virus, 10-fold serial dilution was performed with 1640 complete medium. The diluted cells were added to the virus wells, 100 μL / well, mixed, centrifuged at 32°C, 1500 rpm, for 90 minutes, and cultured in a 37°C, 5% CO2 incubator. After 72 hours, the infection efficiency was measured by flow cytometry. When calculating the titer, wells with an infection rate of 2-30% were selected, and the calculation formula was: Titer (TU / mL) = 1.5×10 4 × positive rate ÷ virus volume (μL) × 1000. The above virus was used to infect T cells to express STAR.

[0297] After obtaining primary T cells by Ficoll separation, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 at an initial culture density of 1×10 6 / mL, and activated in CD3, CD28, and Fibronectin-precoated plates. 24 hours after activation, add the viral solution, centrifuge at 1500 rpm for 90 minutes, and incubate in a CO2 incubator. 24 hours after infection, supplement with X-VIVO medium supplemented with 10% FBS and 100 IU / mL IL-2, and transfer the cells. Subculture every 1-2 days.

[0298] STAR infection efficiency detection: 72 hours after infection, the STAR infection efficiency was analyzed by detecting the ratio of fluorescent label (RFP) by flow cytometry.

[0299] 3.2. CCR8 STAR Killing Screening in Vitro

[0300] Luciferase is a common substance used in cell function research. Enzyme activity is determined by adding luciferase substrate to the system. Luciferase activity is closely related to target gene expression, binding strength, and cell number. In the present invention, a target cell line stably expressing luciferase is established. The amount of luciferase is used to indicate the number of target cells, thereby indicating the killing function of functional cells.

[0301] In the experiment, the NCR802-STAR vector was expressed in T cells, and T cells (MOCK-T) not infected with STAR lentivirus were used as a reference. 293T, 293T-hCCR8, and H9-LUC target cells were constructed at a rate of 4×10 5The density of the holes is laid in a 24-well plate, and the above-mentioned CCR8-STAR positive T cells are mixed with target cells at a ratio of 1:1 and 3:1, and the corresponding number of STAR-T cells is added to the target cells, the co-culture volume is 1 mL, and the co-culture is carried out for 24 hours. After that, the co-culture cell suspension is taken, the luciferase reporter gene detection kit is used to detect the LUC luminescence value, and the killing efficiency of the STAR-T cells on the target cells is calculated.

[0302] As shown in Figure 4 NCR802-STAR has good specific killing and specific recognition on target cells. The specific killing on target cells H9 is the best, and the specific killing on target cells 293T-hCCR8 is also good.

[0303] 3.3. In vivo function evaluation of CCR8 STAR

[0304] NPG immunodeficient mice are used to construct the model. The mice lack T cells, B cells, NK cells, and their macrophages and dendritic cells are also defective. In this experiment, 6-8-week-old female NPG mice are used, and the weight difference of the mice in each batch of experiments is controlled within 2 g. The mice are raised in specific pathogen-free (SPF) independent ventilated cages, and normal diet and pH slightly acidic drinking water are provided to prevent pathogen contamination. All animal operations are carried out after the approval of the animal protocol.

[0305] In order to verify the killing effect of STAR-T cells in vivo and potential safety problems, H9-LUC target cell tumor models are constructed to study the in vivo efficacy and in vivo expansion of NCR802-STAR and STAR-uninfected T cells (MOCK-T). The above-mentioned fluorescently labeled target cells are injected into the tail vein of 6-8-week-old female NPG mice at a dose of 1E6 per mouse. On the 6th day after infusion, NCR802-STAR and STAR-uninfected T cells (MOCK-T) are injected into the tail vein at a dose of 1E6 per mouse, 2E6 per mouse, and 4E6 per mouse. Then on the -1st, 5th, 10th, 14th, 19th, and 27th days, the tumor growth, tumor fluorescence value, body weight change, and peripheral blood flow cytometry (CD3, CD8, mTCRb) are detected by luciferin substrate catalytic luminescence method.

[0306] As shown in Figures 5-7The results show that the T cells of NCR802-STAR in the above-mentioned 4E6 / mouse dose group have tumor inhibition effect on CCR8+ target cells. The T cells of NCR802-STAR have good tumor inhibition effect on CCR8+ target cells before 27 days, and the T cell mice have no obvious weight loss, and the safety is relatively good. In terms of survival detection, the survival of NCR802-STAR is better, and it has remained stable for the first 27 days.

[0307] Example 4, Double-target CCR8-MSLN STAR

[0308] 4.1. Nanobody and STAR targeting MSLN

[0309] The present inventors previously obtained a nanobody NM5 (SEQ ID NO: 34) specifically targeting MSLN against the MSLN target point through a nanobody screening platform, and proved that the NM5 antibody specifically binds to MSLN using a membrane protein array chip technology, suggesting that it has good safety. It was found through BLI affinity detection and competition experiments that NM5 has high affinity for MSLN (KD = 1.14E-08 M) and has a unique near-membrane end recognition epitope, based on which NM5 STAR was constructed. The constant region of the alpha chain of NM5 STAR is based on TRAC-Cys-TM, and the constant region of the beta chain is based on TRBC-Cys-TM, and the constant regions of the alpha chain and the beta chain are directly connected to the OX40 intracellular region, and the NM5 nanobody is fused to the N terminus of the beta chain constant region. The amino acid sequence of the alpha chain of NM5 STAR is shown in SEQ ID NO: 40; the amino acid sequence of the beta chain is shown in SEQ ID NO: 39. The alpha chain and the beta chain of NM5 STAR also contain a GM-CSF signal peptide (SEQ ID NO: 38) at the N terminus, which will be cleaved in the cell after expression.

[0310] 4.2. Double-target CCR8-MSLN STAR vector construction

[0311] Commercially synthesized and assembled to obtain Figure 3 The nucleic acid sequence encoding the structure shown in B1-B2. MSLN nanobody NM5 and CCR8 antibody NCR802 sequence are assembled together into the constant region of STAR molecule (the constant region of the alpha and beta chains of STAR is selected from any one of SEQ ID NO: 9-15 and any one of SEQ ID NO: 16-22, the alpha chain is preferably SEQ ID NO: 11 or SEQ ID NO: 14, and the beta chain is preferably SEQ ID NO: 18, and the C terminus of the constant region of the alpha and / or beta chain of STAR is directly connected to the OX40 costimulatory domain), and inserted into a lentiviral vector using homologous recombination method to construct a complete CCR8-MSLN STAR plasmid. For specific methods, see Example 3.1.

[0312] Table 2 STAR target and structure name

[0313]

[0314]

[0315] 4.3. Double-target CCR8-MSLN STAR co-expression detection

[0316] After obtaining the primary T cells by Ficoll separation method, the primary culture density was 1x10 6 IU / mL IL-2 containing X-VIVO medium, and then activated by adding CD3, CD28 and Fibronectin pre-coated well plate. After 24h activation, virus liquid was added, centrifuged at 1500rpm for 90min, and cultured in CO2 incubator. After 24h infection, X-VIVO medium containing 10% FBS and 100IU / mL IL-2 was supplemented and transferred to well, and later subcultured every 1-2 days.

[0317] The above-mentioned vector packaging lentivirus and infecting T cells, after one week of culture, the expression level of STAR and MSLN on the cells was detected by flow cytometry, and the positive rate of STAR vector infection was detected by mTCRβ. The results showed that NM5STAR, NCR802STAR, NM5β-NCR802αSTAR and NCR802-(EAAAK)3-NM5βSTAR(first peptide chain sequence: NCR802 antibody and NM5 antibody are connected by (EAAAK)3 linker, and then connected with TRBC constant region, transmembrane region and OX40 to form β chain; second peptide chain sequence: STAR TRAC constant region, transmembrane region and OX40 to form α chain) can be well combined with protein.

[0318] 4.4. Double-target CCR8-MSLN STAR in vitro killing screening

[0319] Luciferase is a common substance for cell function research. By adding luciferase substrate in the system, the enzyme activity can be judged, and the luciferase activity is closely related to the expression of target gene, the binding strength and the number of cells. In the present application, a target cell line stably expressing luciferase is established, and the number of target cells is indicated by the amount of luciferase, and then the killing function of functional cells is indicated.

[0320] H9 target cells were constructed, 293T, 293T-hCCR8, 293T-MSLN target cells were constructed, and CCR8-MSLN STAR-T cells were constructed to verify the killing level of CCR8-MSLN STAR-T cells on tumor cells. The above target cells were plated in a 24-well plate at a density of 4E5 / well, CCR8-MSLN STAR vector was expressed in T cells, and the corresponding number of CCR8-MSLN STAR-T cells was added to the target cells at a ratio of CCR8-MSLN STAR positive T cells to target cells of 2:1 and 1:1, the co-culture volume was 1 mL, and the co-culture cells were taken after co-culturing for 24 hours. The luciferase reporter gene detection kit was used to detect the LUC luminescence value, and the killing efficiency of STAR-T cells on target cells was calculated. As shown in Figure 8, it can be found that:

[0321] Under the target cell H9 system, Figure 8A ), NCR802 STAR, NM5β-NCR802αSTAR, NCR802-(EAAAK)3-NM5βSTAR T cells have killing effect on target cells H9, and the killing efficiency NCR802-(EAAAK)3-NM5βSTAR-T>NM5β-NCR802αSTAR-T>NCR802 STAR-T.

[0322] Under the target cell 293T-CCR8 system, Figure 8B ), NCR802 STAR, NM5β-NCR802αSTAR, NCR802-(EAAAK)3-NM5βSTAR T cells have killing effect on target cells 293T-CCR8, and the killing efficiency NCR802-(EAAAK)3-NM5βSTAR-T>NM5β-NCR802αSTAR-T>NCR802 STAR-T.

[0323] Under the target cell 293T-MSLN system, Figure 8D ), the killing efficiency of NM5 STAR, NM5β-NCR802αSTAR, NCR802-(EAAAK)3-NM5βSTAR T cells on target cells 293T-MSLN is basically the same.

[0324] Under the target cell 293T system, Figure 8C ), each STAR-T cell did not show in vitro killing.

[0325] 4.5. In vivo functional verification of double-target CCR8-MSLN STAR

[0326] To verify the killing effect of STAR-T cells in vivo and potential safety issues, a H9-LUC target cell tumor model (CCR8 single positive target cell model) was constructed to study the in vivo efficacy and in vivo expansion of NCR802-STAR and STAR-uninfected T cells (NC). The above-mentioned fluorescently labeled target cells were injected into the tail vein of 6-8-week-old female NPG mice at a dose of 1E6 per mouse. On the 6th day after infusion, NCR802 STAR-T (dose groups 1E6 per mouse, 2E6 per mouse, 4E6 per mouse) and NM5β-NCR802αSTAR-T (dose group 4E6 per mouse) and STAR-uninfected T cells (NC) were injected into the tail vein. Then on the -1st, 5th, 10th, 14th, 19th, and 27th days, the tumor growth, tumor fluorescence value, body weight change, and peripheral blood flow cytometry (CD3, CD8, mTCRb) were detected by luciferin substrate catalytic luminescence method.

[0327] As Figures 9-12 The results show that the NCR802-STAR T cells in the above-mentioned 4E6 per mouse dose group have tumor inhibition effect on CCR8+ target cells. The NCR802-STAR T cells have the best tumor inhibition effect on CCR8+ target cells before 27 days, and the T cell mice have no obvious weight loss and good safety. In terms of survival period detection, the NCR802-STAR has a better survival period and the weight has been stable for 27 days. The in vivo H9 model efficacy of NM5β-NCR802αSTAR-T is comparable to that of NCR802-STAR-T.

[0328] Example 5, Dual Target and CCR8-Claudin18.2 STAR Co-expressing mbIL-15 Screening

[0329] 5.1. Nanobodies and STARs targeting Claudin18.2

[0330] The inventors constructed a nanobody NCLD04 targeting Claudin18.2 and constructed it into NCLD04-STAR-T cells.

[0331] A dual-target CCR8-Claudin18.2 STAR-T was constructed similarly to Example 3, and the dual-target CCR8-Claudin18.2 STAR-T has stronger in vitro and in vivo anti-tumor effect.

[0332] 5.2. Vector construction and virus packaging

[0333] Referring to Example 3, a Claudin18.2-CCR8-STAR structure targeting Claudin18.2 and CCR8 at the same time was constructed as Figure 3C1-C2 are shown. Claudin18.2 Nanobody NCLD04 and antibody NCR802 sequence of CCR8 were co-assembled into STAR molecule constant region (the constant region of α and β chain of STAR is selected from any one of SEQ ID NO: 9-15 and any one of SEQ ID NO: 16-22, the α chain is preferably SEQ ID NO: 11 or SEQ ID NO: 14, and the β chain is preferably SEQ ID NO: 18, and the C-terminal of the constant region of α and / or β chain of STAR is directly connected to the OX40 costimulatory domain), and inserted into a lentiviral vector by homologous recombination method to construct a complete Claudin18.2-CCR8-STAR plasmid (Table 3), and MND is a promoter encoding a nucleic acid molecule.

[0334] Referring to Example 3, a Claudin18.2-CCR8-STAR structure targeting Claudin18.2 and CCR8 at the same time was constructed, and mbIL-15 was connected through furin-P2A on the basis of the STAR (the structure of the vector is shown in Figure 3 D1-D2). The corresponding nucleic acid sequence was synthesized and assembled, and inserted into a lentiviral vector by homologous recombination method to construct a STAR-T plasmid, and the specific test method is described in Example 3.1.

[0335] Table 3 STAR target and structure name

[0336]

[0337] 5.3. CCR8-Claudin18.2 STAR co-expression detection of double targeting and co-expression of mbIL-15

[0338] After obtaining the primary T cells by Ficoll separation method, the cells were cultured in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2, and the initial culture density was 1×10 6 IU / mL IL-2 and transferred to a 6-well plate coated with CD3, CD28 and Fibronectin. After 24 h of activation, the virus solution was added, centrifuged at 1500 rpm for 90 min, and cultured in a CO2 incubator. After 24 h of infection, the X-VIVO medium containing 10% FBS and 100 IU / mL IL-2 was supplemented and transferred to a new well, and the later subculture was performed every 1-2 days.

[0339] The above-mentioned vector packaging lentivirus and infecting T cells, after one week of culture, the expression level of STAR and Claudin18.2 on the cell is detected by flow cytometry, wherein the expression of Claudin18.2 antibody in the STAR structure on the cell membrane is detected by Claudin18.2 Protein antibody staining, and mTCRβ detects the positive rate of STAR vector infection, and the results show that NCLD04STAR, NCR802-STAR, NCLD04β-NCR802αSTAR, NCR802β-NCLD04αSTAR, NCLD04β-NCR802αSTAR-mbIL-15 and NCR802β-NCLD04αSTAR-mbIL-15 are all well combined with the protein (see Figure 13 ).

[0340] 5.4 Dual-target and CCR8-Claudin18.2 STAR co-expressing mbIL-15 in vitro killing screening

[0341] Luciferase is a common substance for cell function research. By adding luciferase substrate in the system, the enzyme activity can be judged, and the luciferase activity is closely related to the expression of the target gene, the binding strength, and the cell number. In the present application, a target cell line stably expressing luciferase is established, and the number of target cells is indicated by the amount of luciferase, and then the killing function of functional cells is indicated.

[0342] Claudin18.2 single-positive target cell 293T-Claudin18.2 target cell is constructed to verify the killing level of CCR8-Claudin18.2 STAR-T cells on Claudin18.2 tumor cells. 293T-Claudin18.2 target cells expressing only Claudin18.2 target are plated in a 24-well plate at a density of 4E5 / well. The above-mentioned STAR vector is expressed in T cells, and the corresponding number of STAR-T cells is added to the target cells according to the ratio of positive T cells to target cells of 3:1 and 1:1, and the co-culture volume is 1mL. After co-culturing for 24 hours, the co-culture cell suspension is taken, and the luciferase reporter gene detection kit is used to detect the LUC luminescence value, and the killing efficiency of STAR-T cells on target cells is calculated.

[0343] Figure 14As shown, in the Claudin18.2 single-positive target cell 293T-Claudin18.2 target cell system, NCLD04-STAR and NCLD04β-NCR802α-STAR and NCR802β-NCLD04α-STAR and NCLD04β-NCR802α-STAR-mbIL-15 and NCR802β-NCLD04α-STAR-mbIL-15 all have good killing efficiency on target cells, and the STAR-T co-expressing mbIL-15 has enhanced killing effect compared with double-target or single-target STAR.

[0344] CCR8 single-positive target cell 293T-hCCR8 and H9 cell lines were constructed to verify the killing level of CCR8-Claudin18.2 STAR-T cells on tumor cells expressing CCR8. Target cells 293T-Claudin18.2 and target cells H9 were plated in 24-well plates at a density of 4E5 / well. The above STAR vectors were expressed in T cells, and the above STAR-T cells were co-cultured with target cells at a ratio of STAR-positive T cells to target cells of 3:1 and 1:1, with a co-culture volume of 1 mL. After co-culturing for 24 hours, the co-culture cell suspension was taken, and the luciferase reporter gene detection kit was used to detect the LUC luminescence value, and the killing efficiency of STAR-T cells on target cells was calculated.

[0345] Figure 15A As shown, in the CCR8 single-positive target cell 293T-hCCR8 cell line, NCR802-STAR and NCLD04β-NCR802α-STAR and NCR802β-NCLD04α-STAR and NCLD04β-NCR802α-STAR-mbIL-15 and NCR802β-NCLD04α-STAR-mbIL-15 all have good killing efficiency on target cells, and the STAR-T co-expressing mbIL-15 has enhanced killing effect compared with double-target or single-target STAR.

[0346] CCR8 single-positive target cell H9 cell line with a target ratio of 1:1 Figure 15B ), NCR802-STAR and NCLD04β-NCR802α-STAR and NCR802β-NCLD04α-STAR and NCLD04β-NCR802α-STAR-mbIL-15 and NCR802β-NCLD04α-STAR-mbIL-15 all have good killing efficiency on target cells.

[0347] 5.5 Double-target and CCR8-Claudin18.2 STAR cells co-expressing mbIL-15 cytokine secretion detection

[0348] After the above experiment, the T cells are co-cultured with target cells, and the supernatant is collected. The secretion levels of IFN-γ, IL-2, and TNF-α are detected by ELISA.

[0349] A large amount of cytokines are released during the activation of T cells to help T cells kill target cells or promote the expansion of T cells themselves. Common ones are TNF-α, IFN-γ, and IL-2. After T cells are stimulated by target cells or antigens, the T cells are collected, centrifuged, and the supernatant is taken. The TNF-α, IFN-γ, and IL-2 ELISA kits use Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA (item numbers are 88-7025, 88-7346, and 88-7316, respectively). The specific steps are as follows: dilute 10X Coating Buffer with ddH2O to 1X, add the coating antibody (250X), mix well, and then add 100 μL / well to a 96-well plate (special for ELISA). Seal with plastic wrap at 4°C overnight, wash 3 times with 1X PBST (also known as Wash Buffer, 1X PBS with 0.05% Tween 20), 260 μL / well each time, dilute 5X ELISA / ELISPOT Diluent with ddH2O to 1X, add to the 96-well plate, 200 μL / well, and stand at room temperature for 1 h. Wash once with PBST, dilute the standard curve (ranges are 2-250, 4-500, and 4-500, respectively), and dilute the sample 20-50 times with 1X Diluent. Add the sample and the standard curve, 100 μL / well, two duplicate wells, incubate at room temperature for 2 h, wash 3 times with PBST, add the Detection antibody diluted with 1X Diluent, incubate for 1 h, wash 3 times with PBST, then add HRP diluted with 1X Diluent, incubate for 30 min, wash 6 times, add TMB for color development, the color development time is not more than 15 min, add 2N H2SO4 to stop, and detect the light absorption at 450 nm.

[0350] Figure 16 shows that for 293T-hClaudin18.2 target cells, the factor secretion of NCLD04β-NCR802α-STAR is significantly improved than that of NCR802β-NCLD04α-STAR, and the combination of NCLD04β-NCR802α-STAR and mbIL-15 can significantly improve the secretion of IL-2. Similarly, for 293T-hCCR8 target cells, the factor secretion of NCR802β-NCLD04α-STAR is significantly improved than that of NCLD04β-NCR802α-STAR, and the combination of NCR802β-NCLD04α-STAR and mbIL-15 can significantly improve the secretion of IL-2.

[0351] 5.6 Dual-target and CCR8-Claudin18.2 STAR co-expressing mbIL-15 in vivo functional verification

[0352] To further verify the in vivo inhibitory effect of CCR8-Claudin18.2 STAR-T cells in a mouse model of Claudin18.2-positive tumor cells, SNU1-Claudin18.2 fluorescently labeled target cells were constructed and subcutaneously inoculated into 6-8-week-old female NPG mice at a dose of 2E6 per mouse. On the 8th day after infusion, NCLD04-STAR, NCR802-STAR, NCLD04β-NCR802α-STAR, NCR802β-NCLD04α-STAR, NCLD04β-NCR802α-STAR-mbIL-15, and NCR802β-NCLD04α-STAR-mbIL-15 were infused via the tail vein at a dose of 3E6 per mouse. The tumor growth, tumor fluorescence value, and body weight changes were detected by luciferin substrate catalytic luminescence method at 1 day before reinfusion, and 4, 7, 14, 21, 27, 34, and 42 days after reinfusion, respectively.

[0353] In the in vivo killing effect and potential safety problem test in the gastric cancer tumor model, as shown in Figure 17 , Figures 19-21The results show that NCLD04-STAR, NCLD04β-NCR802α-STAR, NCR802β-NCLD04α-STAR, NCLD04β-NCR802α-STAR-mbIL-15 and NCR802β-NCLD04α-STAR-mbIL-15 have good tumor inhibition effect on SNU1-Claudin18.2 target cells, among which NCR802β-NCLD04α-STAR-mbIL-15, NCLD04β-STAR and NCLD04β-NCR802α-STAR-mbIL-15 cells have the best tumor inhibition effect on target cells; NCLD04β-NCR802α-STAR cells and NCR802β-NCLD04α-STAR cells have moderate tumor inhibition effect on target cells, and NCR802-STAR has the worst tumor inhibition effect on target cells. At the same time, the body weight of several T cells does not decrease significantly (see Figure 18 ), and the safety is relatively good. NCLD04β-NCR802α-mbIL-15 and NCR802β-NCLD04α-STAR-mbIL-15 have the strongest in vivo expansion ability of STAR-T cells, and can maintain a higher proportion of CD8 positive T cells, and the mouse toxicity reaction is high.

[0354] Example 6, CCR8-CAR-T and double-target CCR8-MSLN CAR-T

[0355] 6.1. CCR8-CAR-T and double-target CCR8-MSLN CAR vector construction and virus packaging

[0356] The VHH sequence of the hCCR8 positive antibody NCR802 obtained by the above sequencing is assembled with other modules of the CAR molecule (including but not limited to hinge region, transmembrane region, costimulatory domain, signal transduction domain, VHH region of antibody targeting tumor antigen and / or linker sequence) to construct CARs with different structures, and inserted into a lentiviral vector by homologous recombination to construct a CCR8 CAR plasmid.

[0357] The CAR structure targeting CCR8 and MSLN at the same time is as shown in Figure 22 . After connecting MSLN nanobody NM5 and CCR8 antibody NCR802 through Linker such as (G4S)4, (G4S)3, (EAAAK)3, they are assembled into a CAR molecule structure, and inserted into a lentiviral vector by homologous recombination to construct a complete CCR8-MSLN-CAR plasmid.

[0358] Lentix-293T cells were prepared according to 5×10 5Cells were inoculated into 10 cm culture dishes at a density of approximately 80% using a 1:1 ratio of PMD2.G:PRSV-Rev:PMD1g:transformation plasmid. The volume-to-mass ratio of PEI-Max to plasmid was 3:1. The medium was changed after 12-16 hours, and viral fluids were collected after 48 and 72 hours, respectively.

[0359] The virus was diluted 10-fold in a 96-well plate, and then TCR-knockout Jurkat-C5 cells were plated at 1.5×10 5 100 μL / mL was added to the virus wells, 100 μL per well was centrifuged at 32°C, 1500 rpm for 90 minutes, and cultured in an incubator. After 72 hours, the infection efficiency was measured by flow cytometry. The wells with an infection rate of 2-30% were selected for titer calculation. The titer (TU / mL) was 1.5×10 4 × positive rate ÷ virus volume (μL) × 1000.

[0360] 6.2. CCR8-MSLN CAR-T Cell Infection Efficiency Detection

[0361] After obtaining PBMCs by Ficoil separation, the cells were counted and 1.5 times CD3 / CD28 Dynabeads were added and incubated for 45 minutes. 6 The cells were cultured at a density of 100 μg / mL and infected with the virus at an MOI of 2 after 24 hours. The cells were then cultured every other day. 72 hours after infection, the ratio of fluorescent label (RFP) and the antibody assay for VHH were used to analyze the CAR infection efficiency. The results showed (see Figure 23 ), single-target NM5-CAR and NCR802-CAR and dual-target CCR8-MSLN-CAR can be successfully loaded onto the membrane and bind well to proteins, and can be used for subsequent experiments.

[0362] 6.3. CCR8-MSLN CAR-T Cell Killing Efficiency Detection

[0363] 293T, 293T cells overexpressing hCCR8, and 293T cells overexpressing hMSLN were constructed and plated in a 24-well plate at a density of 1.5E5 / well. After 24 hours, the corresponding number of CAR-T cells were added to the target cells at a ratio of 3:1 and 1:1 between CAR-positive T cells and target cells. After 24 hours of culture, the killing efficiency of CAR-T cells on target cells was detected (see Figure 24A -C).

[0364] Among the target cells 293T, the killing effect on single and double target CART cells was weak.

[0365] The overexpressed hCCR8 293T target cells were killed by both single-target NCR802-CAR-T and double-target CAR-T cells. The killing efficiency of CAR-T cells on target cells was ranked as follows: NM5-NCR802-CAR was basically equivalent to NCR802-NM5-CAR, and greater than NCR802-CAR.

[0366] All CAR-T cells had killing efficiency on the overexpressed hMSLN 293T target cells. The killing efficiency of CAR-T cells on target cells was ranked as follows (from large to small): NM5-NCR802-CAR was greater than NCR802-NM5-CAR, which was greater than NM5-CAR, which was greater than NCR802-CAR.

[0367] Example 7, CCR8-CAR-T and double-target CCR8-Claudin18.2 CAR-T

[0368] 7.1. CCR8-CAR-T and double-target CCR8-Claudin18.2 CAR vector construction and virus packaging

[0369] The VHH sequence of the hCCR8 positive antibody NCR802 obtained by the above sequencing was assembled with other modules of the CAR molecule (including but not limited to hinge region, transmembrane region, costimulatory domain, signal transduction domain, VHH region of antibody targeting tumor antigen and / or linker sequence) to construct CARs with different structures, and inserted into a lentiviral vector to construct a CCR8 CAR plasmid by homologous recombination.

[0370] The CAR structure targeting CCR8 and Claudin18.2 at the same time was constructed as shown in Figure 25 The Claudin18.2 nanobody NCLD04 and the antibody NCR802 of CCR8 were connected by (G4S)4 or (G4S)3 or (EAAAK)3, and then assembled into a CAR molecule structure, and inserted into a lentiviral vector to construct a complete Claudin18.2-CCR8-CAR plasmid by homologous recombination.

[0371] Lentix-293T cells were inoculated into 10 cm dishes at a concentration of 5×10 5 The cells were transfected when the cell density reached about 80%. The ratio of the four plasmids was PMD2.G: PRSV-Rev: PMDlg: transformation plasmid = 1:1:2:4, and the volume-to-mass ratio of PEI-Max and plasmid was 3:1. The medium was changed after 12-16 hours, and the virus liquid was collected at 48 hours and 72 hours, respectively.

[0372] The virus was diluted 10 times in a 96-well plate, then the TCR-knockout Jurkat-C5 cells were added to the virus well at 1.5x10 5 rpm for 90 minutes, and cultured in the incubator. After 72 hours, the infection efficiency was detected by flow cytometry. The well plate with an infection rate of 2-30% was selected for titer calculation. Titer (TU / mL) = 1.5x10 4 x positive rate ÷ virus volume (μL) x 1000.

[0373] 7.2. CCR8-Claudin18.2 CAR-T cell infection efficiency detection

[0374] After obtaining PBMC by Ficoil separation method, cell counting was performed, 1.5 times of CD3 / CD28 Dynabeads was added for incubation for 45 minutes, and the culture density was 1.2x10 6 / mL. After 24 hours, virus infection was performed according to MOI = 2. After 24 hours, cell replacement was performed, and later, subculture was performed every other day. After 72 hours of infection, the proportion of fluorescent labels (RFP) was detected by flow cytometry, and the CAR infection efficiency was analyzed by VHH antibody. All single-target and double-target CAR-T structures can successfully be membrane-bound and well combined with proteins, which can be used for subsequent experiments.

[0375] 7.3. CCR8-Claudin18.2 CAR-T cell killing efficiency detection

[0376] 293T, 293T target cells overexpressing hCCR8, and 293T target cells overexpressing hClaudin18.2 were constructed, and the above three target cells were plated in a 24-well plate at a density of 1.5E5 / well. After 24 hours, the corresponding amount of CAR-T cells was added to the target cells according to the ratio of CAR positive T cells to target cells of 3:1 and 1:1. After 24 hours of culture, the killing efficiency of CAR-T cells on target cells was detected (see Figure 27).

[0377] On target cells 293T( Figure 27A ), NCLD04-CAR-T had no killing effect on target cells, and NCR802-CAR, NCR802-NCLD04-CAR-T, and NCR802-NCLD04-CAR-T had weak killing efficiency on target cells.

[0378] On 293T target cells overexpressing hClaudin18.2( Figure 27B), single-target NCLD04-CAR-T and double-target CAR-T cells both have killing efficiency. The killing efficiency of CAR-T cells on target cells is in the order of NCLD04-CAR > NCLD04-NCR802-CAR > NCR802-NCLD04-CAR > NCLD04-CAR; NCR802-CAR-T has no killing.

[0379] For 293T target cells overexpressing hCCR8 Figure 27C ), single-target NCLD04-CAR-T and double-target CAR-T cells both have killing efficiency. The killing efficiency of CAR-T cells on target cells is in the order of NCLD04-CAR > NCLD04-NCR802-CAR > NCR802-NCLD04-CAR > NCLD04-CAR; NCR802-CAR-T has no killing.

[0380] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Although the specific embodiments of the present application are described above, those skilled in the art should understand that this is only an example, and the protection scope of the present application is defined by the appended claims. Those skilled in the art can make various changes or modifications to the disclosed technology without departing from the scope of the technical solutions of the present application, and these changes and modifications all fall within the protection scope of the present application.

Claims

1. A single-domain antibody that specifically binds to CCR8, comprising a CDR1, a CDR2, and a CDR3 selected from the sequence of SEQ ID NO:

8.

2. The single-domain antibody according to claim 1, comprising CDR1 as shown in SEQ ID NO: 5, CDR2 as shown in SEQ ID NO: 6, and CDR3 as shown in SEQ ID NO:

7.

3. The single-domain antibody according to claim 1 or 2, comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8, preferably, it comprises the amino acid sequence shown in SEQ ID NO:

8.

4. The single-domain antibody according to any one of claims 1 to 3, wherein the antibody is a murinized antibody or a humanized antibody.

5. A synthetic T cell receptor antigen receptor (STAR) targeting CCR8, wherein the target binding region comprises the single domain antibody specifically binding to CCR8 according to any one of claims 1 to 4.

6. The STAR according to claim 5, wherein: The STAR comprises a first peptide chain and a second peptide chain: i) the first peptide chain comprises a first target binding region and a first constant region, and the second peptide chain comprises a second target binding region and a second constant region; or, ii) the first peptide chain comprises a first constant region, the second peptide chain comprises a second constant region, and the first peptide chain or the second peptide chain comprises a first target binding region; wherein the first target binding region and / or the second target binding region comprises one or more antigen binding regions, and the multiple antigen binding regions are the same or different; The antigen binding region in the first target binding region and / or the second target binding region comprises the CCR8 single domain antibody according to any one of claims 1 to 4.

7. The STAR according to claim 6, wherein: The antigen binding region in the first target binding region and / or the second target binding region further comprises an antibody or antigen binding fragment thereof that specifically binds to MSLN or Claudin18.2; Preferably, the antibody or antigen-binding fragment thereof is selected from scFv, nanobody or single domain antibody.

8. The STAR according to any one of claims 5 to 7, wherein: The STAR includes any one of the following groups: a) the first peptide chain comprises a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, at least one CCR8 single-domain antibody according to any one of claims 1 to 4 and a second constant region; b) the first peptide chain comprises, from N-terminus to C-terminus, the CCR8 single-domain antibody according to any one of claims 1 to 4, and a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, an antibody or antigen-binding fragment thereof that specifically binds to MSLN or Claudin18.2, and a second constant region; c) the first peptide chain comprises a first constant region; the second peptide chain comprises, from N-terminus to C-terminus, at least one CCR8 single-domain antibody according to any one of claims 1 to 4, an antibody that specifically binds to MSLN or Claudin18.2, or an antigen-binding fragment thereof, and a second constant region; In each group a) to c) above, the first constant region of the first peptide chain is the TCRα chain constant region or the TCRβ chain constant region, and the second constant region of the second peptide chain is the TCRβ chain constant region or the TCRα chain constant region; the constant regions of the first peptide chain and the second peptide chain are not both TCRα chain constant regions, or are not both TCRβ chain constant regions.

9. The STAR according to claim 8, wherein the antibody or antigen-binding fragment thereof that specifically binds to MSLN comprises CDR1 as shown in SEQ ID NO: 31, CDR2 as shown in SEQ ID NO: 32, and CDR3 as shown in SEQ ID NO: 33, preferably, the antibody or antigen-binding fragment thereof that specifically binds to MSLN comprises a single domain antibody represented by the amino acid sequence as shown in SEQ ID NO: 34; The antibody or antigen-binding fragment thereof that specifically binds to Claudin18.2 comprises CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO:

43. Preferably, the antibody or antigen-binding fragment thereof that specifically binds to Claudin18.2 comprises a single-domain antibody represented by the amino acid sequence shown in SEQ ID NO:

44.

10. STAR according to claims 5-9, characterized in that The first constant region is a TCRα chain constant region or a TCRβ chain constant region, preferably a modified TCRα chain constant region or a TCRβ chain constant region; The second constant region is a TCRα chain constant region or a TCRβ chain constant region, preferably a modified TCRα chain constant region or a TCRβ chain constant region; Preferably, the constant region of the TCRα chain is selected from the constant region of the TCRα chain of a wild-type human or wild-type mouse; Preferably, the constant region of the TCRβ chain is selected from the constant region of the TCRβ chain of a wild-type human or wild-type mouse.

11. The STAR according to claim 10, wherein: The modified TCR α chain constant region is derived from a mouse wild-type TCR α chain constant region, and comprises an amino acid mutation selected from the following group or a combination thereof relative to the mouse wild-type TCR α chain constant region: i) T48C amino acid substitution; ii) S112L, M114I and / or G115V amino acid substitutions; iii) E6D and K13R amino acid substitutions and amino acids 15-18 deleted; iv) K122R amino acid substitution; v) lacks the intracellular domain of the constant region, amino acids 136-137 are deleted; And / or, the modified TCR β chain constant region is derived from a mouse wild-type TCR β chain constant region, which comprises an amino acid mutation selected from the following group or a combination thereof relative to the mouse wild-type TCR β chain constant region: i) S56C amino acid substitution; ii) amino acids 150, 168, and 170 were substituted with R; iii) R3K, T6F, K9E, S11A, L12V amino acid substitutions, and / or deletions of amino acids 17, 21-25; iv) The intracellular domain of the constant region is missing, and amino acids 167-172 of the intracellular domain are deleted.

12. The STAR according to claim 10, wherein: The modified TCR α chain constant region is derived from a human wild-type TCR α chain constant region, and comprises an amino acid mutation selected from the following groups or a combination thereof relative to the human wild-type TCR α chain constant region: i) T47C amino acid substitution; ii) P90S, E91D, S92V, S93P amino acid substitutions; iii) S115L, G118V amino acid substitutions; And / or the modified TCR β chain constant region is derived from a human wild-type TCR β chain constant region, which comprises an amino acid mutation selected from the following group or a combination thereof relative to the human wild-type TCR β chain constant region: i) S56C amino acid substitution; ii) E17K, S21A, F132I, E135A and / or Q138H amino acid substitutions.

13. The STAR according to claims 5-12, wherein the amino acid sequence of the constant region of the modified TCR α chain is selected from the sequence shown in any one of SEQ ID NOs: 11-15, and / or the amino acid sequence of the constant region of the modified TCR β chain is selected from the sequence shown in any one of SEQ ID NOs: 18-22.

14. The STAR according to any one of claims 5 to 13, wherein the first peptide chain and / or the second peptide chain is connected to at least one exogenous intracellular functional domain at its C-terminus, such as the intracellular domain of a co-stimulatory molecule, preferably the intracellular domain of OX40, and more preferably, the intracellular domain of OX40 comprises the amino acid sequence of SEQ ID NO:

23.

15. The STAR according to any one of claims 5 to 14, wherein the exogenous intracellular functional domain is directly linked or linked via a linker to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain, Preferably, the exogenous intracellular functional domain is connected to the C-terminus of the constant region of the first peptide chain and / or the second peptide chain with the intracellular region deleted through a linker. Preferably, the linker is a (G4S)n linker or a (EAAAK)n linker, wherein n represents an integer of 1-10, preferably, n is 3 or 4.

16. The STAR according to any one of claims 5 to 15, wherein the STAR is co-expressed with membrane-bound IL-15 protein (mbIL-15).

17. The STAR according to claim 15 or 16, wherein a) the STAR comprises a first peptide chain represented by SEQ ID NO: 48 and a second peptide chain represented by SEQ ID NO: 47; b) the STAR comprises a first peptide chain represented by SEQ ID NO: 50 and a second peptide chain represented by SEQ ID NO: 49; c) the STAR comprises a first peptide chain represented by SEQ ID NO: 52 and a second peptide chain represented by SEQ ID NO: 51; d) the STAR comprises a first peptide chain represented by SEQ ID NO: 54 and a second peptide chain represented by SEQ ID NO: 53; e) the STAR comprises the first peptide chain shown in SEQ ID NO: 56 and the second peptide chain shown in SEQ ID NO: 55; or f) The STAR comprises the first peptide chain shown in SEQ ID NO: 58 and the second peptide chain shown in SEQ ID NO:

57.

18. The antigen receptor STAR according to claim 16 or 17, wherein i) the amino acid sequence of IL-15 is shown in SEQ ID NO: 27; ii) the amino acid sequence of the IL-15Ra extracellular domain is shown in SEQ ID NO: 28; iii) the amino acid sequence of the linker connecting the extracellular domain of IL-15Ra to IL-15 is shown in SEQ ID NO: 24-25, 29; and / or iv) The amino acid sequence of mbIL-15 is shown in SEQ ID NO:

30.

19. A chimeric antigen receptor (CAR) targeting CCR8, comprising an extracellular antigen binding region, wherein: The extracellular antigen binding region comprises the CCR8 single-domain antibody according to any one of claims 1 to 4, and the CAR comprises, from N-terminus to C-terminus, an extracellular antigen binding region, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

20. The CAR of claim 19, wherein the extracellular antigen binding region further comprises an antigen binding region that specifically binds to another antigen, preferably, the antigen binding region that specifically binds to another antigen comprises a single-chain antibody (scFv) or a single-domain antibody that specifically binds to the other antigen.

21. The CAR according to claim 20, wherein the other antigen is Claudin18.2, preferably the antigen-binding region that specifically binds to Claudin18.2 comprises CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 43, preferably, the antigen-binding region that specifically binds to Claudin18.2 comprises the amino acid sequence shown in SEQ ID NO: 44; Alternatively, the other antigen is MSLN, preferably, the antigen binding region that specifically binds to MSLN comprises CDR1 shown in SEQ ID NO: 31, CDR2 shown in SEQ ID NO: 32 and CDR3 shown in SEQ ID NO: 33, preferably, the antigen binding region that specifically binds to MSLN comprises the amino acid sequence shown in SEQ ID NO:

34.

22. The CAR according to claim 20 or 21, wherein the CAR comprises an amino acid sequence selected from any one of SEQ ID NOs: 59 to 64.

23. An isolated nucleic acid molecule encoding the single domain antibody of any one of claims 1-4, or the STAR of any one of claims 5-18, or the CAR of any one of claims 19-20.

24. An expression vector comprising the nucleic acid molecule of claim 23, wherein the nucleic acid molecule is operably linked to an expression control element such as a promoter.

25. The expression vector according to claim 24, wherein the expression vector comprises: 1) a nucleotide sequence encoding a fusion polypeptide of the first peptide chain of the STAR and the second peptide chain of the STAR linked by a self-cleaving peptide; or 2) A nucleotide sequence encoding a fusion polypeptide of the first peptide chain of STAR, the second peptide chain of STAR and mbIL-15 connected by a self-cleaving peptide.

26. The expression vector according to claim 25, wherein the self-cleaving peptide is a 2A polypeptide, preferably, the self-cleaving peptide is a Furin-2A polypeptide, such as the Furin-P2A polypeptide shown in SEQ ID NO:

26.

27. A host cell obtained by transforming a cell with the nucleic acid molecule according to claim 23 or the expression vector according to any one of claims 24 to 26.

28. An isolated therapeutic immune cell comprising the STAR of any one of claims 5-18, or the CAR of any one of claims 19-22.

29. The therapeutic immune cell according to claim 28, wherein the immune cell is a T cell or a NK cell, preferably a T cell.

30. A method for preparing the therapeutic immune cell according to claim 28 or 29, comprising Step 1) providing initial immune cells; Step 2) introducing the expression vector according to any one of claims 24 to 26 into the starting immune cells; and Step 3) harvesting the immune cells obtained in step 2).

31. A method for producing a single domain antibody that specifically binds to CCR8, comprising: (i) culturing the host cell of claim 27 under conditions suitable for expression of the nucleic acid molecule or expression vector, and (ii) isolating and purifying the single domain antibody that specifically binds to CCR8 and is expressed by the host cell.

32. A pharmaceutical composition comprising the single domain antibody of any one of claims 1-4, the STAR of any one of claims 5-18, the CAR of any one of claims 19-22, the therapeutic immune cell of claim 28 or 29, and / or the expression vector of any one of claims 24-26, and a pharmaceutically acceptable carrier, preferably for treating a disease in a subject.

33. Use of the single domain antibody of any one of claims 1-4, the STAR of any one of claims 5-18, the CAR of any one of claims 19-22, the therapeutic immune cell of claim 28 or 29, the expression vector of any one of claims 24-26 and / or the pharmaceutical composition of claim 32 in the preparation of a medicament for treating a disease in a subject.

34. The use according to claim 33, wherein the disease is a CCR8-related disease, such as a CCR8-related autoimmune disease, a hematological tumor or a solid tumor; Preferably, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), polymyositis and dermatitis, systemic scleroderma, Sjögren's syndrome, autoimmune hemolytic anemia, and rheumatoid arthritis; Preferably, the blood tumor or solid tumor is selected from the group consisting of epithelial cell cancer, glioblastoma, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, digestive tract cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; More preferably, the blood tumor or solid tumor is selected from the group consisting of epithelial cell cancer, glioblastoma, colorectal cancer, breast cancer, gastric cancer, metastatic brain cancer, metastatic liver cancer, lung cancer, digestive tract cancer, mesothelioma, pancreatic cancer, ovarian cancer and B-cell malignancies.

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