STAR and application of immune cells containing STAR in treatment and prevention of tumors

Abstract

The invention relates to the technical field of biological medicine, in particular to the field of cell therapy, and particularly relates to application of STAR and immune cells comprising the STAR to treatment and / or prevention of tumors, an antigen binding region of the STAR comprises one or more than two of an antibody, a ligand or an antibody fusion body which binds to LILRB4, the antibody such as scFv or VHH, and the ligand and the antibody fusion body can be combined with the LILRB4. Wherein the LILRB4 is used as a tumor antigen and / or a receptor in an immunosuppressive active cell. According to the invention, the targeted killing effect of immune cells on tumors is enhanced by targeting tumor cells and / or reducing immune escape caused by immunosuppressive active cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the field of cell therapy, and specifically to the application of STAR and immune cells containing STAR in the treatment and prevention of tumors. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy is an anticancer immunotherapy. Unlike the way natural T cells recognize tumor cells, CAR-T cells do not rely on MHC molecules (such as HLA) to recognize tumor cells. CAR molecules are mainly composed of an extracellular region responsible for recognizing target antigens, a transmembrane region, and an intracellular region responsible for transmitting T cell activation signals after stimulation. When CAR molecules are transfected into the patient's T cells through transfection technology, the patient's T cells express tumor antigen receptors. After purification and amplification, CAR-T cells have been used to treat B cell lymphoma and blood tumors. However, CAR-T therapy has encountered difficulties in the treatment of solid tumors. There are multiple factors that have failed to achieve good results in the treatment of solid tumors. One of the important reasons is that the function of CAR-T cells is inhibited in the tumor microenvironment and T cells are prone to exhaustion and apoptosis. However, recent studies have shown that the suppressive immune cells (MDSC and Treg cells, etc.) present in the tumor microenvironment have a strong inhibitory effect on a variety of tumor immunotherapies, including cell therapy, which greatly limits its therapeutic potential. The T cell receptor (TCR) complex molecule contains multiple chains. The TCRα chain and TCRβ chain are responsible for recognizing MHC-peptide molecules, and the other 6 CD3 subunits bind to the TCRα / β chain to perform signal transduction functions. 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 TCR signal is slower than the CAR signal, the TCR signal is more persistent. Therefore, the signal transduction function of the natural TCR can alleviate T cell dysfunction and enable it to better play an anti-solid tumor role. However, TCR's recognition of tumor cells depends on MHC molecules, and its affinity is also lower than that of CAR.

[0003] Based on the high similarity between the extracellular region of TCR and the Fab domain of antibody, the TCR variable domain sequence is replaced with the antibody variable domain sequence (such as scFv) to obtain a synthetic T cell receptor antigen receptor (Snythetic TCR and Antigen Receptor, STAR). STAR combines the advantages of TCR and CAR. It has both the specificity of antibodies and the superior signal transduction function of natural TCR. It can mediate complete T cell activation and has greatly improved safety and efficacy. Therefore, it has become a promising new type of cellular immunotherapy. Summary of the invention

[0004] Based on the technical advantages of STAR, the present invention assembles a peptide or protein that specifically binds to LILRB4 in the antigen binding region of one chain (either chain α and β or either chain γ and δ) in TCR, thereby designing, constructing and expressing a novel STAR that (i) targets LILRB4 as a tumor antigen and / or (ii) targets LILRB4 as a receptor in cells with immunosuppressive activity. Since the immunosuppressive activity is reduced or removed, the targeted killing of tumors by immune cells (such as T cells, NK cells and macrophages, etc.) is enhanced, which is further used in the treatment and / or prevention of tumors. Specifically, the present invention solves the technical problems existing in the prior art through the following technical solutions.

[0005] In a first aspect of the present invention, a STAR or a nucleic acid encoding the same, a STAR complex or a nucleic acid encoding the same, a vector expressing STAR or a STAR complex, or an immune cell expressing STAR or a STAR complex is provided for use in the preparation of a drug for treating and / or preventing tumors. STAR treats and / or prevents tumors by targeting LILRB4, wherein LILRB4 (i) serves as a target antigen for the tumor; and / or (ii) serves as a receptor in cells with immunosuppressive activity.

[0006] Preferably, the STAR (i) treats and / or prevents tumors by killing tumor cells; and / or (ii) kills immunosuppressive active cells;

[0007] Preferably, the STAR enhances the killing of tumors by immune cells by killing immunosuppressive active cells.

[0008] Preferably, the immunosuppressive active cells include MDSC cells or Treg cells.

[0009] Preferably, the enhancement of the killing of tumors by immune cells includes increasing the secretion of cytokines. Further preferably, the cytokines include one or more of IFN-γ, IL-2, TNF-α or IL-10.

[0010] Preferably, the STAR includes:

[0011] The N-terminus of the constant domain of the TCR alpha chain or a variant thereof is linked to at least one antigen binding region.

[0012] At least one functional domain is linked to the C-terminus of the constant domain of the TCR alpha chain or a variant thereof.

[0013] The N-terminus of the constant domain of the TCR β chain or a variant thereof is linked to at least one antigen binding region.

[0014] At least one functional domain is linked to the C-terminus of the constant domain of the TCR β chain or a variant thereof.

[0015] Preferably, the STAR comprises an α chain and a β chain.

[0016] Preferably, the α chain comprises at least one functional domain from the C-terminus to the N-terminus, a constant domain of a TCR α chain or a variant thereof, and preferably also comprises an antigen binding region.

[0017] In a specific embodiment of the present invention, the α chain comprises at least one functional domain, a constant domain of a TCR α chain or a variant thereof, and an antigen binding region from the C-terminus to the N-terminus.

[0018] Preferably, the β chain comprises the constant domain of the TCR β chain or its variant, the antigen binding region from the C-terminus to the N-terminus, and preferably further comprises at least one functional domain.

[0019] In a specific embodiment of the present invention, the β chain comprises at least one functional domain, a constant domain of a TCR β chain or a variant thereof, and an antigen binding region from the C-terminus to the N-terminus.

[0020] Preferably, the antigen binding region comprises one or more of an antibody, a ligand or an antibody fusion that specifically binds to LILRB4; the antibody preferably comprises VHH or scFv.

[0021] Preferably, the antibody fusion comprises a VHH, scFv or ligand that can specifically bind to LILRB4, and a VHH, scFv or ligand that specifically binds to other target antigens.

[0022] In a specific embodiment of the present invention, the antibody fusion comprises, from N-terminus to C-terminus: a VHH that specifically binds to other target antigens, a linker, and a scFv that specifically binds to LILRB4.

[0023] In a specific embodiment of the present invention, the antibody fusion comprises, from N-terminus to C-terminus: a scFv that specifically binds to other target antigens, a linker, and a VHH that specifically binds to LILRB4.

[0024] Preferably, the functional domain can be connected to the C-terminus of the constant domain of the TCRα chain or its variant and / or the C-terminus of the constant domain of the TCRβ chain or its variant. Preferably, the C-terminus of the constant domain sequence of the TCRα chain modified by the intracellular domain (preferably the intracellular domain is deleted, for example, the 136-137 position is deleted) is connected to the functional domain. And / or, the C-terminus of the constant domain sequence of the TCRβ chain modified by the intracellular domain (preferably the intracellular domain is deleted, for example, the 167-172 position is deleted) is connected to the functional domain.

[0025] Preferably, the VHH comprises NLB4 and / or NLB14.

[0026] Preferably, the NLB4 comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.

[0027] Preferably, the NLB14 comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO:4, CDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0028] Preferably, the amino acid sequence of NLB4 comprises SEQ ID NO: 7; or, comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homologous to the amino acid sequence shown in SEQ ID NO: 7.

[0029] Preferably, the nucleotide sequence encoding the NLB4 comprises SEQ ID NO: 122; or, comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homologous to the nucleotide sequence shown in SEQ ID NO: 122.

[0030] Preferably, the amino acid sequence of NLB14 comprises SEQ ID NO: 8; or, comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homologous to the amino acid sequence shown in SEQ ID NO: 8.

[0031] Preferably, the nucleotide sequence encoding the NLB14 comprises SEQ ID NO: 123; or, comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homologous to the nucleotide sequence shown in SEQ ID NO: 123.

[0032] Preferably, the constant domain of the TCRα chain or its variant and the antigen binding region, the constant domain of the TCRα chain or its variant and the functional domain, the constant domain of the TCRβ chain or its variant and the antigen binding region, the constant domain of the TCRβ chain or its variant and the functional domain, and the functional domains are directly connected or connected through a linker.

[0033] Preferably, the linker comprises one or more of a rigid linker, a flexible linker, a cleavable linker or a meaningless amino acid.

[0034] Preferably, the amino acid sequence of the rigid linker includes one or more of SEQ ID NOs: 25-35.

[0035] Preferably, the flexible linker comprises a peptide segment rich in glycine and / or serine. For example, the flexible linker comprises one or more of SEQ ID NOs: 36-87, or the flexible linker comprises SEQ ID NO: 88.

[0036] Preferably, the cleavable linker comprises one or more of SEQ ID NOs: 89-93.

[0037] In one embodiment of the present invention, the linker is selected from: (G4S) m , (EA3K) n and (SEQ ID NO: 102) p , wherein m, n and p are each independently an integer of 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably m is 1-6, n is 1-3, and p is 1-3; more preferably m is 1, 3 or 5, n is 3, and p is 1.

[0038] According to the needs of a specific embodiment, the STAR may also contain other peptides or proteins that bind to LILRB4, and / or antibodies that bind to other target antigens, such as scFv, VHH, ligand or antibody fusion.

[0039] Preferably, the functional domain includes one or more of a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof.

[0040] Preferably, the co-stimulatory molecules include one or more of 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, CD40 or NKp80;

[0041] Preferably, the co-stimulatory molecules include: OX40 as shown in SEQ ID NO:9, CD40 as shown in SEQ ID NO:10, ICOS as shown in SEQ ID NO:11, CD28 as shown in SEQ ID NO:12, 4-1BB as shown in SEQ ID NO:13, and CD27 as shown in SEQ ID NO:14.

[0042] In a specific embodiment of the present invention, the co-stimulatory molecule is OX40, in particular OX40 as shown in SEQ ID NO:9.

[0043] Preferably, the co-inhibitory molecules include one or more of TIM3, PD1, CTLA4 or LAG3;

[0044] Preferably, the cytokine receptor includes one or more of an interleukin receptor, an interferon receptor, a tumor necrosis factor superfamily receptor, a colony stimulating factor receptor, a chemokine receptor or a growth factor receptor, such as one or more of IL-2β, IL-7α or IL-21;

[0045] In a specific embodiment of the present invention, the cytokine receptors include: IL-2β as shown in SEQ ID NO:94, IL-7α as shown in SEQ ID NO:95, and IL-21 as shown in SEQ ID NO:96.

[0046] Preferably, the intracellular protein includes an immune cell regulatory factor, preferably a T cell regulatory factor, such as a domain of NIK.

[0047] Preferably, the functional domain may be a fusion protein of a cytokine receptor and a human STAT5 activation module.

[0048] Preferably, the cytokine receptor is directly linked to the human STAT5 activation module or is linked via a linker.

[0049] Preferably, the amino acid sequence of the human SATA5 activation module includes SEQ ID NO:97.

[0050] Preferably, the functional domain is a fusion protein of the intracellular end of the human IL-2β receptor and the human STAT5 activation module, and preferably its amino acid sequence includes SEQ ID NO:98.

[0051] Preferably, the functional domain is a fusion protein of the intracellular end of the human IL-7α receptor and the human STAT5 activation module, and preferably its amino acid sequence includes SEQ ID NO: 99.

[0052] Preferably, the variant of the constant domain of the TCRα chain or the variant of the constant domain of the TCRβ chain comprises a mutation described in any one of the following groups:

[0053] 1) N-terminal modification;

[0054] 2) Cysteine ​​substitution; or,

[0055] 3) C-terminal modification;

[0056] Preferably, the N-terminal modification includes N-terminal truncation, humanization mutation or mouseization mutation;

[0057] Preferably, the C-terminal modification includes a hydrophobic amino acid substitution in the transmembrane domain or a modification in the intracellular domain (preferably, the modification in the intracellular domain includes a deletion in the intracellular domain sequence, such as a deletion in positions 136-137 of the TCRα chain intracellular domain, and / or a deletion in positions 167-172 of the TCRβ chain intracellular domain; or, the modification in the intracellular domain includes a lysine substitution).

[0058] Preferably, the sequence of the constant domain of the TCRα chain or its variant includes any one of SEQ ID NOs: 15-19, 100-101, 103, 110, and 112.

[0059] In a specific embodiment of the present invention, the sequence of the constant domain of the TCRα chain or its variant is SEQ ID NO: 18.

[0060] Preferably, the sequence of the constant domain of the TCRβ chain or its variant includes any one of SEQ ID NOs: 20-24, 104, 111, and 113.

[0061] In a specific embodiment of the present invention, the sequence of the constant domain of the TCRβ chain or its variant is SEQ ID NO: 20.

[0062] Preferably, the STAR includes any one of the following groups:

[0063] a) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, and the constant domain of the TCRα chain or its variant from the C-terminus to the N-terminus;

[0064] b) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, and the constant domain of the TCRα chain or its variant from the C-terminus to the N-terminus;

[0065] c) the β chain includes the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain includes OX40, a linker, the constant domain of the TCRα chain or its variant, and NLB14 from the C-terminus to the N-terminus;

[0066] d) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, the constant domain of the TCRα chain or its variant, and NLB4 from the C-terminus to the N-terminus;

[0067] e) the β chain comprises OX40, the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, the constant domain of the TCRα chain or its variant, and NLB4 from the C-terminus to the N-terminus;

[0068] f) the β chain comprises OX40, the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain comprises OX40, the constant domain of the TCRα chain or its variant, and NLB14 from the C-terminus to the N-terminus;

[0069] g) the β chain comprises OX40, a linker, a constant domain of a TCRβ chain or a variant thereof, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, a constant domain of a TCRα chain or a variant thereof, and NLB4 from the C-terminus to the N-terminus;

[0070] h) The β chain comprises OX40, a linker, a constant domain of a TCRβ chain or a variant thereof, and NLB4 from C-terminus to N-terminus; the α chain comprises OX40, a linker, a constant domain of a TCRα chain or a variant thereof, and NLB14 from C-terminus to N-terminus.

[0071] Preferably, the STAR complex comprises STAR and CD3 molecules, and the CD3 molecules preferably comprise one or more of CD3ε, CD3γ, CD3δ or CD3ζ.

[0072] Preferably, the amino acid sequence of CD3ε comprises SEQ ID NO:116.

[0073] Preferably, the amino acid sequence of CD3γ comprises SEQ ID NO:114.

[0074] Preferably, the amino acid sequence of CD3δ comprises SEQ ID NO:115.

[0075] Preferably, the amino acid sequence of CD3ζ comprises SEQ ID NO:117.

[0076] Preferably, the vector expressing STAR or STAR complex comprises, from 5' end to 3' end:

[0077] A nucleotide sequence encoding an α chain, a connecting peptide, and a nucleotide sequence encoding a β chain; or,

[0078] a nucleotide sequence encoding the β chain, a connecting peptide, and a nucleotide sequence encoding the α chain;

[0079] Preferably, the connecting peptide comprises a 2A peptide, such as Furin-P2A. Preferably, the amino acid sequence of Furin-P2A comprises SEQ ID NO:105.

[0080] Preferably, the immune cells include one or more of T cells, NK cells, NKT cells, TIL cells, mast cells, macrophages, dendritic cells, CIK cells or stem cell-derived immune cells.

[0081] Preferably, the T cells include one or more of memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), non-infiltrating regulatory T cells (Tregs), effector memory T cells (Tem), γδT cells or αβT cells.

[0082] In a specific embodiment of the present invention, the immune cells are T cells.

[0083] Preferably, the method for preparing the immune cell expressing STAR or STAR complex comprises introducing a nucleic acid encoding STAR or STAR complex or a vector expressing STAR or STAR complex into the immune cell.

[0084] Preferably, the tumor comprises a blood tumor or a solid tumor;

[0085] Preferably, the tumor includes lymphoma (preferably including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell lymphoma or Waldenstrom's macroglobulinemia), leukemia (preferably including acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia (such as M4 or M5 type), chronic myeloid leukemia (such as chronic myelomonocytic leukemia) or plasma cell leukemia), multiple myeloma, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer , colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer (e.g., non-small cell 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, mesothelioma, B-cell malignancies, myelodysplastic syndrome, or sarcoma (e.g., osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma), one or more of the following:

[0086] Preferably, the chronic myeloid leukemia (eg, chronic myelomonocytic leukemia) has a tendency to progress to acute myeloid leukemia.

[0087] In a specific embodiment of the present invention, the tumor is multiple myeloma or leukemia (such as acute myeloid leukemia or chronic myeloid leukemia, and the chronic myeloid leukemia is preferably chronic myelomonocytic leukemia).

[0088] The second aspect of the present invention provides a STAR or STAR complex according to the first aspect.

[0089] In a third aspect of the present invention, there is provided a synthetic T cell receptor antigen receptor (STAR) targeting LILRB4, comprising:

[0090] a) a first polypeptide chain comprising a first antigen binding region and a first T cell receptor domain, wherein the first antigen binding region comprises a constant domain of a first TCR subunit, and the first T cell receptor domain comprises a first transmembrane domain and a first intracellular domain of the first TCR subunit, wherein the first polypeptide chain lacks the antigen binding region of the first TCR subunit;

[0091] b) a second polypeptide chain comprising a second antigen binding region and a second T cell receptor domain, wherein the second antigen binding region comprises a constant domain of a second TCR subunit, and the second T cell receptor domain comprises a second transmembrane domain and a second intracellular domain of the second TCR subunit, wherein the second polypeptide chain lacks the antigen binding region of the second TCR subunit;

[0092] The first antigen binding region and / or the second antigen binding region comprises a peptide or protein capable of specifically binding to LILRB4, and the peptide or protein capable of specifically binding to LILRB4 is preferably located at the N-terminus of the constant domain of the first TCR subunit and / or the N-terminus of the constant domain of the second TCR subunit;

[0093] And wherein i) the first TCR subunit is a TCRα chain and the second TCR subunit is a TCRβ chain, or ii) the first TCR subunit is a TCRβ chain and the second TCR subunit is a TCRα chain; or iii) the first TCR subunit is a TCRγ chain and the second TCR subunit is a TCRδ chain, or iv) the first TCR subunit is a TCRδ chain and the second TCR subunit is a TCRγ chain.

[0094] Preferably, the TCRα chain, TCRβ chain, TCRγ chain or TCRδ chain are independently derived from human or mouse.

[0095] Preferably, the amino acid sequence of the constant domain of the human TCR α chain comprises SEQ ID NO:110.

[0096] Preferably, the amino acid sequence of the constant domain derived from the murine TCR α chain comprises SEQ ID NO:103.

[0097] Preferably, the amino acid sequence of the constant domain of the human TCRβ chain comprises SEQ ID NO:111.

[0098] Preferably, the amino acid sequence of the constant domain derived from the murine TCRβ chain comprises SEQ ID NO:104.

[0099] Preferably, the amino acid sequence of the constant domain of the human TCRγ chain comprises SEQ ID NO:118.

[0100] Preferably, the amino acid sequence of the constant domain derived from the murine TCRγ chain comprises SEQ ID NO:119.

[0101] Preferably, the amino acid sequence of the constant domain of the human TCRδ chain comprises SEQ ID NO:120.

[0102] Preferably, the amino acid sequence of the constant domain derived from the murine TCRδ chain comprises SEQ ID NO:121.

[0103] Preferably, the peptide capable of specifically binding to LILRB4 is a short peptide or polypeptide, and the protein capable of specifically binding to LILRB4 is selected from the following group: scFv, VHH, ligand and antibody fusion, preferably, the peptide or protein capable of specifically binding to LILRB4 binds to the same or different regions of LILRB4, such as the same or different epitopes.

[0104] Preferably, the first antigen binding region comprises a VHH that specifically binds to LILRB4, wherein the VHH comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; or the VHH comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6.

[0105] Preferably, the first antigen binding region comprises a VHH that specifically binds to LILRB4, wherein the VHH comprises or consists of the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. Preferably, LILRB4 (i) serves as a target antigen for tumors; and / or (ii) serves as a receptor in cells with immunosuppressive activity, preferably a receptor in myeloid-derived suppressor cells (MDSC) or Treg.

[0106] Preferably, the first intracellular domain and / or the second intracellular domain comprises a functional domain selected from the group consisting of a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof;

[0107] Preferably, the costimulatory molecule is selected from the group consisting of one or more of 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, CD40, or NKp80, more preferably, the costimulatory molecule is OX40;

[0108] Preferably, the co-inhibitory molecule is selected from the following group: one or more of TIM3, PD1, CTLA4 or LAG3;

[0109] Preferably, the cytokine receptor is selected from the group consisting of one or more of interleukin receptors, interferon receptors, tumor necrosis factor superfamily receptors, colony stimulating factor receptors, chemokine receptors, growth factor receptors or other membrane proteins; such as one or more of IL-2β, IL-7α or IL-21;

[0110] Preferably, the intracellular protein includes an immune cell regulatory factor, preferably a T cell regulatory factor, such as a domain of NIK;

[0111] Preferably, the functional domain may be a fusion protein of a cytokine receptor and a human STAT5 activation module.

[0112] Preferably, the functional domain comprises or consists of an amino acid sequence selected from the group consisting of any one or more of SEQ ID NOs: 9-14 and 94-99.

[0113] Preferably, the constant domain of the first TCR subunit and the constant domain of the second TCR subunit comprise a mutation selected from the group consisting of:

[0114] (1) N-terminal truncation;

[0115] (2) N-terminal disulfide modification; and

[0116] (3) C-terminal modification.

[0117] Preferably, the TCRα chain has undergone a mutation selected from the group consisting of: T48C, S112L, M114 I, G115V, E6D, K13R, Δ15-18 and Δ136-137, and / or the TCRβ chain has undergone a mutation selected from the group consisting of: S56C, R3K, T6F, K9E, S11A, L12V, Δ17+Δ21-25 and Δ167-172.

[0118] Preferably, the TCRα chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:15-19, 100-101 or 103, and / or the TCRβ chain comprises or consists of the amino acid sequence shown in any one of SEQ ID NO:20-24 or 104.

[0119] Preferably, the antibody fusion capable of specifically binding to LILRB4 comprises a scFv capable of specifically binding to LILRB4 and a VHH, scFv or ligand that binds to other target antigens, or the antibody fusion capable of specifically binding to LILRB4 comprises a VHH capable of specifically binding to LILRB4 and a VHH, scFv or ligand that binds to other target antigens.

[0120] Preferably, the antibody fusion capable of specifically binding to LILRB4 comprises, from N-terminus to C-terminus: a VHH that binds to other target antigens, a linker and a scFv that can specifically bind to LILRB4, or the antibody fusion capable of specifically binding to LILRB4 comprises, from N-terminus to C-terminus: a scFv that binds to other target antigens, a linker and a VHH that can specifically bind to LILRB4.

[0121] Preferably, the linker is selected from the group consisting of a rigid linker, a flexible linker, a cleavable linker or nonsense amino acids.

[0122] Preferably, the linker is selected from the group consisting of: (G4S) m , (EA3K) n and (SEQ ID NO: 102) p , wherein m, n and p are each independently an integer of 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably m is 1-6 or 1-5, n is 1-3, and p is 1-3; more preferably m is 1, 3 or 5, n is 3, and p is 1.

[0123] Preferably, the amino acid sequence of the rigid linker includes one or more of SEQ ID NOs: 25-35;

[0124] Preferably, the flexible linker comprises a peptide segment rich in glycine and / or serine; more preferably, the flexible linker comprises one or more of SEQ ID NOs: 36-87, or the flexible linker comprises SEQ ID NO: 88;

[0125] Preferably, the cleavable linker comprises one or more of SEQ ID NOs: 89-93.

[0126] The fourth aspect of the present invention provides a STAR complex, wherein the STAR complex comprises the above-mentioned STAR and a CD3 molecule, wherein the CD3 molecule preferably comprises one or more of CD3ε, CD3γ, CD3δ or CD3ζ.

[0127] Preferably, the amino acid sequence of CD3ε comprises SEQ ID NO:116.

[0128] Preferably, the amino acid sequence of CD3γ comprises SEQ ID NO:114.

[0129] Preferably, the amino acid sequence of CD3δ comprises SEQ ID NO:115.

[0130] Preferably, the amino acid sequence of CD3ζ comprises SEQ ID NO:117.

[0131] The fifth aspect of the present invention provides a nucleic acid encoding the above-mentioned STAR or STAR complex.

[0132] The sixth aspect of the present invention provides a vector which expresses the above-mentioned STAR or STAR complex, or which comprises the above-mentioned nucleic acid, wherein the nucleic acid is preferably operably linked to a regulatory sequence.

[0133] Preferably, the vector is a eukaryotic vector or a prokaryotic vector, preferably a plasmid or a viral vector, and more preferably a lentiviral vector.

[0134] Preferably, the regulatory sequence includes one or more of a promoter, a terminator, a leader sequence, an enhancer, a transposon, an intron, a polyadenylation sequence, a replication origin or a multiple cloning site; preferably, the regulatory sequence includes one or more of a hEF1a-HTLV promoter, a WPRE element, an LTR, a SV40 polyA sequence, a SV40 origin or a multiple cloning site.

[0135] The seventh aspect of the present invention provides an immune cell, wherein the immune cell expresses the aforementioned STAR or STAR complex, or comprises the aforementioned nucleic acid or the aforementioned vector.

[0136] Preferably, the immune cells include: one or more of T cells, NK cells, NKT cells, TIL cells, mast cells, macrophages, dendritic cells, CIK cells or stem cell-derived immune cells.

[0137] Preferably, the T cells include: one or more of memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), non-infiltrating regulatory T cells (Tregs), effector memory T cells (Tem), γδT cells or αβT cells.

[0138] The eighth aspect of the present invention provides a method for preparing the above-mentioned immune cells, which comprises introducing the above-mentioned nucleic acid or the above-mentioned vector into immune cells, and inducing the immune cells to express the above-mentioned STAR or STAR complex.

[0139] The ninth aspect of the present invention provides a pharmaceutical composition or kit, which comprises the aforementioned STAR or STAR complex, the aforementioned nucleic acid, the aforementioned vector and / or the aforementioned immune cell, and preferably further comprises a pharmaceutically acceptable excipient.

[0140] In one embodiment, the pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, wetting agents, surfactants, lubricants or disintegrants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0141] Preferably, the pharmaceutical composition is suitable for parenteral or enteral administration, such as intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).

[0142] The tenth aspect of the present invention provides a use of the above-mentioned STAR or STAR complex, the above-mentioned nucleic acid, the above-mentioned vector, and the above-mentioned immune cell in the preparation of a drug, wherein the drug is used to treat or improve tumors, autoimmune diseases and / or immunosuppression caused by immunosuppressive active cells in a subject.

[0143] Preferably, the immunosuppressive active cells include regulatory T cells (Treg) or myeloid-derived suppressor cells (MDSC).

[0144] Preferably, the tumor is a blood tumor or a solid tumor;

[0145] Preferably, the tumor is selected from the group consisting of lymphoma, non-small cell lung cancer, leukemia, multiple myeloma, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric 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;

[0146] More preferably, the tumor is selected from the group consisting of gastric cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer and B-cell malignancies.

[0147] Preferably, the leukemia is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, plasma cell leukemia, and chronic myeloid leukemia; more preferably, the acute myeloid leukemia is M4 or M5 type acute myeloid leukemia; more preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia;

[0148] Preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia;

[0149] Preferably, the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma;

[0150] In a specific embodiment of the present invention, the tumor is multiple myeloma or leukemia (such as acute myeloid leukemia or chronic myeloid leukemia, and the chronic myeloid leukemia is preferably chronic myelomonocytic leukemia).

[0151] The eleventh aspect of the present invention provides a method for treating a disease, which comprises administering a therapeutically effective amount of the above-mentioned STAR or STAR complex, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned immune cell or the above-mentioned pharmaceutical composition to a subject in need.

[0152] Preferably, the effective amount includes 1×10 5 Immune cells / kg-1×10 8 immune cells / kg, preferably 1×10 6 Immune cells / kg-1×10 7 Immune cells / kg, for example 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 ,7×10 5 , 8×10 5 ,9×10 5 , 1×10 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 ,7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×106 ,9×10 6 , 9.5×10 6 , 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 ,7×10 7 , 8×10 7 ,9×10 7 , 1×10 8 immune cells / kg.

[0153] Preferably, the method comprises administering the above-mentioned immune cells derived from autologous or allogeneic sources.

[0154] Preferably, the method comprises first performing chemotherapy and then administering the above-mentioned immune cells (eg, infusing immune cells).

[0155] Preferably, the disease includes tumors, autoimmune diseases or immunosuppression caused by immunosuppressive active cells.

[0156] Preferably, the immunosuppressive active cells include regulatory T cells (Treg) or myeloid-derived suppressor cells (MDSC).

[0157] Preferably, the tumor is a blood tumor or a solid tumor;

[0158] Preferably, the tumor is selected from the group consisting of lymphoma, non-small cell lung cancer, leukemia, multiple myeloma, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric 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;

[0159] More preferably, the tumor is selected from the group consisting of gastric cancer, mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer and B-cell malignancies.

[0160] Preferably, the leukemia is selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, plasma cell leukemia, and chronic myeloid leukemia; more preferably, the acute myeloid leukemia is M4 or M5 type acute myeloid leukemia; more preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia;

[0161] Preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia;

[0162] Preferably, the sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma and chondrosarcoma;

[0163] In a specific embodiment of the present invention, the tumor is multiple myeloma or leukemia (such as acute myeloid leukemia or chronic myeloid leukemia, and the chronic myeloid leukemia is preferably chronic myelomonocytic leukemia). DETAILED DESCRIPTION OF THE INVENTION

[0165] 1. Definition

[0166] Other terms used in the present invention are common terms used in the field of molecular biology and have the same meanings as those understood in the art.

[0167] The short peptides described in the present invention are also called oligopeptides or low-molecular-weight peptides, which are peptides formed by 2-20 amino acid residues, preferably 2-12 amino acid residues, more preferably 2-10 amino acid residues, and further preferably 3-9 amino acid residues; the polypeptides described in the present invention are peptides formed by more than 20 amino acid residues.

[0168] The term "vector" used in the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or can be an RNA (such as mRNA) that can be translated. In some preferred embodiments, the vector is a viral vector, such as a lentiviral vector.

[0169] The "regulatory sequence" and "regulatory element" described in the present invention are used interchangeably and refer to a nucleotide sequence located upstream (5' non-coding sequence), in the middle or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing or stability or translation of the relevant coding sequence. An expression regulatory element refers to a nucleotide sequence that can control the transcription, RNA processing or stability or translation of a nucleotide sequence. A regulatory sequence may include, but is not limited to, a promoter, a leader sequence, a transposon, a terminator, a multiple cloning site, a replication origin, an intron, an enhancer or a polyadenylation sequence.

[0170] The term "operably linked" as used herein refers to the linking of the regulatory element to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleic acid sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0171] "T cells" of the present invention can be obtained from many 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, T cells can be derived from healthy donors or from patients diagnosed with cancer. In some embodiments, cells can be part of a mixed population of cells showing different phenotypic characteristics. For example, T cells can be obtained by isolating peripheral blood mononuclear cells (PBMC), then activating and amplifying them with specific antibodies.

[0172] In some embodiments of various aspects of the invention, the T cells are derived from autologous cells of the subject. As used herein, "autologous" refers to cells, cell lines or cell populations used to treat the subject that are derived from the subject. In some embodiments, the T cells are derived from allogeneic cells, such as from a donor that is compatible with the human leukocyte antigen (HLA) of the subject. Standard protocols can be used to convert cells from donors into non-alloreactive cells and replicate as needed to produce cells that can be administered to one or more subjects.

[0173] "Subject" as used herein refers to an organism that suffers from or is susceptible to a disease (such as cancer) that can be treated by the cells, methods, or pharmaceutical compositions of the present invention. Non-limiting examples include humans or non-human animals, wherein the non-human animals are non-human mammals, such as cows, rabbits, pigs, rats, mice, dogs, monkeys, sheep (e.g., goats, sheep), deer, and other non-mammals. In a preferred embodiment, the subject is a human.

[0174] The "pharmaceutically acceptable excipients" described in the present invention include any and all solvents, diluents, adhesives, wetting agents, surfactants, lubricants or disintegrants, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible.

[0175] The "effective amount" of the present invention refers to the amount of a substance, compound, material or cell that is at least sufficient to produce a therapeutic effect after being applied to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block or partially block the symptoms of a disease or condition. For example, an "effective amount" of the STAR or STAR complex, immune cell (preferably T cell) or pharmaceutical composition of the present invention leads to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the pain of the disease. For example, for the treatment of tumors, an "effective amount" of the STAR or STAR complex, immune cell (preferably T cell) or pharmaceutical composition of the present invention inhibits tumor cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit tumor cell growth, and this inhibition can be determined in vitro by tests known to those skilled in the art.

[0176] In practice, the dosage level of the active ingredient (e.g., STAR or STAR complex, immune cell) in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected depends on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention being applied, the route of administration, the time of administration, the excretion rate of the particular compound being applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition being applied, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

[0177] The term "include" or "comprising" as used in the present invention refers to an open description. When used to describe a protein or nucleic acid sequence in the present invention, the protein or nucleic acid may be composed of the sequence, or may 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 invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0178] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be understood by those skilled in the art. Reference is made to, for example, standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual"; Lewin et al., "Genes VIII"; and Roitt et al., "Immunology" (8th edition), as well as the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art. Reference is also made to, for example, standard manuals, the above-mentioned general prior art and other references cited therein.

[0179] The technical solution of the present invention has the following benefits or technical advantages:

[0180] The present invention provides a STAR targeting tumor antigen LILRB4 and / or receptor LILRB4 in immunosuppressive active cells, and also provides a STAR complex, an immune cell expressing STAR or STAR complex, which can be used for disease treatment. Its advantages are that it can specifically target immunosuppressive active cells (such as MDSC cells or Treg cells) in tumor tissues while killing tumor cells, which can reduce or relieve immunosuppressive effects, improve tumor immunosuppressive microenvironment and body immunity, and enhance the in vivo persistence and function of immune cell products, thereby improving the clinical efficacy of tumor treatment. Specifically:

[0181] (1) The STAR and STAR-T products of the present invention have the advantages of both CAR-T and TCR-T, with high affinity comparable to that of CAR-T cells and high signal transduction capacity and high sensitivity to antigens similar to those of TCR-T cells. They also avoid the defects of both CAR-T and TCR-T cells, avoiding both the dependence of TCR-T cells on HLA and the more serious self-activation in CAR-T cells, making them less susceptible to exhaustion.

[0182] (2) The STAR and STAR-T products of the present invention can use LILRB4 as a tumor target antigen, as a receptor in immunosuppressive cells, or both. They can not only kill tumor cells, but also reduce immunosuppression in tumor cells, thereby further improving tumor killing ability;

[0183] (3) The present invention further optimizes the STAR structure by further coupling the co-stimulatory molecule OX40. The results at the cellular level show that after the bifunctional STAR of the present invention is coupled to OX40 in its intracellular domain, it can significantly increase the proliferation level of STAR-T cells and the secretion level of IL-2 under the stimulation of target cells.

[0184] (4) In the prior art, in order to improve the tumor immunosuppressive microenvironment while killing the tumor, patients need to receive separate treatment or medication, which affects the tumor treatment effect, increases the patient burden, and also increases the risk of adverse drug reactions. The STAR-T cells of the present invention can achieve the effect of improving the tumor immunosuppressive microenvironment while killing the tumor in the form of a single drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0186] Figure 1A : Schematic diagram of the structure of STAR targeting LILRB4;

[0187] Figure 1B : Schematic diagram of the structure of STAR targeting LILRB4, where LS represents GM-CSF signal peptide;

[0188] Figure 2A : Detection of LILRB4 expression in NCL-H929 cells and MM.1S cells;

[0189] Figure 2B : Detection of LILRB4 expression in U266B1;

[0190] Figure 3 :The in vitro efficacy of LILRB4 STAR-T was detected by flow cytometry;

[0191] Figure 4A :LILRB4 STAR-T's killing ability and cytokine release level on NCI-H929 cells

[0192] Figure 4B :The killing ability of LILRB4 STAR-T on U266B1 cells and the level of cytokine release;

[0193] Figure 5A : Tumor volume curves of the treatment group and the control group;

[0194] Figure 5B :Survival curves of mice in the treatment group and the control group;

[0195] Figure 5C : Body weight curves of mice in the treatment group and the control group;

[0196] Figure 5D : Tumor imaging of the control group at the end point;

[0197] Figure 6: Experimental flow chart to verify the function of LILRB4 STAR-T targeting MDSCs;

[0198] Fig. 7A :CD33 + CD11b + The expression of LILRB4 and CLL1 in MDSC and MV4-11 cells was compared by flow cytometry.

[0199] Figure 7B : Mean fluorescence intensity ratio (MFIR) of LILRB4 and CLL1 expression in MDSCs and MV4-11, respectively;

[0200] Fig. 8A :From left to right, they are all cells, all single cells, CD8 + Single Cell and CTR + Fluorescence flow cytometry images of MDSCs in cells;

[0201] Figure 8B : The degree of T cell suppression at different MDSC:T ratios;

[0202] Figure 8C :CD8 at different MDSC:T ratios + Comparison of T cell proliferation % and induced IFN-γ concentration;

[0203] Fig.9A :The control group and experimental group had a significant difference in total cells, total single cells, CD8 + Single Cell and CTR + Fluorescence flow cytometry images of MDSCs in cells;

[0204] Fig. 9B : % lysis of MDSCs in the control group and experimental group at different E:T ratios after treatment for different time periods;

[0205] Fig. 9C : IFN-γ concentrations induced by different combinations and ratios determined by ELISA;

[0206] Fig.9D : IFN-γ concentrations induced by different combinations and ratios determined by ELISA;

[0207] Fig.9E : IL-10 concentrations induced by different combinations and ratios determined by ELISA;

[0208] Fig.9F : IL-10 concentrations induced by different combinations and ratios determined by ELISA;

[0209] Fig.10 : OX40 connection diagram of different connection methods;

[0210] Fig.11 : Upper membrane expression of LILRB4 STAR-T with three different OX40 linkages;

[0211] Fig. 12A : In vitro sequential killing assay of LILRB4 STAR-T with three different OX40 linkages;

[0212] Fig. 12B : Cytokine secretion detection of LILRB4 STAR-T with three different OX40 connection modes;

[0213] Fig.13A : Tumor growth of mice in each group;

[0214] Fig. 13B :The weight changes of mice in each group

[0215] Fig. 13C : Changes in tumor fluorescence values ​​of mice in each group;

[0216] Fig.14 : IIT clinical data results. DETAILED DESCRIPTION

[0217] The present invention will be further described below in conjunction with specific embodiments, but these specific embodiments cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and the changed and modified embodiments still fall within the scope of protection of the present invention.

[0218] Experimental Materials and Methods

[0219] Vector construction

[0220] The lentiviral vectors and lentiviral packaging plasmids used in the examples of the present application are purchased from commercial companies or synthesized by commercial companies. The gene fragments used in the examples of the present application include signal peptides, antibody binding regions (i.e., antibodies, such as VHH), hinge regions, TCR constant domains, tag proteins, etc., all of which are synthesized by commercial companies. By means of synthetic primer PCR, one or more segments of the target fragments are connected to obtain the corresponding functional sequence. The lentiviral vector used in the present invention is pHAGE-hEF1α-RFP, and the pHAGE-hEF1A-WPRE-AMP vector is obtained by restriction endonuclease SpeI / SalI. The fragment gene is obtained by synthesis and PCR methods, and the complete vector is obtained by homologous recombination method under the action of recombinase.

[0221] The α chain constant domain is of human or mouse origin, and the wild type is named TRAC.

[0222] The sequence of the human α chain constant domain includes SEQ ID NO:110.

[0223] The sequence of the mouse α chain constant domain includes any one of SEQ ID NOs: 15-19, 100-101, 103, and 112.

[0224] The β-chain constant domain is of human or mouse origin, and the wild type is named TRBC.

[0225] The sequence of the human-derived β chain constant domain includes SEQ ID NO:111.

[0226] The mouse-derived β chain constant domain includes any one of SEQ ID NOs: 20-24, 104, and 113.

[0227] The co-stimulatory molecule is OX40.

[0228] Lentiviral packaging

[0229] Lentix-293T cells were cultured at 5×10 5 / mL was inoculated into a 10cm culture dish and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 80% (observed under a microscope), transfection was performed. The four plasmids were mixed evenly with 500uL serum-free DMEM in a ratio of PMD2.G:REV:PMDLG:transfer plamid=1:1:2:4. 54uL PEI-max was mixed evenly with 500uL serum-free DMEM and allowed to stand at room temperature for 5min (the volume-to-mass ratio of PEI-Max to plasmid was 3:1). The PEI-max mixture was slowly added to the plasmid mixture, gently blown, mixed, and allowed to stand at room temperature for 15min. The final mixture was slowly added to the culture medium, mixed thoroughly, and returned to the incubator for continued culture for 12h-16h, then switched to 6% FBSDMEM culture medium for continued culture, and the 48h and 72h virus liquids were collected.

[0230] T cell culture and infection

[0231] 1) Jurkat T cell line culture method

[0232] Jurkat T cell line was cultured in RPMI 1640 medium containing 10% FBS. The culture density was 3×10 5 / ml, the maximum is no more than 3×10 6 / ml, subculture every 1-2 days, take the required amount of cells after cell counting, add culture medium to adjust to the above density, and culture in a CO2 incubator.

[0233] 2) Infection method of Jurkat T cell line

[0234] Cell count, take 1×10 6 / ml cells were centrifuged and replaced with 1ml RPMI 1640 medium containing 10% FBS, and added to a 24-well plate. Virus solution (MOI = 1) was added, and the cells were centrifuged at 1500rpm for 90min and placed in a CO2 incubator for culture. After 12h of infection, the medium was completely replaced with fresh RPMI 1640 medium containing 10% FBS, and the positive rate was tested at 72h.

[0235] 3) Method for culturing primary human T cells

[0236] After obtaining primary T cells by Ficoll separation, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / ml IL-2. The initial culture density was 1×10 6 / ml, and added to the pre-coated well plate with CD3 and RetroNectin (final concentration of 5μg / ml). The later culture density was 5×10 5 / ml, the maximum is no more than 3×10 6 / ml, and subcultured every 1-2 days.

[0237] 4) Method for infection of primary human T cells

[0238] After 48 hours of primary T cell culture, virus solution was added, MOI = 20, 1500 rpm, centrifuged for 90 minutes, and placed in a CO2 incubator for culture. After 24 hours of infection, X-VIVO medium containing 10% FBS and 100 IU / ml IL-2 was added and the cells were transfected. After 72 hours, the infection efficiency was detected by label protein or antibody.

[0239] 5) Infection efficiency detection method

[0240] After 72 hours of infection, the cells were evenly blown and counted, and 5×10 5 / ml, centrifuge, discard the supernatant, add the corresponding antibody for incubation in PBS+2% FBS+2mM EDTA staining solution for 30 minutes, then add PBS for washing twice and detect on the machine.

[0241] In vitro functional assay of STAR-T cells

[0242] Positive T cells were co-cultured with target cells expressing Luciferase at a specified effector-target ratio.

[0243] After 24 hours of co-culture, gently blow the cell suspension evenly, take 150 μL of cell suspension from each well and add it to a white 96-well plate, take 2 replicates from each well, and add luciferase substrate. Oscillate (low speed), incubate for 10 minutes, and use a multifunctional microplate reader to detect the chemiluminescence value. Calculate cell killing: killing efficiency = 100% - (effect cell-target cell well value / control cell-target cell well value). Alternatively, after 24 hours of co-culture, collect the cell culture supernatant and detect the cytokine content using a commercial kit.

[0244] Example 1. Constant domain of STAR structure

[0245] The constant domain of the STAR structure in the present invention can be derived from humans or mice. The constant domain of human origin can be modified by murinization, and the constant domain of mouse origin can be modified by humanization. Preferably, the constant domain can also include cysteine ​​point mutation, hydrophobic amino acid mutation of the α chain constant domain, and constant domain N-terminal modification to improve STAR function. Specifically:

[0246] First, if the constant domain is of human origin, it can be moused first: Since the constant domain sequences of human, primate and mouse TCRα / β chains (mouse TCRAC / mouse TCRBC) are highly functionally conserved and have the same key amino acid sequences, they can be replaced with each other. The interchange increases the efficiency of correct pairing of STAR molecules on the one hand, and reduces the possibility of unknown specificity caused by mismatches, thereby increasing safety on the other.

[0247] Secondly, cysteine ​​point mutations were introduced into disulfide bonds: Threonine T at position 48 was mutated to Cysteine ​​C in the constant domain of the TCRα chain, and Serine S at position 56 was mutated to Cysteine ​​C in the constant domain of the TCRβ chain. These two newly added cysteines will form a disulfide bond between the two chains of STAR, reduce the mismatch between the two chains of STAR and the endogenous TCR chain, and help the STAR molecule form a more stable complex.

[0248] Design of hydrophobic amino acid replacement in the transmembrane region of STAR: mutations were made in the amino acid region from 111 to 119 of the transmembrane domain of the constant domain of the TCRα chain, changing the serine S at position 112 to leucine L, the methionine M at position 114 to isoleucine I, and the glycine G at position 115 to valine V. The overall amino acid sequence of this region changed from LSVMGLRIL to LLVIVLRIL. This design increases the hydrophobicity of the transmembrane region, offsets the instability caused by the positive charge carried by the TCR transmembrane region, and allows the STAR molecule to exist more stably on the cell membrane, thereby obtaining better functions.

[0249] In order to further optimize the design of STAR molecules, the N-terminus of the constant domain of the STAR molecule was specifically rearranged based on the murinization of the constant domain, cysteine ​​point mutations, and hydrophobic amino acid mutations in the α-chain constant domain to obtain better results. Rearrangement means deleting part of the sequence and performing humanization mutations on part of the sequence. The significance of humanization mutations is to minimize the non-human sequence in the STAR molecule while ensuring the function of the STAR molecule, so as to minimize the possibility of STAR-T cells being rejected by the receptor in clinical applications. To this end, the N-terminal 18 amino acids of the TCRα chain constant domain were further modified, including the replacement of the 6th amino acid such as E by D, the 13th K by R, and the 15th-18th amino acids were deleted. The N-terminal 25 amino acids of the TCRβ chain constant domain were further modified, including the replacement of the amino acid at position 3 such as R by K, the amino acid at position 6 such as T by F, the K at position 9 by E, the S at position 11 by A, the L at position 12 by V, and the amino acids at positions 17 and 21-25 were deleted.

[0250] In addition, costimulatory molecules such as OX40 cytoplasmic regions can be connected to the C-terminus of the α chain constant domain and / or the β chain constant domain to further enhance the function of STAR. The costimulatory molecule can be directly connected or connected to the C-terminus of the α chain constant domain and / or the β chain constant domain by a linker (e.g., connected to a (G4S) 3 linker). In addition to the above-mentioned modifications, the constant domain connected to the costimulatory molecule can also be deleted relative to the wild-type constant domain. The natural intracellular domain, which further improves the function of STAR. For example, the α chain constant domain can be missing amino acids 136-137; and / or, the β chain constant domain can be missing amino acids 167-172.

[0251] The sequence of the α chain constant domain includes any one of SEQ ID NO: 15-19, 100-101, 103, 110 or 112.

[0252] The sequence of the β chain constant domain includes any one of SEQ ID NO: 20-24, 104, 111 or 113.

[0253] Example 2. Preparation and screening of VHHs targeting LILRB4 and structural design of LILRB4 STAR

[0254] 1. Immunization of alpacas with human LILRB4 protein

[0255] The commercially available human LILRB4 protein extracellular region (100 μg) was used to immunize healthy alpacas. The adjuvants included complete Freund's adjuvant (CFA, Sigma) and incomplete Freund's adjuvant (IFA, Sigma). The expressed and purified human LILRB4 protein extracellular region was diluted with PBS and then mixed with the corresponding adjuvant in a 1:1 ratio. The antigen and adjuvant were completely mixed to form a stable emulsion. The antigen mixture was extracted with a syringe and injected subcutaneously at multiple points under the skin of the alpaca's neck, with 100-200 μL injected at each point. The specific animal immunization process is as follows:

[0256] 1) Day 1 (1st immunization): LILRB4 antigen (100 μg) mixed with complete Freund's adjuvant (CFA) was injected subcutaneously to immunize alpacas;

[0257] 2) Day 14 (second immunization): LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was injected subcutaneously to immunize alpacas;

[0258] 3) Day 28 (third immunization): LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was injected subcutaneously again to immunize the alpaca;

[0259] 4) Day 42 (1st serum collection): 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;

[0260] 5) Day 42 (4th immunization): LILRB4 antigen (100 μg) mixed with incomplete Freund's adjuvant (IFA) was injected subcutaneously again to immunize the alpaca and enhance the immune effect;

[0261] 6) Day 53 (second serum collection): 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;

[0262] 7) Days 54, 57, and 60: 30-40 mL of alpaca blood samples were collected from the alpaca hind leg vein for PBMC isolation.

[0263] 2. PBMC Isolation

[0264] 1) Isolate PBMC in a biosafety cabinet, combine the blood in the anticoagulant tube into a 50 mL centrifuge tube (30 mL), add PBS to a total volume of 50 mL, and mix gently.

[0265] 2) Take another new 50mL centrifuge tube and add Ficoll separation solution, 15mL / tube. Layer the blood sample on the Ficoll liquid surface, 25mL / tube. The operation process should be steady to form a layer of Ficoll and blood to prevent mixing.

[0266] 3) Adjust the centrifuge speed to 0, RT (room temperature), centrifugal force 800g, and centrifuge for 30 minutes. After centrifugation, take the sample out of the centrifuge and separate the samples into layers as follows: upper aqueous phase - buffy coat layer - Ficoll layer - red blood cell layer, of which PBMC is in the buffy coat layer. Aspirate the buffy coat layer and transfer it to a new 50mL centrifuge tube.

[0267] 4) Add PBS to 50 mL into the sample tube, mix well, centrifuge at 2000 rpm, RT (room temperature) for 5 min, discard the supernatant, and add 5 mL PBS to resuspend the cell pellet.

[0268] 5) Repeat step 4 and cool down to 4°C.

[0269] 6) Add 5 mL of PBS to resuspend the cell pellet, add PBS to 40 mL, and count the cells.

[0270] 7) Centrifuge at 1500 rpm and 4°C for 5 min, discard the supernatant, add 1 mL PBS, resuspend the cells, and blow evenly; add 20 mL Trizol, mix well, let stand at room temperature for 5 min, lyse the cells, and dispense 1 mL into RNase-free 1.5 mL EP tubes, freeze at -80°C for RNA extraction.

[0271] 3. RNA Extraction and Reverse Transcription

[0272] 1) Take the sample out from -80℃, thaw at room temperature, add 200μL chloroform, and let it stand at room temperature for 3min. Centrifuge the sample at 4℃, 12000g for 15min, and the sample will be separated into the upper aqueous phase, the middle layer and the organic layer. Transfer the upper layer to a new RNase-free tube, add 1μL glycogen and 500μL isopropanol, and let it stand at 4℃ overnight.

[0273] 2) Centrifuge the sample at 4°C, 12000g for 20 minutes, remove the supernatant, add 1mL of pre-cooled 75% alcohol to wash the precipitate, centrifuge again to remove the alcohol, and air dry. Add 15μL of RNase-free water to each tube to dissolve the RNA precipitate and perform reverse transcription.

[0274] 3) Promega reverse transcription kit was used to synthesize cDNA (20 μL system).

[0275] Step 1: Take a certain amount of template RNA and add Oligo(dT), see Table 1;

[0276] Table 1

[0277] Components Volume (μL) RNA(<5μg / reaction) 11 Oligo(dT)15Primer 1 Total volume 12

[0278] Step 2: Denature the mixture of template RNA and Oligo (dT) at 65°C for 5 minutes and place it back on ice.

[0279] Step 3: During pre-denaturation, RT-Mix can be prepared in advance, 8 μL per tube. The components and volumes are shown in Table 2.

[0280] Table 2

[0281] Components Volume (μL) 5×Reaction Buffer 4μL RiboLockRnaseInhibitor(20U / μL) 1μL 10mM dNTP Mix 2μL RevertAid M-MuLVRT (200 U / μL) 1μL Total volume 8μL

[0282] Step 4: Set up the reverse transcription program, extension, and reverse transcriptase inactivation. After the program is completed, cDNA is obtained.

[0283] 4. Phage Library Construction

[0284] 1) PCR to obtain VHH sequence

[0285] The VHH sequence was obtained through two rounds of PCR, and homology arms of the vector were added to both ends of the sequence.

[0286] 2) First round of PCR

[0287] Step 1: Prepare the reaction system as shown in Table 3.

[0288] Table 3

[0289] Components Volume (μL) cDNA 1-2 Primer-F 1 Primer-R 1 PrimeSTAR Max DNA polymerase 15 <![CDATA[H2O]]> 11-12 Total volume 30

[0290] Step 2: PCR conditions are shown in Table 4.

[0291] Table 4

[0292]

[0293] The PCR products were subjected to gel electrophoresis, and the target band at 0.7 kb was cut out for product recovery.

[0294] 3) Second round of PCR

[0295] Step 1: Prepare the reaction system as shown in Table 5.

[0296] Table 5

[0297] Components Volume (μL) Template, first round PCR product 1-3(60ng) VHH-F 1 VHH-R 1 PrimeSTAR Max DNA polymerase 15 <![CDATA[H2O]]> 10-12 Total volume 30

[0298] Step 2: PCR conditions are shown in Table 6.

[0299] Table 6

[0300]

[0301] The PCR product was subjected to gel electrophoresis, and the target band at 400 bp was cut out for product recovery.

[0302] 4) Vector PCR

[0303] The vector region of phagemid was obtained by PCR and used to express the VHH sequence.

[0304] Step 1: Prepare the reaction system as shown in Table 7.

[0305] Table 7

[0306] Components Volume (μL) pADL Plasmid 1(10ng) pADL-Vector-F 1 pADL-Vector-R 1 PrimeSTAR Max DNA polymerase 15 <![CDATA[H2O]]> 10-12 Total volume 30

[0307] Step 2: PCR conditions are shown in Table 8.

[0308] Table 8

[0309]

[0310] The PCR product was subjected to gel electrophoresis, and the 4000 bp target band was cut out for product recovery.

[0311] 5) Ligation, purification and concentration of ligation products

[0312] The VHH fragment was ligated to the phagemid vector and the ligation product was subsequently concentrated.

[0313] Step 1: Prepare the reaction system as shown in Table 9.

[0314] Table 9

[0315] Components Usage NEBuilder HiFi DNA Assembly Master Mix 10μL VHH fragment 0.133 pmols Vector fragment 0.067pmols <![CDATA[H2O]]> margin Total volume 20

[0316] Step 2: Incubate the above mixture at 50°C for 2 h and cool on ice.

[0317] Step 3: Purify the ligation product, remove salt ions, proteins and other components in the ligation system, and concentrate the volume to 1 / 10 of the original volume.

[0318] 5. Electroporation and library construction

[0319] 1) Take a tube of competent E. coli and thaw it on ice.

[0320] 2) Take 2 μL of the ligation product or positive control, add it to the competent medium, and gently blow it evenly. Let it stand on ice for 1-2 minutes, transfer it to a pre-cooled electroporation cup, and perform electroporation.

[0321] 3) After electroporation, immediately add 1 mL of 37°C 2YT-G, blow and rinse the electroporation cup with a pipette tip, transfer the electroporated bacterial solution to a 15 mL centrifuge tube or a 2 mL EP tube, and resuscitate in a 37°C water bath until all samples are electroporated. (2YT-G: 2×YT medium containing 2% glucose) Transfer to a 37°C shaker, 220 rpm, and resuscitate for 1 hour.

[0322] 4) Aspirate 5 μL of the above bacterial solution and dilute it 10^2-10^5 times, apply it to 2YT-A (2YT plate containing 100 μg / mL ampicillin), and incubate it in a 37°C incubator overnight for colony counting.

[0323] 5) Inoculate the remaining bacterial liquid into 2YT-AG medium, shake to the logarithmic phase, add helper phage for infection, and shake at 30°C, 220 rpm for 12-16 hours. (2YT-AG: 2×YT medium containing 2% Glucose and 100 μg / mL ampicillin)

[0324] 6) Collect and concentrate the phages and determine the titer.

[0325] 6. Phage library antibody screening

[0326] The phage library obtained in step 4 above was subjected to three rounds of antibody screening, each of which included a positive selection and a negative selection. The phages were first incubated with the antigen peptide, and the phages that could not bind were discarded, leaving the phages that bound to the antigen peptide, and then incubated with BSA for negative selection, leaving the phages that could not bind to BSA.

[0327] 1) Coat the plate. Dilute the antigen to a concentration of 2 ng / μL with PBS and add it to a 96-well plate at 100 μL / well. Prepare 2% BSA with PBS and add it to the corresponding negative selection wells at 100 μL / well. Seal with plastic wrap and incubate at 4°C overnight.

[0328] 2) Discard the coating solution, add 200 μL of washing solution (washing solution: 1% Tween 20 / PBS, pH 7.4), and wash three times.

[0329] 3) Blocking: Add 2% BSA blocking solution to all wells, 100 μL / well, seal with plastic wrap, and incubate at 37°C for 1 h.

[0330] 4) Discard the supernatant, add 200 μL of washing solution, and wash three times.

[0331] 5) Add phage to the positive selection wells, 1×10 12 phages / well, dilute to 100 μL, seal with plastic wrap, and incubate at 37°C for 1 h.

[0332] 6) Discard the supernatant, add 200 μL of washing solution, and wash 10 times.

[0333] 7) Elution-neutralization: Add 200 μL of elution buffer to the positively selected wells and neutralize to pH 7-7.4.

[0334] 8) Negative selection. Add the above eluate to the negative selection wells, seal with plastic wrap, incubate at 37°C for 1 hour, aspirate and retain the supernatant, and perform titer detection.

[0335] 9) Take a small amount of phage after one round of panning, dilute it and apply it to 2×YT-A plates, and incubate it at 37°C overnight; count the colonies the next day and calculate the titer; and select single clones for sequencing to analyze sequence diversity and enrichment.

[0336] 10) All remaining phages are used for TG1 infection.

[0337] 11) Infection with M13KO7. Dilute the phage, add M13KO7 to the bacterial solution, and incubate in a 37°C water bath for 30 min; replace with 2×YT-AK medium, and shake at 30°C, 220 rpm for 14-16 h.

[0338] 12) Concentrate the phages and test the phage titer before proceeding to the next round of screening.

[0339] The amount of coated antigen in the second and third rounds of screening was reduced, and the number of washes was increased after the phage was incubated with the positive wells. The other steps were the same as the above steps.

[0340] 7. Combined detection and sequence acquisition

[0341] The phages obtained from three rounds of screening were co-infected with M13KO7 helper phage on TG1, coated on 2YT-AK plates, single clones were picked for phage expansion, and phages were collected for binding detection to determine the usable phage / antibody.

[0342] 1) Coat the plate. Dilute the antigen to 1 ng / μL with coating solution, 100 μL / well; add 2% BSA to the negative control well; seal with plastic wrap and incubate at 4°C overnight.

[0343] 2) Discard the coating solution in the plate and add 200-250 μL of washing solution and wash three times.

[0344] 3) Add 200 μL of 2% BSA to all wells and block at room temperature for 1 h.

[0345] 4) Discard the blocking solution in the plate, add 200 μL of washing solution, and wash once.

[0346] 5) Add 100 μL of phage to each of the positive and negative wells and incubate at 37°C for 1 h.

[0347] 6) Discard the phages in the plate, add 200 μL of washing solution, and wash three times.

[0348] 7) Dilute anti-M13-HRP antibody to 50 ng / well and incubate at room temperature for 1 hour.

[0349] 8) Discard the antibody in the plate, add 200 μL of washing solution, and wash 5 times.

[0350] 9) Add 100 μL of TMB colorimetric solution to each well and react at room temperature until the OD value is between 2 and 3.

[0351] 10) Add stop solution to the color development system, 50 μL per well.

[0352] 11) Measure the absorbance at 450 nm using a spectrophotometer. Send the monoclonal bacterial solution corresponding to the positive wells for sequencing to determine the VHH sequence.

[0353] 12) The obtained antibodies were named NLB1-NLB23 respectively.

[0354] 8. Detection of the obtained antibodies

[0355] 1) The in vitro co-culture killing efficiency of the obtained antibodies was detected. The results showed that NLB3, 4, 5, 6, 7, 8, 9, 10, 11, and 14 specifically recognized LILRB4 target cells; NLB1, 2, 12, 13, 21, 22, and 23 did not recognize LILRB4; and NLB15, 16, 17, 18, 19, and 20 had non-specific recognition and killing of LILRB4-negative cells.

[0356] 2) Taking NLB4 and NLB14 as examples, the affinity, competitive binding ability, epitope recognition detection, binding specificity and safety evaluation of the obtained antibodies were determined. The results showed that the affinities of NLB4 and NLB14 to LILRB4 protein were 1.32nM and 0.493nM respectively; there was no competitive binding between NLB4 and NLB14; NLB4 or NLB14 could recognize the full-length extracellular region of LILRB4, but could not bind to a single Ig domain or stock region; NLB4 and NLB14 significantly bound to LILRB4, but not to other proteins in the same family; and NLB4 and NLB14 had no non-specific binding to 9 tissue cell lines (lung, muscle, blood vessel, prostate, liver, kidney, brain, intestine, skin).

[0357] 9. Cell-level functional screening

[0358] 1) Construction of STAR targeting LILRB4 (its structure is as follows Figure 1A and Figure 1B The positive antibody heavy chain (VHH) sequence obtained from the above screening was assembled with the STAR molecule constant domain, and inserted into the lentiviral vector using the homologous recombination method to construct a complete STAR plasmid.

[0359] 2) Packaging virus

[0360] Lentix-293T cells were cultured at 5×10 5 / mL was inoculated into a 10cm culture dish and cultured in a 37°C, 5% CO2 incubator. When the cell density reached about 80% (observed under a microscope), transfection was performed. The four plasmids were mixed evenly with 500μL serum-free DMEM according to the ratio of PMD2.G:PRSV-Rev:PMDlg:transfer plamid=1:1:2:4. 54μL PEI-max was mixed evenly with 500μL serum-free DMEM and allowed to stand at room temperature for 5min (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, mixed, and allowed to stand at room temperature for 15min. The final mixture was slowly added to the culture medium, mixed thoroughly, returned to the incubator and continued to be cultured for 12h-16h, then switched to 6% FBSDMEM culture medium for continued culture, and the 48h and 72h virus liquids were collected.

[0361] 3) Virus titer measurement

[0362] TCR knockout Jurkat-C4 cells were cultured at 1.5×10 5 / mL was inoculated in a flat-bottomed 96-well plate, and 100μL of 1640 medium containing 10% FBS and 0.2μL 1000×polybrene was added to each well. When diluting the virus, 10 times dilution was performed with 1640 complete medium. The diluted cells were added to the virus wells, 100μL / well, mixed, centrifuged at 32℃, 1500rpm, 90min, and cultured in a 37℃, 5% CO2 incubator. After 72h, the infection efficiency was measured by flow cytometry. When calculating the titer, the wells with an infection rate of 2-30% were selected. The calculation formula is: 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.

[0363] 4) Isolation, activation and infection of primary human T cells

[0364] After obtaining primary T cells using the Ficoil separation method, they were cultured in X-VIVO medium containing 10% FBS and 100 IU / mL IL-2. The initial culture density was 1×10 6 / mL, add CD3, CD28 and Fibronectin pre-coated wells for activation. 24h after activation, add virus solution, centrifuge at 1500rpm for 90min, and place in CO2 incubator for culture. 24h after infection, supplement with X-VIVO medium containing 10% FBS and 100IU / mL IL-2 and transfer, and subculture every 1-2 days.

[0365] 5) Determination of killing efficiency by co-culture of T cells and target cells in vitro

[0366] One day in advance, target THP1 cells or 293T cells overexpressing LILRB4 were plated in a 24-well plate at a density of 1E5 / well and cultured overnight. According to the ratio of STAR-positive T cells to target cells of 0.5:1, a corresponding number of STAR-T cells were added to the target cells, and the killing effect of STAR-T cells on target cells was detected after 24 or 48 hours of culture.

[0367] like Figure 1A and Figure 1B As shown, schematic diagrams of the structures of various single-epitope and dual-epitope STARs targeting LILRB4 are listed.

[0368] Among them, the single-epitope STAR includes NLB4 STAR-a(G4S)3OX40 (amino acid sequence is SEQ ID NO: 106, and the nucleotide sequence encoding it is SEQ ID NO: 124) and NLB14 STAR-a(G4S)3OX40 (amino acid sequence is SEQ ID NO: 107, and the nucleotide sequence encoding it is SEQ ID NO: 125).

[0369] The dual-epitope STARs include NLB4 / NLB14 STAR-a(G4S)3OX40 (amino acid sequence is SEQ ID NO: 108, and the nucleotide sequence encoding it is SEQ ID NO: 126) and NLB4 / (myc)NLB14 STAR-a(G4S)3OX40 (amino acid sequence is SEQ ID NO: 109, and the nucleotide sequence encoding it is SEQ ID NO: 127).

[0370] In the embodiments of the present invention, the NLB4 / NLB14 single-target dual-epitope STAR-T cells constructed by antibodies NLB4 (amino acid sequence SEQ ID NO: 7) and NLB14 (amino acid sequence SEQ ID NO: 8) were used as an example for subsequent verification.

[0371] Among them, the STAR structure used in Example 3-8 is: NLB4 is connected to the N-terminus of the β chain constant domain (SEQ ID NO: 20), NLB14 is connected to the N-terminus of the α chain constant domain (SEQ ID NO: 18), and OX40 is connected to the C-terminus of the α chain constant domain (SEQ ID NO: 18) via a (G4S)3 linker.

[0372] It should be understood that any STAR-T cell that specifically binds to LILRB4 is within the scope of protection of the present invention. It should also be understood that the STAR targeting LILRB4 of the present invention may also include structures that recognize other antigens.

[0373] Example 3. Construction of tumor target cells

[0374] In order to detect the killing effect of LILRB4 STAR-T on tumor cells, this example takes myeloma as an example and detects the expression of LILRB4 antigen in various myeloma cell lines (including NCI-H929, MM.1S, and U266B1) by flow cytometry. Figure 2A and 2B As shown, LILRB4 antigen expression was higher in NCI-H929, MM.1S, and U266B1.

[0375] Example 4. In vitro efficacy of LILRB4 STAR-T (flow cytometry)

[0376] In this example, NCI-H929 was selected as a target cell, and flow cytometry was used to detect the killing efficiency of LILRB4 STAR-T cells on the target cells.

[0377] 1. Co-culture of T cells and target cells in vitro

[0378] The target cells were pretreated with 5 μM fluorescent dye CFSE (hydroxyfluorescein diacetate succinimidyl ester) and stained for 20 min at 37°C in the dark. The target cells were plated at a density of 5E5 / well in a 24-well plate, and the corresponding number of STAR-T cells were added to the target cells according to the ratio of STAR-positive T cells to target cells of 1:1 and 3:1.

[0379] 2. Detection Method

[0380] After STAR-T cells and target cells were co-cultured for 24 hours and 48 hours, the co-cultured cells were collected and pre-treated with Zombieviolet Fixable Viability Kit (1:200 dilution) to mark dead cells. They were stained for 30 minutes in the dark, washed twice with PBS, and fixed with cell fixative. The killing efficiency of STAR-T cells on target cells was detected by flow cytometry. The killing efficiency was calculated by CFSE + Zombie violet% in cells.

[0381] 3. Experimental results

[0382] like Figure 3 As shown, LILRB4 STAR-T cells showed higher killing efficiency against NCI-H929.

[0383] Example 5. In vitro killing evaluation of LILRB4 STAR-T

[0384] In this example, NCI-H929 and U266B1 were selected as target cells, and the killing efficiency of LILRB4 STAR-T cells on NCI-H929 target cell line was detected by luciferase method, and the killing efficiency of LILRB4 STAR-T cells on U266B1 target cells was detected by flow cytometry. The specific lysis (%) and the concentrations (pg / mL) of induced IFN-γ, IL-2 and TNF-α after treatment with the control group (Mock-T) and the experimental group (LILRB4-STAR) for a certain period of time (24h or 48h) were tested on NCI-H929 and U266B1 cells at different E:T ratios.

[0385] 1. Co-culture of T cells and target cells in vitro

[0386] The target cells NCI-H929 were plated at a density of 4E5 / well in a 24-well plate. A corresponding number of STAR-T cells were added to the target cells at a ratio of 0.3:1 and 1:1 between STAR-positive T cells and target cells. The killing efficiency of STAR-T cells on target cells and the level of cytokine release were detected after 24 hours of culture.

[0387] Target cells U266B1 were plated at a density of 4E5 / well in a 24-well plate. A corresponding number of STAR-T cells were added to the target cells at a ratio of 0.3:1 and 1:1 between STAR-positive T cells and target cells. The killing efficiency of STAR-T cells on target cells and the level of cytokine release were detected after 24 and 48 hours of culture.

[0388] 2. Detection Method

[0389] 1) Killing efficiency detection: luciferase method

[0390] After STAR-T was co-cultured with NCI-H929 expressing luciferase, the cell suspension was gently mixed, 150 μL of cell suspension was pipetted into two replicates of 96 white bottom opaque detection well plates, 70 μL Bio-Lite Luciferase Assay System (Vazyme) was added to each well, and the well was shaken at 300 rpm in the dark for 10 minutes at room temperature. The chemiluminescence value was detected using a multifunctional microplate reader, and the gain value was fixed at 100. Cell killing calculation: killing efficiency = 100% - (effect cell - target cell well value) / (control cell - target cell well value).

[0391] 2) Killing efficiency detection: flow cytometry

[0392] After STAR-T cells and U266B1 cells were co-cultured for 24 hours and 48 hours, the killing efficiency of STAR-T cells on target cells was detected by flow cytometry. The flow cytometry detection method was the same as that in Example 3.

[0393] 3) Cytokine release level detection

[0394] After T cells and target cells were co-cultured, the supernatant was collected. The TNF-α, IFN-γ, and IL-2 ELISA kits used Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA (Cat. No. 88-7025, 88-7346, and 88-7316, respectively). The specific steps are as follows: dilute 10× Coating Buffer to 1× with ddH2O, add coating antibody (250×), mix well, and add to a 96-well plate (for ELISA), 100 μL / well. After sealing with plastic wrap, place at 4°C overnight, wash 3 times with 1×PBST (also known as Wash Buffer, 1×PBS with 0.05% Tween 20), 260μL / well each time, dilute 5×ELISA / ELISPOT Diluent to 1× with ddH2O, add to 96-well plate, 200μL / well, and let stand at room temperature for 1h. Wash once with PBST, standard dilution (range: 2-250, 4-500, 4-500), and dilute the sample 20-50 times with 1×Diluent. Add samples and standard curves, 100 μL per well, duplicate wells, incubate at room temperature for 2 hours, wash three times with PBST, add detection antibody diluted with 1×Diluent, incubate for 1 hour, wash three times with PBST, then add HRP diluted with 1×Diluent, incubate for 30 minutes, wash six times, add TMB for color development, the color development time should not exceed 15 minutes, add 2N H2SO4 for termination, and detect light absorption at 450nm.

[0395] 3. Experimental results

[0396] like Figure 4A and 4B As shown, LILRB4 STAR-T has significant in vitro killing effect and higher cytokine release levels on both NCI-H929 and U266B1 cells.

[0397] Example 6. In vivo efficacy of LILRB4 STAR-T on MM

[0398] Taking multiple myeloma as an example, the in vivo efficacy of the experimental group (LILRB4 STAR-T) and the control group (Mock-T) was measured under the following experimental conditions, including the determination of the tumor growth volume curve ( Figure 5A ), mouse survival curve ( Figure 5B ), mouse weight change curve ( Figure 5C ) and the control group at the end point of tumor imaging ( Figure 5D ):

[0399] Group: YTS104 (i.e. LILRB4 STAR-T), Mock-T;

[0400] NPG female mice, 9 / group;

[0401] Target cells: NCI-H929; 1E7 / cell; subcutaneous inoculation;

[0402] T cell inoculation: 1E7 / mouse; tumor reaches 100 mm 3 .

[0403] During the experiment, mouse No. 48 in the control group died, and the tumor volume of other mice in the group grew to 2000 mm. 3 Until the end of the experiment, there was no tumor recurrence in the experimental group.

[0404] like Figures 5A-5D As shown, the tumor volume of the control group mice began to increase exponentially around 10-15 days, while the tumor volume of the experimental group mice basically remained at 0; the median survival of the control group mice was only 22 days, while all the experimental group mice survived on day 25; during the 22 days of the experiment, there was no significant difference in the weight of the mice in the control and experimental groups. The results show that LILRB4STAR-T can effectively treat tumors (especially multiple myeloma) in mice.

[0405] Example 7. Induced expression and functional detection of MDSC

[0406] The experimental process for verifying the function of LILRB4 STAR-T in targeting MDSC is as follows Figure 6 Specifically, peripheral blood mononuclear cells (PBMC) were subjected to CD14 + CD14 + The resulting cells were treated with different cell growth and regulatory factors (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF) / interleukin-6 (IL-6) / prostaglandin E2 (PGE2)) for 7 days, followed by M-MDSC isolation to obtain CD33 + CD11b + The obtained MDSC cells were used for LILRB4 expression detection and human MDSC / T inhibition assay.

[0407] 1) Validation of LILRB4 expression on MDSCs

[0408] CD33 was compared by flow cytometry + CD11b +The expression of LILRB4 and CLL1 and their subtypes in MDSC and MV4-11 cells was compared. In addition, the expression levels of LILRB4 and CLL1 in the two cells were compared by the mean fluorescence intensity ratio (MFIR). Fig. 7A As shown in the upper left panel of the figure, in MDSC and MV4-11 cells, LILRB4 expression is significantly higher than that of its subtypes, making it easy to separate. Figure 7B As shown, the expression levels of LILRB4 in MDSCs and MV4-11 cells were significantly higher than that of CLL1.

[0409] 2) Human MDSC / T inhibition assay

[0410] Fluorescence flow cytometry was used to analyze the total cells in the blood, the total single cells in the blood, and the CD8 + Single cell and blood CTR + Furthermore, the degree of MDSC inhibition on T cells at different MDSC:T ratios and the effect on CD8 + Effect of IFN-γ concentration on T cell proliferation and induction. Fig. 8A As shown, in all cells, all single cells, CD8 + Single Cell and CTR + MDSCs are present in all cells. Figure 8B As shown in the figure, as the proportion of MDSC increases, the amount of T cells decreases significantly, indicating that MDSC has a significant inhibitory effect on T cells. Figure 8C As shown, as the proportion of MDSCs increased, the CD8+ T cell proliferation % and the induced IFN-γ concentration were significantly reduced, indicating that T cells were significantly inhibited.

[0411] Example 8. Experiment on killing of MDSC by LILRB4 STAR-T

[0412] Fluorescence flow cytometry was used to analyze the total cells in the blood, total single cells in the blood, and CD8 + Single cell and blood CTR + The killing of MDSC in cells was determined. In addition, the effects of different E:T ratios on the lysis of MDSC cells after treatment for a period of time (24h or 48h) in the control group and the experimental group were compared, as well as the effects on the induced IFN-γ concentration and IL-10 concentration.

[0413] 1. Co-culture of T cells and target cells in vitro

[0414] Collect MDSC cells and centrifuge at 1200 rpm for 5 minutes at room temperature, remove the supernatant, add PBS and adjust the cell concentration to 1×10 6 Cells / ml, then diluted CellTrace FarRed dye (1:1000 dilution) was added and the cells were gently resuspended. The cells were incubated at 37°C in the dark for 20-30 minutes. Then PBS was added to a final volume of 10 ml, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded and the cells were washed once with preheated culture medium, the cells were counted and the cell concentration was adjusted, and 5E4 / well were plated. The control target cell MV4-11 was plated at the same density. STAR-T cells and Mock-T cells were co-cultured with target cells at an effector-target ratio of 1:1 and 5:1 for 24 hours.

[0415] 2. Detection Method

[0416] 1) Killing efficiency detection

[0417] After STAR-T cells and target cells were co-cultured for 24 hours, the co-cultured cells were collected and pre-treated with PI staining to mark dead cells. The cells were stained in the dark for 30 minutes, washed twice with PBS, and fixed with cell fixative. The killing efficiency of STAR-T cells on target cells was detected by flow cytometry. The killing efficiency was calculated by CFSE + PI% in cells.

[0418] 2) Cytokine release level detection

[0419] After the T cells were co-cultured with the target cells, the supernatant was collected and the secretion levels of IFN-γ and IL-10 were detected by ELISA. The detection method was the same as that in Example 4.

[0420] 3. Experimental results

[0421] like Fig.9A As shown in Figure 2, the killing effect of the experimental group on MDSC was significantly stronger than that of the control group. Fig. 9B and Figure 9C-9F As shown, under all E:T ratios and treatment time conditions, the experimental group showed significantly higher MDSC lysis% and induced IFN-γ and IL-10 concentrations relative to the control group. These results demonstrate that LILRB4 STAR-T exhibits a significant killing effect on MDSC cells with immunosuppressive activity, thereby further improving the drug's tumor killing ability.

[0422] Example 9. Optimization of the connection mode of the co-stimulatory domain of the LILRB4 STAR-T structure

[0423] In order to enhance T cell toxicity and T cell proliferation persistence, the present invention introduces the human co-stimulatory molecule OX40 sequence into the C-terminus of the αβ chain constant domain of STAR, respectively, to explore its effect on the dual-epitope targeted LILRB4 STAR-T cell function. OX40 can be connected in series to the C-terminus of the TCR α chain, or the C-terminus of the β chain, or the C-terminus of the α chain and the β chain (co-STAR). In addition, the co-stimulatory molecule OX40 domain can be directly connected to the C-terminus of the TCR constant domain or connected through G4S / (G4S)3. Fig.10 LILRB4 STAR with different OX40-linked optimized structures. Including:

[0424] 1) NLB4 / NLB14-STAR-ab(G4S)3OX40, referred to as STAR-ab(G4S)3OX40 or ab(G4S)3OX40 (its amino acid sequence is shown in SEQ ID NO: 129): its structure is: NLB4 is connected to the N-terminus of the β chain constant domain (SEQ ID NO: 20), OX40 is connected to the C-terminus of the β chain constant domain (SEQ ID NO: 20) through a (G4S)3 linker, NLB14 is connected to the N-terminus of the α chain constant domain (SEQ ID NO: 18), and OX40 is connected to the C-terminus of the α chain constant domain (SEQ ID NO: 18) through a (G4S)3 linker.

[0425] 2) NLB4 / NLB14-STAR-abOX40, referred to as STAR-abOX40 or abOX40 (its amino acid sequence is shown in SEQ ID NO: 128): its structure is: NLB4 is connected to the N-terminus of the β chain constant domain (SEQ ID NO: 20), OX40 is directly connected to the C-terminus of the β chain constant domain (SEQ ID NO: 20), NLB14 is connected to the N-terminus of the α chain constant domain (SEQ ID NO: 18), and OX40 is directly connected to the C-terminus of the α chain constant domain (SEQ ID NO: 18).

[0426] 3) NLB4 / NLB14-STAR-a(G4S)3OX40, referred to as STAR-a(G4S)3OX40 or a(G4S)3OX40 (its amino acid sequence is shown in SEQ ID NO: 108): its structure is: NLB4 is connected to the N-terminus of the β chain constant domain (SEQ ID NO: 20), NLB14 is connected to the N-terminus of the α chain constant domain (SEQ ID NO: 18), and OX40 is connected to the C-terminus of the α chain constant domain (SEQ ID NO: 18) via a (G4S)3 linker.

[0427] Subsequent validation was performed using LILRB4 STAR-T cells with the three OX40-linked structures described above. The control group included: T cells not infected with STAR (MOCK-T).

[0428] 1. Expression on membrane

[0429] For STARs targeting LILRB4 with different OX40 connection strategies, primary T cells were infected with lentivirus, and their membrane expression was detected by flow cytometry. RFP fluorescent protein was co-expressed at the end of STAR through IRES, and the membrane expression of LILRB4 Dual STAR was determined by anti-mouse TCRβ chain antibody and RFP expression efficiency. The experimental results are shown in Fig.11 As shown, different OX40-linked structures of LILRB4 STAR-T did not affect LILRB4 protein binding.

[0430] 2. In vitro killing efficiency of three OX40-linked structures of LILRB4 STAR-T

[0431] 1) Continuous killing in vitro

[0432] NCI-H929-luc target cells and MV4-11-LUC target cells expressing LILRB4 targets were plated in a 24-well plate at a density of 1E6 / well. LILRB4 STAR-T cells with different OX40 connection structures were added to the target cells at a density of 5E5 / well of STAR-T cells with a co-culture volume of 1 mL. After 24 hours of co-culture, the co-culture cell suspension was taken. The co-culture was continued and the co-culture cell suspension was removed every 24 hours. A total of 7 co-culture cell suspensions were taken. The LUC luminescence value was detected using a luciferase reporter gene detection kit, and the killing efficiency of STAR-T cells on target cells was calculated as follows: Fig. 12A The results showed that STAR-ab(G4S)3OX40 and STAR-abOX40 had comparable continuous killing abilities against target cells, and both were superior to the continuous killing ability of STAR-a(G4S)3OX40. In other words, the continuous killing ability of STAR-T linked to two OX40s was superior to that of STAR-T linked to one OX40.

[0433] 2) Cytokine secretion detection

[0434] During the activation of T cells, a large number of cytokines are released to help T cells kill target cells or promote the expansion of T cells themselves. Common ones include IFN-γ, IL-2 and TNF-α. After the above experiment, T cells are stimulated with target cells or antigens, and then collected, centrifuged and the supernatant is taken. The secretion levels of IFN-γ, IL-2 and TNF-α are detected by ELISA. The IFN-γ ELISA kit uses Human IL-2 Uncoated ELISA, Human TNF-α Uncoated ELISA, and Human IFN-γ Uncoated ELISA (Cat. No. 88-7025, 88-7346, 88-7316, respectively). The specific steps are: dilute 10X Coating Buffer to 1X with ddH2O, add coating antibody (250X), mix well and add to 96-well plate (ELISA only), 100μL / well. After sealing with plastic wrap, place at 4°C overnight, wash 3 times with 1X PBST (also known as Wash Buffer, 1X PBS with 0.05% Tween20), 260μL / well each time, dilute 5XELISA / ELISPOT Diluent to 1X with ddH2O, add to 96-well plate, 200μL / well, and let stand at room temperature for 1h. Wash once with PBST, standard dilution (range: 2-250, 4-500, 4-500), and dilute the sample 20-50 times with 1×Diluent. Add samples and markers, 100 μL per well, duplicate wells, incubate at room temperature for 2 hours, wash three times with PBST, add detection antibody diluted with 1×Diluent, incubate for 1 hour, wash three times with PBST, then add HRP diluted with 1xDiluent, incubate for 30 minutes, wash six times, add TMB for color development, the color development time should not exceed 15 minutes, add 2N H2SO4 for termination, and detect light absorption at 450nm.

[0435] like Fig. 12B As shown, when T cells were co-cultured with target cells NCI-H929-luc, they could significantly stimulate T cells to secrete IFN-γ. Among them, in LILRB4-STAR-T cells, the cytokine secretion concentration of STAR-ab(G4S)3OX40 was the highest, followed by STAR-abOX40, and the cytokine secretion concentration of STAR-a(G4S)3OX40 of single OX40 was lower.

[0436] 3. In vivo efficacy evaluation

[0437] In order to verify the killing effect of STAR-T cells in vivo and potential safety issues, a MV4-11-LUC target cell mixed model was constructed. LILRB4-STAR-T cell groups: STAR-ab(G4S)3OX40, STAR-abOX40 and STAR-a(G4S)3OX40 and T cells not infected with STAR (MOCK-T) were selected to study the effects of in vivo efficacy and in vivo proliferation. Fluorescently labeled MV4-11-LUC target cells were injected into the tail vein of female NPG mice aged 6 to 8 weeks at a dose of 2E6 / mouse. On the 8th day after tumor formation, the above STAR-T cells were infused into the tail vein at a dose of 4E6 / mouse. Then, the tumor growth, tumor fluorescence value and body weight changes were detected by luciferin substrate catalytic luminescence method on days -1, 5, 8, 14, 20, 27, 33 and 41. Fig.13A , Fig. 13B and Fig. 13C The results showed that STAR-abOX40 had the best inhibitory effect on target cell tumors, followed by STAR-ab(G4S)3OX40, and the weight of mice did not decrease significantly, and the safety was relatively good. The in vivo efficacy of STAR-T connected to two OX40s was better than that of STAR-T connected to one OX40.

[0438] Example 10. Clinical treatment of patients with relapsed or refractory multiple myeloma

[0439] 1. Study Population

[0440] 1. Selection criteria:

[0441] Subjects must meet all of the following inclusion criteria to be included in this study.

[0442] 1) Aged ≥18 years and ≤75 years, regardless of gender;

[0443] 2) Patients diagnosed with relapsed / refractory multiple myeloma according to the diagnostic criteria of multiple myeloma of the International Myeloma Working Group (IMWG 2014);

[0444] 3) positive expression of LILRB4 in bone marrow specimens;

[0445] 4) The presence of one or more measurable lesions at screening, defined as any of the following: 1) serum M protein ≥ 0.5 g / dL (≥ 5 g / L), 2) urine M protein level ≥ 200 mg / 24 hours; 3) serum free light chain (sFLC) ≥ 100 mg / L and abnormal serum κ / λ free light chain ratio (< 0.26 or > 1.65);

[0446] 5) Good organ function:

[0447] (1) Routine blood test: absolute neutrophil count (ANC) ≥ 1.0 × 109 / L; absolute lymphocyte count (ALC)

[0448] ≥0.3×109 / L; hemoglobin (Hb) ≥60g / L; platelet count (PLT) ≥50×109 / L, the proportion of plasma cells in bone marrow nuclei

[0449] In <50% of subjects, platelet count was ≥75 × 109 / L;

[0450] (2) Liver function: total bilirubin ≤1.5×upper limit of normal (ULN), aspartate aminotransferase (AST), alanine aminotransferase (ALT)

[0451] ≤2.5×ULN;

[0452] (3) Renal function: creatinine clearance (Cockcroft-Gault formula) ≥40 mL / min;

[0453] (4) Coagulation function: International normalized ratio (INR) ≤ 1.5 × ULN, and activated partial prothrombin time (APTT)

[0454] ≤1.5×ULN;

[0455] (5) Cardiac function: left ventricular ejection fraction (LVEF) ≥50%;

[0456] (6) Pulmonary function: defined as ≤ grade 1 dyspnea and blood oxygen saturation ≥ 92%.

[0457] 6) ECOG score ≤ 1 point. The ECOG score is a scoring system used to assess the daily activities and self-care abilities of cancer patients;

[0458] 7) Expected survival period ≥ 12 weeks;

[0459] 8) Female subjects of childbearing age or male subjects whose partners are women of childbearing age agree to use effective contraceptive methods throughout the trial and within 12 months after cell infusion;

[0460] 9) The subjects voluntarily participated in the study, signed the informed consent, had good compliance, and cooperated with the follow-up.

[0461] 2. Exclusion criteria

[0462] Subjects who meet any of the following criteria cannot be enrolled in this study.

[0463] 1) Allergic history to any component of the cell product;

[0464] 2) Patients who have used CAR-T cell therapy or any other gene transduction therapeutic products within 3 months of signing the informed consent form, except for those whose CAR-T cannot be detected or whose CAR-T is below the detection limit;

[0465] 3) Patients with plasma cell leukemia, Waldenstrom's macroglobulinemia, POEMS syndrome, or primary light chain amyloidosis;

[0466] 4) Those who have had any of the following cardiovascular and cerebrovascular diseases within 6 months before screening:

[0467] (1) Congestive heart failure (New York Heart Association [NYHA] ≥ III), congenital long QT syndrome, left anterior hemiblock (bifascicular block), asymptomatic right bundle branch block allowed; myocardial infarction, unstable angina, coronary angioplasty, stent implantation, coronary artery / peripheral artery bypass grafting;

[0468] (2) cerebrovascular accident (CVA), transient ischemic attack;

[0469] (3) Severe arrhythmias requiring treatment (such as sustained ventricular tachycardia, ventricular fibrillation, torsade de pointes, etc.);

[0470] (4) suffering from uncontrolled hypertension (systolic blood pressure greater than 160 mmHg and / or diastolic blood pressure greater than 100 mmHg), a history of hypertensive crisis or hypertensive encephalopathy;

[0471] (5) Pulmonary embolism within 6 months before screening, or a history of deep vein thrombosis in the lower extremities, or active lung disease and / or pneumonia, or a history of interstitial lung disease;

[0472] (6) Hepatitis B surface antigen (HBsAg) positive; hepatitis B core antibody (HBcAb) positive and peripheral blood HBV DNA positive; hepatitis C virus (HCV) antibody positive and HCV RNA positive; syphilis Treponema pallidum antibody positive;

[0473] (7) Patients with known systemic lupus erythematosus, active or uncontrolled autoimmune diseases (such as Crohns disease, rheumatoid arthritis, autoimmune hemolytic anemia, etc.), primary or secondary immunodeficiency (such as HIV infection or severe infectious diseases, etc.);

[0474] (8) Patients with other uncured malignant tumors that are unstable in control and affect the long-term survival of the subjects, excluding cured cervical carcinoma in situ, non-invasive basal cell or squamous cell skin cancer, or other localized prostate cancer that has been radically treated, ductal carcinoma in situ after radical surgery and has not recurred for at least 5 years;

[0475] (9) Patients currently suffering from or with a history of central nervous system diseases, such as epileptic seizures, stroke, severe brain damage, aphasia, paralysis, dementia, Parkinson's disease, mental illness, etc.;

[0476] (10) central nervous system (CNS) involvement or symptoms of CNS involvement (including cranial neuropathy and extensive lesions or spinal cord compression);

[0477] (11) Patients who had received solid organ transplantation or allogeneic hematopoietic stem cell transplantation (allo-HSCT) within 6 months before screening or autologous stem cell transplantation within 3 months before apheresis;

[0478] (12) Patients with acute or chronic graft-versus-host disease (GVHD) during screening;

[0479] (13) The following anti-MM treatments have been used within the specified time before apheresis:

[0480] a. Use of any immunosuppressant or radiotherapy within 2 weeks before apheresis;

[0481] b. Received cytotoxic or proteasome inhibitors or small molecule targeted therapy within 2 weeks or 3 half-lives (whichever is shorter) before apheresis;

[0482] c. Received any monoclonal antibody or other macromolecular therapy within 4 weeks or 3 half-lives (whichever is shorter) before apheresis;

[0483] d. Received immunomodulators within 1 week;

[0484] (14) History of live vaccination within 4 weeks before signing the ICF;

[0485] (15) Those with mental illness or a history of substance abuse;

[0486] (16) Pregnancy or lactation;

[0487] (17) If participating in other interventional clinical studies before apheresis, the drug elution requirements before apheresis must be met;

[0488] (18) The researcher believes that there are other factors that make the subject unsuitable for inclusion or affect the subject's participation in or completion of the study.

[0489] 2. Experimental Design

[0490] This study is a dose-escalating, open-label Phase I clinical trial conducted in patients with relapsed / refractory multiple myeloma. The purpose is to evaluate the preliminary efficacy, pharmacokinetic (PK) / pharmacodynamic (PD) characteristics and immunogenicity of the STAR-T cells of this application in subjects with relapsed / refractory multiple myeloma.

[0491] The study is divided into two phases: dose escalation and dose expansion. The dose escalation is a 3+3 design, and the dose expansion plan selects 1-2 doses for expansion to further evaluate the safety, efficacy and PK / PD characteristics of STAR-T cells.

[0492] Dose escalation stage: The 3+3 dose escalation design principle is adopted, and the dose groups are set as follows, with dose group 1 as the starting dose:

[0493] Dose group 1: 1.0×10 6 STAR + T cells / kg

[0494] Dose group 2: 3.0×10 6 STAR + T cells / kg

[0495] Dose group 3: 6.0×10 6 STAR + T cells / kg

[0496] Dose group 4: 1.0×10 7 STAR + T cells / kg

[0497] The design principle of dose escalation: If no dose-limiting toxicity (DLT) occurs in 3 subjects in a dose group, the dose will be increased to the next group. If 1 of 3 subjects in a dose group has DLT, 3 additional subjects will be added to the dose group and infused with the same dose and method; if no DLT occurs in the additional 3 subjects, the dose will be increased to the next group. If 2 of 3 subjects in a dose group or 1 of the additional 3 subjects has DLT, the dose escalation will be stopped and the previous dose will be the maximum tolerated dose (MTD).

[0498] While ensuring the safety of the subjects, dose groups 1 and 2 can adopt an accelerated titration strategy to avoid excessive exposure to low-dose levels. If a subject enrolled in the accelerated titration dose group develops DLT, the regimen will be directly switched to 3+3 ramp-up.

[0499] If in dose group 4 (1.0×10 7 STAR + T cells / kg), if the clinically recommended dose has not yet been found, the SRC may decide to continue to increase the dose after discussion (the planned increase in dose is 2.0×10 7 STAR + T cells / kg).

[0500] Dose escalation for the same subject is not allowed. In principle, the last subject in the previous dose group should be observed for 4 weeks after receiving cell reinfusion, and the first subject in the next dose group is allowed to receive cell reinfusion.

[0501] Dose expansion phase: Based on the cumulative safety, preliminary efficacy, and possible PK and PD characteristics of STAR-T cells in the dose escalation phase, it is planned to select 1-2 doses for dose expansion to further evaluate the safety and clinical efficacy of STAR-T cell therapy. The research process includes the screening period (3 weeks before apheresis), apheresis and baseline assessment period (D-13 to D-6), lympholysis chemotherapy pretreatment and rest observation period (D-5 to D-1), cell reinfusion and DLT observation period (D0 to D28 after reinfusion) and follow-up period (D29 to 24 months after reinfusion).

[0502] Routine laboratory tests (blood routine, blood biochemistry, coagulation function, etc.) were performed on the day of cell infusion and on the 4th, 7th, 10th, 14th, 21st, and 28th days after infusion to detect STAR-T ratio, cytokines, lymphocyte subsets (if conditions permit), etc., and DLT observation and evaluation were performed. Adverse events (AEs) and serious adverse events (SAEs) were recorded during treatment, with a focus on cytokine release syndrome (CRS)

[0503] and immune cell-associated neurotoxicity (ICANS).

[0504] After the 28-day DLT observation period, the follow-up period begins, and the follow-up lasts until 24 months after cell infusion. During the follow-up period, the disease status, survival, and expansion, persistence, and safety of the subjects' STAR-T cells need to be regularly monitored and evaluated. Starting from the 28th day, the follow-up visit points are 2, 3, 4, 5, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, and 24 months.

[0505] 3. Dosage Design

[0506] This study proposes 1.0×10 6 The starting dose was 1.0×10 7 STAR-T cell / kg was used as the maximum escalating dose, and the dose group was set at 1.0×10 6 STAR-T cells / kg, 3.0×10 6 STAR-T cells / kg, 6.0×10 6 STAR-T cells / kg, 1.0×10 7 STAR-T cells / kg.

[0507] The dose expansion phase will select 1-2 doses for dose expansion based on the accumulated safety, preliminary efficacy, and possible PK and PD characteristics of the dose escalation phase to further evaluate the safety and clinical efficacy of STAR-T cell therapy.

[0508] IV. Definition of DLT

[0509] DLT is defined as any of the following STAR-T cell-related adverse events that occur within 28 days after the subject receives cell reinfusion in the dose escalation trial. Cytokine release syndrome (CRS) and ICANS are graded using the ASTCT 2018 criteria, and the remaining AEs will be evaluated using the CTCAE version 5.0 criteria, including:

[0510] 1. Grade 4 or 5 CRS and ICANS caused by STAR-T cells;

[0511] 2. Grade 3 CRS and ICANS caused by STAR-T cells and not relieved to below grade 2 within 7 days after onset;

[0512] 3. Grade 4 hematological AEs caused by STAR-T cells fail to recover to Grade 2 or baseline (whichever is more severe) within 28 days after cell transfusion; 4. Grade 3 or higher non-hematological toxicities caused by STAR-T cells fail to recover to Grade 2 or baseline (whichever is more severe) within 28 days after cell transfusion.

[0513] whichever is more serious);

[0514] 5. Any unexpected toxicity that requires termination of treatment at the discretion of the investigator or collaborating unit.

[0515] The following anticipated events arising from cell therapy will not be considered DLTs:

[0516] Hematological toxicity: such as grade 4 lymphocytopenia;

[0517] Non-hematologic toxicity: Fever of any grade, including febrile neutropenia. Grade 4 infusion reaction, reversible to ≤ 2 within 8 hours

[0518] Grade 3 or 4 tumor lysis syndrome (TLS) ≤ 2 weeks; Grade 3 bone pain caused by cell proliferation in the bone marrow cavity ≤ 2 weeks; Grade 3 or below diarrhea, nausea, vomiting, fatigue, chills lasting < 72 hours; Grade 3 or 4 alopecia; Grade 3 or 4 abnormal laboratory indicators with no clinical significance; Note: For safety reasons, if the subject develops the above DLT symptoms, the subject may be given corresponding symptomatic supportive treatment.

[0519] 5. Single sampling

[0520] According to the relevant operation guidelines of the single collection room of the research center, the total number of mononuclear cells collected should not be less than 3×10 9 Cell or volume at 90-

[0521] 120mL. Before apheresis, the subject needs to undergo routine blood tests and other related tests, and must meet the apheresis requirements of the research center.

[0522] The cells are stored at 2-8°C and transported to the cell preparation center by a qualified logistics company for STAR-T cell preparation.

[0523] 6. Chemotherapy Pretreatment

[0524] Before the administration of lymph node clearance medication, the subjects must complete relevant examinations according to the visit plan, and the examination results must be assessed by the researchers as being suitable for lymph node clearance chemotherapy. There must be no uncontrolled active infection before chemotherapy, and the body temperature must be ≤38°C within 48 hours before medication (excluding tumor-related fever).

[0525] On D-5, D-4 and D-3 before cell infusion, the subjects need to receive fludarabine + cyclophosphamide (FC regimen) chemotherapy pretreatment. The specific chemotherapy regimen is as follows:

[0526] (1) Fludarabine 25-30 mg / m 2 / d, intravenous drip × 3 days (maximum dose not exceeding 50 mg)

[0527] (2) Cyclophosphamide 250-300 mg / m 2 / d, intravenous drip × 3 days

[0528] Note: For the chemotherapy pretreatment dosage of the subjects, the researchers can adjust it as appropriate after comprehensively considering the actual tolerance of the subjects.

[0529] If serum creatinine or creatinine clearance or glomerular filtration rate is abnormal on the day of chemotherapy pretreatment (before drug administration), that is, serum creatinine >1.5 times ULN, or creatinine clearance (Cockcroft-Gault formula) ≤30mL / min or radioisotope glomerular filtration rate (GFR)

[0530] ≤30mL / min / 1.73m 2 , chemotherapy should be temporarily withheld. If serum creatinine > 2 mg / dL for more than 3 days, the subject will be excluded from the study.

[0531] VII. Clinical Evaluation

[0532] 1. Safety evaluation

[0533] Safety evaluation included physical examination, vital sign recording, clinical laboratory tests, neurological examination and rating, electrocardiogram, fingertip oxygen saturation, and AE / SAE reports collected at each visit.

[0534] The researchers will evaluate whether the test results of each visit of the subjects exceed the normal reference value range and determine whether they are clinically significant. This judgment should be based on the characteristics of the laboratory test and the degree of abnormality. Among them, the researchers believe that the abnormalities that are clinically significant and / or related to STAR-T cell infusion should be taken seriously and continue to be followed. If there is any objection to the study results, the abnormal results can be repeated to rule out errors in the results caused by the laboratory or sample collection.

[0535] 2.PK / PD evaluation

[0536] In principle, sample testing and analysis should be carried out in accordance with the test procedures and schedule.

[0537] The main research sample is peripheral blood, and the main detection indicators are the proportion of STAR-T in peripheral blood, the number of STAR gene copies and the level of cytokines. Flow cytometry will be used to detect the number and proportion of STAR-T cells in peripheral blood, and the phenotype of immune cells can be evaluated if conditions permit. The Q-PCR method will be used to detect the number of STAR gene copies in peripheral blood to simultaneously monitor the expansion of STAR-T cells.

[0538] Serum cytokines will be used as markers of immune activation to analyze the potential correlation between cytokine production and efficacy, as well as the severity of CRS. It is planned to use ELISA or flow cytometry to detect the content of cytokines in serum.

[0539] 3. Effectiveness evaluation

[0540] The criteria for MM efficacy evaluation will be determined using the International Myeloma Working Group (IMWG) efficacy criteria, which are divided into strict complete remission (sCR), complete remission (CR), very good partial remission (VGPR), partial remission (PR), minimal remission (MR), stable disease (SD), and progressive disease (PD).

[0541] For subjects whose efficacy is evaluated as CR and sCR, MRD will also be evaluated. The second-generation flow cytometry (NGF) is used to detect the subjects' bone marrow samples to monitor MRD. MRD efficacy is divided into MRD negative and MRD positive.

[0542] During the trial, serum and urine protein (serum free light chains, serum and urine protein electrophoresis and immunofixation, 24-hour urine protein quantification, immunoglobulin), serum calcium, etc. will be measured according to the time set in this study; imaging examinations can choose CT, X-ray or MRI to evaluate systemic bone lesions, and perform imaging examinations on extramedullary plasmacytomas (if the subject has extramedullary lesions at baseline); bone marrow puncture / biopsy will be performed at baseline, PD, CR / sCR, after achieving CR / sCR, and at exit visits. Imaging examinations for relapse after MRD negativity are performed using whole-body PET-CT.

[0543] 8. Clinical Results

[0544] Since the project started, a total of several subjects have been enrolled. Taking the recent 4 subjects as an example, one of them has achieved sustained remission for the ninth month and is being followed up; one of them has achieved two months of disease stability SD, one of them has achieved three months of disease stability SD, and one of them was discharged from the group due to disease progression on the 28th day. The specific situation is as follows:

[0545] Subject number: 02. Gender / age: female / 72; Diagnosis: relapsed MM; Number of treatment lines: 5; Dose group: 1E6 / kg;

[0546] STAR product: STAR-a(G4S)3OX40; bridging therapy: none; reinfusion after lympholysis chemotherapy; CRS: none; tumor assessment: VGPR / M9; status: sustained remission in the ninth month, ongoing follow-up. The subject's urine protein is shown in Table 10 below.

[0547] Table 10

[0548]

[0549]

[0550] Subject number: 03. Gender / age: female / 52; Current diagnosis: relapsed and refractory MM; Number of treatment lines: 4; Dose group: 3E6 / kg; STAR product: STAR-a(G4S)3OX40; Bridging therapy: None; Re-infusion after lympholysis chemotherapy; CRS: None; Tumor assessment: SD / M1, SD / M2, PD / M3;

[0551] The urine protein of the subject is shown in Table 11 below.

[0552] Table 11

[0553]

[0554] Subject number: 04. Gender / age: male / 60; Current diagnosis: relapsed and refractory MM; Number of treatment lines: 6; Dose group: 6E6 / kg; STAR product: STAR-abOX40; Bridging therapy: None; Re-infusion after lympholysis chemotherapy; CRS: None; Tumor assessment: PD / D14;

[0555] The urine protein of the subject is shown in Table 12 below.

[0556] Table 12

[0557]

[0558]

[0559] Subject number: 07. Gender / age: female / 54; Current diagnosis: relapsed MM; Number of treatment lines: 3; Dose group: 1E7 / kg; STAR product: STAR-abOX40; Bridging therapy: None; Re-infusion after lympholysis chemotherapy; CRS: Grade 2 CRS; Tumor assessment: SD / D28, SD / M2, SD / M3;

[0560] The urine protein of the subject is shown in Table 13 below.

[0561] Table 13

[0562]

[0563] Example 11. Exploratory clinical study on the safety and efficacy of LILRB4 STAR-T cells in the treatment of relapsed / refractory acute myeloid leukemia / chronic myelomonocytic leukemia

[0564] 1. Study Population

[0565] 1. Selection criteria:

[0566] Subjects must meet all of the following inclusion criteria to be included in this study.

[0567] (1) Aged ≥18 years and ≤60 years, regardless of gender;

[0568] (2) Patients with acute myeloid leukemia or chronic myelomonocytic leukemia;

[0569] Patients with relapsed or refractory (R / R) AML who are diagnosed with acute myeloid leukemia (AML) according to the 2016 World Health Organization (WHO) criteria and meet any of the following criteria according to the "Guidelines for the Diagnosis and Treatment of Relapsed or Refractory Acute Myeloid Leukemia in China (2021 Edition)":

[0570] Patients who have not responded to two courses of standard treatment (including cytarabine and an anthracycline or anthraquinone drug); patients who have relapsed within 12 months after complete remission (CR) after consolidation and intensive treatment; patients who have relapsed after 12 months but have not responded to conventional treatment; patients who have relapsed 2 or more times; patients with persistent extramedullary leukemia. Note: Relapse definition: leukemia cells appear in peripheral blood again after CR or primitive cells in the bone marrow are greater than 5% (excluding other reasons such as bone marrow regeneration after consolidation chemotherapy) or leukemia cell infiltration appears outside the bone marrow.

[0571] According to the 2016 World Health Organization (WHO) classification criteria for hematopoietic and lymphoid tissue tumors and the "Chinese Guidelines for the Diagnosis and Treatment of Chronic Myelomonocytic Leukemia (2021 Edition)", chronic myelomonocytic leukemia is diagnosed, and relapse and refractory disease are defined as follows: failure to achieve remission or relapse after remission after at least 2 complete cycles of one treatment (such as demethylating drugs) or allogeneic hematopoietic stem cell transplantation (allo-HSCT).

[0572] (3) ECOG score 0-2;

[0573] (4) Bone marrow sample LILRB4 positive (flow cytometry);

[0574] (5) Good organ function: Liver function: total bilirubin ≤1.5×upper limit of normal (ULN), aspartate aminotransferase (AST), alanine aminotransferase (ALT) ≤3×ULN; Renal function: serum creatinine (Cr) ≤1.5×ULN or creatinine clearance (Cockcroft-Gault formula) ≥60 mL / min;

[0575] Cardiac function: left ventricular ejection fraction (LVEF) ≥ 50%; Pulmonary function: defined as ≤ grade 1 dyspnea and blood oxygen saturation > 92%.

[0576] (6) Female subjects of childbearing age or male subjects whose partners are women of childbearing age agree to use effective contraceptive methods throughout the trial and within 12 months after cell infusion;

[0577] (7) The subjects voluntarily participated in this study, signed the informed consent, showed good compliance, and cooperated with the follow-up.

[0578] 2. The donor must meet the following conditions:

[0579] (1) Aged 12 or above;

[0580] (2) HLA matching is at least half-matched;

[0581] (3) adequate venous access for peripheral blood leukocyte separation;

[0582] (4) Voluntarily sign a written informed consent.

[0583] 2. Exclusion criteria

[0584] 1. Subjects who meet any of the following criteria are not eligible for this study

[0585] (1) Patients who have used CAR-T cell therapy or any other gene transduction therapeutic products within 3 months of signing the informed consent, except for those whose CAR-T cannot be detected or whose CAR-T is below the detection limit; or patients who participated in interventional clinical studies of anti-tumor drugs within 4 weeks before signing the informed consent;

[0586] (2) Patients with a history of any of the following cardiovascular and cerebrovascular diseases within 6 months before screening: 1) congestive heart failure (New York Heart Association [NYHA] ≥ III), myocardial infarction, unstable angina, congenital long QT syndrome, left anterior hemiblock (bifascicular block), coronary angioplasty, stent implantation, coronary artery / peripheral artery bypass grafting, cerebrovascular accident (CVA), transient ischemic attack or pulmonary embolism, asymptomatic right bundle branch block is allowed; 2) severe arrhythmias requiring treatment (such as sustained ventricular tachycardia, ventricular fibrillation, torsade de pointes, etc.); 3) uncontrolled hypertension (systolic blood pressure greater than 160 mmHg and / or diastolic blood pressure greater than 100 mmHg), history of hypertensive crisis or hypertensive encephalopathy;

[0587] (3) Subjects who are positive for hepatitis B surface antigen (HBsAg), positive for hepatitis B core antibody (HBcAb) and whose peripheral blood HBV DNA copy number is higher than the detection limit, subjects who are positive for hepatitis C virus (HCV) antibody and whose HCV RNA copy number is higher than the detection limit, and subjects who are positive for Treponema pallidum antibody test;

[0588] (4) Patients with known systemic lupus erythematosus, active or uncontrolled autoimmune diseases (such as Crohns disease, rheumatoid arthritis, autoimmune hemolytic anemia, etc.), primary or secondary immunodeficiency (such as HIV infection or severe infectious diseases, etc.);

[0589] (5) Patients with other uncured malignant tumors that are unstable in control and affect the long-term survival of the subjects, excluding cured cervical carcinoma in situ, non-invasive basal cell or squamous cell skin cancer, or other localized prostate cancer that has been radically treated, ductal carcinoma in situ after radical surgery and has not recurred for at least 5 years;

[0590] (6) Patients currently suffering from or with a history of central nervous system diseases, such as epileptic seizures, stroke, severe brain damage, aphasia, paralysis, dementia, Parkinson's disease, mental illness, etc.;

[0591] (7) Patients with active central nervous system leukemia (CNSL) at screening;

[0592] (8) Those with a history of solid organ transplantation or hematopoietic stem cell transplantation (HSCT) within 6 months before screening;

[0593] (9) Patients with acute or chronic graft-versus-host disease (GVHD) during screening;

[0594] (10) Patients who have used any of the following drugs or treatments within the specified time before apheresis: any immunosuppressant within 2 weeks before apheresis; any chemotherapy or small molecule targeted therapy within 2 weeks or 3 half-lives before apheresis (whichever is shorter); any monoclonal antibody, antibody-drug conjugate (ADC), bispecific antibody within 4 weeks or 3 half-lives before apheresis (whichever is shorter);

[0595] Anti-macromolecule therapy;

[0596] (11) Those with mental illness or a history of substance abuse;

[0597] (12) Pregnant or lactating subjects;

[0598] (13) The researcher believes that there are other factors that make the subject unsuitable for inclusion or affect the subject's participation in or completion of the study.

[0599] 2. Donor exclusion criteria

[0600] Any of the following shall be used as a donor / third party exclusion criterion:

[0601] (1) Have a history of mental disorder that may affect compliance with this protocol or be unable to sign the informed consent form;

[0602] (2) A history of hypertension that cannot be controlled by medication, stroke, or severe heart disease;

[0603] (3) Anemia or thrombocytopenia;

[0604] (4) HBsAg positive, or HBcAb positive and peripheral blood HBV DNA positive; HCV antibody positive and HCV RNA positive; Treponema pallidum antibody positive; HIV antibody positive;

[0605] (5) The existence or suspicion of uncontrollable fungal, bacterial, viral or other infections;

[0606] (6) Pregnancy;

[0607] Any other situation that the researcher deems unsuitable to be a donor / third party.

[0608] 3. Research Design

[0609] This study is a single-center prospective exploratory study to evaluate the safety, tolerability and efficacy of LILRB4 STAR-T cells in subjects with relapsed / refractory acute myeloid leukemia / chronic myelomonocytic leukemia. The research product is: LILRB4 STAR-T cells, dosage: intravenous infusion, packaging bag specifications: 50mL or 250mL cryopreservation bag.

[0610] The study is generally divided into two phases: dose escalation and dose expansion. The dose escalation is a 3+3 design, and the dose expansion plan selects 1-2 doses for expansion to further evaluate the safety, efficacy and PK / PD characteristics of LILRB4 STAR-T cells.

[0611] The research process mainly includes screening period, single collection, lymphocyte ablation chemotherapy pretreatment, cell infusion-observation period and follow-up period. Routine laboratory tests (blood routine, blood biochemistry, coagulation function, etc.), STAR-T ratio, cytokine level, lymphocyte subsets (if conditions permit), etc. will be performed on the day of cell infusion and on the 4th, 7th, 10th, 14th, 21st and 28th days after infusion, and DLT observation and evaluation will be performed. Adverse events and serious adverse events will be recorded during treatment, with a focus on cytokine release syndrome (CRS) and immune cell-associated neurotoxicity (ICANS).

[0612] After the 28-day DLT observation period, the follow-up period begins, and the follow-up lasts until 12 months after cell infusion. During the follow-up period, the disease status, survival, and expansion, persistence, and safety of the subjects' LILRB4 STAR-T cells need to be regularly monitored and evaluated. Starting from the 28th day, the follow-up period visit points are 2 to 12 months (once a month).

[0613] Bridging therapy: Based on clinical needs, investigators are allowed to perform bridging chemotherapy after apheresis to control disease progression in subjects with high tumor burden or rapid progression.

[0614] IV. Dosage Design

[0615] The study was divided into two phases: dose escalation and dose expansion.

[0616] Dose escalation stage: The 3+3 dose escalation design principle is adopted, and the dose groups are:

[0617] Dose group A: 1.0×10 6 STAR-T cells / kg

[0618] Dose group B: 6.0×10 6 STAR-T cells / kg

[0619] Dose group C: 1.0×10 7STAR-T cells / kg

[0620] The design principle of dose escalation: the subjects will enter 3 different dose groups in sequence. While ensuring the safety of the subjects, dose groups A and B can adopt an accelerated titration strategy and only enroll 1 subject to avoid excessive ineffective exposure to low-dose levels. If this 1 subject has a DLT, it will be switched to a "3+3" design. Starting from 3+3, if the first 3 subjects included do not have a DLT, they will be increased to the next dose group; if 1 of the 3 subjects in a dose group has a DLT, 3 additional subjects will be added to the group and infused with the same dose and method. If no DLT occurs in the 3 additional cases, the dose will be increased to the next group; if in a dose group, ≥2 of the first 3 cases or ≥1 of the 3 additional cases have a DLT, the dose escalation will be stopped. Dose escalation for the same subject is not allowed.

[0621] If in dose group C (1.0×10 7 STAR + T cells / kg), if the clinically recommended dose has not been found, the Safety Monitoring Committee (SRC) may decide to continue to increase the dose after discussion. In principle, the last subject in the previous dose group should be observed for at least 4 weeks after receiving cell reinfusion, and the first subject in the next dose group is allowed to undergo cell reinfusion.

[0622] Dose expansion phase: Based on the cumulative safety, preliminary efficacy, and possible PK and PD characteristics of the LILRB4 STAR-T cell dose escalation phase, it is planned to select 1-2 doses for dose expansion to further evaluate the safety and clinical efficacy of LILRB4 STAR-T cell therapy.

[0623] 5. Single sampling

[0624] According to the apheresis operation guidelines of the research center, the total number of autologous or donor mononuclear cells collected should not be less than 2×10 9 The cell volume is about 110-120mL. Before single collection, the subject needs to undergo blood routine tests and other related tests, which must meet the research center's single collection requirements. After single collection, the cells are stored at 2-8°C and transported to the cell preparation center by a qualified logistics company for LILRB4 STAR-T cell preparation.

[0625] 6. Chemotherapy Pretreatment

[0626] On Day-5, Day-4 and Day-3 before cell infusion, the subjects need to receive chemotherapy pretreatment with fludarabine + cyclophosphamide (FC regimen). The specific chemotherapy regimen is as follows:

[0627] Fludarabine 25-30 mg / m 2 / d, intravenous drip × 3 days

[0628] Cyclophosphamide 250–500 mg / m 2 / d, intravenous drip × 3 days

[0629] Note: For the chemotherapy pretreatment dosage of the subjects, the researchers can adjust it as appropriate after comprehensively considering the actual tolerance of the subjects.

[0630] BSA is calculated using the Mosteller formula. 2 ) = ([Height (cm) × Weight (kg)] / 3600) 1 / 2 .

[0631] If serum creatinine, creatinine clearance or glomerular filtration rate is abnormal on the day of chemotherapy pretreatment (before drug administration), that is, serum creatinine >1.5 times ULN, or creatinine clearance (Cockcroft-Gault formula) ≤30mL / min or radioisotope glomerular filtration rate (GFR) ≤30mL / min / 1.73m 2 , chemotherapy should be temporarily withheld. If serum creatinine > 2 mg / dL for more than 3 days, the subject will be excluded from the study.

[0632] VII. Clinical Evaluation

[0633] 1. Safety evaluation

[0634] Safety evaluation included physical examination, vital sign recording, clinical laboratory tests, neurological examination and rating, electrocardiogram, fingertip oxygen saturation, and AE / SAE reports collected at each visit.

[0635] The researchers will evaluate whether the test results of each visit of the subjects exceed the normal reference value range and determine whether they are clinically significant.

[0636] This decision should be based on the nature and extent of the laboratory abnormalities.

[0637] Abnormalities associated with STAR-T cell infusion should be taken seriously and monitored continuously. If there is any objection to the research results, the abnormal results can be repeated to rule out errors caused by the laboratory or sample collection.

[0638] 2.PK / PD evaluation

[0639] In principle, sample testing and analysis should be carried out in accordance with the test procedures and schedule.

[0640] The main research sample is peripheral blood, and the main detection indicators are the proportion of LILRB4 STAR-T in peripheral blood, the number of STAR gene copies, and the level of cytokines. Flow cytometry will be used to detect the number and proportion of LILRB4 STAR-T cells in peripheral blood, and the immune cell phenotype can be evaluated if conditions permit. The Q-PCR method will be used to detect the number of STAR gene copies in peripheral blood to simultaneously monitor the expansion of LILRB4 STAR-T cells.

[0641] Serum cytokines will be used as markers of immune activation to analyze the potential correlation between cytokine production and efficacy, as well as the severity of CRS. It is planned to use ELISA or flow cytometry to detect the content of cytokines in serum.

[0642] The PK and PD collection time points include: within 24 hours before cell infusion, after cell infusion: 4 days (±1 day), 7 days (±1 day), 10 days

[0643] days (±1 day), 14 days (±1 day), 21 days (±1 day), 28 days (1 month, +2 days), 2 months (±7 days), 3 months (±7

[0644] The test results were repeated once a month until 12 months, disease progression, new anti-tumor treatment (including hematopoietic stem cell transplantation), death of the subject, withdrawal of informed consent, loss to follow-up, termination of the study, or two consecutive lower limits of detection (whichever occurred first).

[0645] If you miss the PK blood collection, you must collect it as soon as possible and clearly indicate the exact time of blood collection.

[0646] See Table 14 below for AML efficacy evaluation criteria

[0647] Table 14

[0648]

[0649] 8. Clinical Results

[0650] Since the project started, a total of several subjects have been enrolled. For example, three recent subjects received SD / NR tumor assessments on D14 / D28, one of whom received two months of stable disease and CRi on D28, and one of whom received three months of stable disease.

[0651] SD, 1 of them was out of the group due to disease progression on day 28. The details are as follows:

[0652] Subject number: BR07. Gender / age: female / 57; Diagnosis: r / r CMML-AML; Transplantation: one transplantation; BM before transfusion (%): 3.62%; Dose group: 6E6 / kg; STAR product: STAR-abOX40; Cell source: donor (for patients after transplantation, single blood samples from donors were collected); Transfusion after lympholysis chemotherapy; CRS: Grade 2 CRS after transfusion; Tumor assessment: SD / CRi; The subject's urine protein is shown in Table 15 below. CD123 positivity rate: 92.21%.

[0653] Test results

[0654] Table 15

[0655]

[0656]

[0657] IIT clinical data showed that the STAR-T cells prepared in this application significantly improved the PK of BR-07 patients (the proportion of STAR+ cells in peripheral blood increased significantly), significantly reduced tumor cells in the bone marrow (<5%), and achieved D28 CRi, see Fig.14 .

[0658] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the specific implementation of the present invention is described above, it should be understood by those skilled in the art that this is only an example, and the protection scope of the present invention is defined by the attached claims. Those skilled in the art may make various changes or modifications to the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A STAR or a nucleic acid encoding the STAR, a STAR complex or a nucleic acid encoding the STAR, a vector expressing the STAR or the STAR complex, or an immune cell expressing the STAR or the STAR complex for use in the preparation of a drug for treating and / or preventing tumors, characterized in that: The STAR achieves treatment and / or prevention of tumors by targeting LILRB4; The LILRB4 (i) serves as a target antigen of the tumor; and / or (ii) serves as a receptor in immunosuppressive active cells; preferably, the immunosuppressive active cells include MDSC cells or Treg cells.

2. The use according to claim 1, characterized in that: The STAR achieves treatment and / or prevention of tumors by (i) killing tumor cells; and / or (ii) killing immunosuppressive active cells; Preferably, the STAR enhances the killing of tumors by immune cells by killing immunosuppressive active cells.

3. The use according to claim 1 or 2, characterized in that: The STAR comprises an α chain and a β chain. The α chain comprises at least one functional domain, a constant domain of a TCR α chain or a variant thereof from the C-terminus to the N-terminus, and preferably also includes an antigen binding region; The β chain comprises the constant domain of the TCR β chain or its variant, the antigen binding region from the C-terminus to the N-terminus, and preferably further comprises at least one functional domain; Preferably, the antigen binding region comprises one or more of an antibody, a ligand or an antibody fusion that specifically binds to LILRB4; the antibody preferably comprises VHH or scFv.

4. The use according to claim 3, characterized in that: The VHH comprises NLB4 and / or NLB14; The NLB4 comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; The NLB14 comprises CDR1-3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6; Preferably, the amino acid sequence of NLB4 comprises SEQ ID NO: 7; Preferably, the amino acid sequence of NLB14 comprises SEQ ID NO:

8.

5. The use according to claim 3 or 4, characterized in that: in, The constant domain of the TCR α chain or its variant and the antigen binding region, the constant domain of the TCR α chain or its variant and the functional domain, the constant domain of the TCR β chain or its variant and the antigen binding region, the constant domain of the TCR β chain or its variant and the functional domain, and the functional domains are directly connected or connected through a linker; Preferably, the linker comprises one or more of a rigid linker, a flexible linker, a cleavable linker or a meaningless amino acid.

6. The use according to any one of claims 3 to 5, characterized in that: The functional domain includes one or more of a co-stimulatory molecule or a fragment thereof, a co-inhibitory molecule or a fragment thereof, a cytokine receptor or a fragment thereof, or an intracellular protein or a fragment thereof; Preferably, the co-stimulatory molecules include one or more of 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, CD40 or NKp80; Preferably, the co-inhibitory molecules include one or more of TIM3, PD1, CTLA4 or LAG3; Preferably, the cytokine receptor includes one or more of an interleukin receptor, an interferon receptor, a tumor necrosis factor superfamily receptor, a colony stimulating factor receptor, a chemokine receptor or a growth factor receptor, such as one or more of IL-2β, IL-7α or IL-21; Preferably, the intracellular protein includes an immune cell regulatory factor, preferably a T cell regulatory factor, such as a domain of NIK; Preferably, the functional domain may be a fusion protein of a cytokine receptor and a human STAT5 activation module.

7. The use according to any one of claims 3 to 6, characterized in that: The variant of the constant domain of the TCRα chain or the variant of the constant domain of the TCRβ chain comprises a mutation described in any one of the following groups: 1) N-terminal modification; 2) Cysteine ​​substitution; or, 3) C-terminal modification; Preferably, the N-terminal modification includes N-terminal truncation, humanization mutation or mouseization mutation; Preferably, the C-terminal modification includes a transmembrane domain hydrophobic amino acid substitution or an intracellular domain modification (preferably, the intracellular domain modification includes a deletion of the intracellular domain sequence or a lysine substitution); Preferably, the sequence of the constant domain of the TCRα chain or its variant comprises any one of SEQ ID NOs: 15-19, 100-101, 103, 110, 112; and / or the sequence of the constant domain of the TCRβ chain or its variant comprises any one of SEQ ID NOs: 20-24, 104, 111, 113; Further preferably, the sequence of the constant domain of the TCRα chain or its variant comprises SEQ ID NO: 18; and / or, the sequence of the constant domain of the TCRβ chain or its variant comprises SEQ ID NO:

20.

8. The use according to claim 7, characterized in that: The STAR includes any one of the following groups: a) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, and the constant domain of the TCRα chain or its variant from the C-terminus to the N-terminus; b) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, and the constant domain of the TCRα chain or its variant from the C-terminus to the N-terminus; c) the β chain includes the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain includes OX40, a linker, the constant domain of the TCRα chain or its variant, and NLB14 from the C-terminus to the N-terminus; d) the β chain comprises the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, the constant domain of the TCRα chain or its variant, and NLB4 from the C-terminus to the N-terminus; e) the β chain comprises OX40, the constant domain of the TCRβ chain or its variant, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, the constant domain of the TCRα chain or its variant, and NLB4 from the C-terminus to the N-terminus; f) the β chain comprises OX40, the constant domain of the TCRβ chain or its variant, and NLB4 from the C-terminus to the N-terminus; the α chain comprises OX40, the constant domain of the TCRα chain or its variant, and NLB14 from the C-terminus to the N-terminus; g) the β chain comprises OX40, a linker, a constant domain of a TCRβ chain or a variant thereof, and NLB14 from the C-terminus to the N-terminus; the α chain comprises OX40, a linker, a constant domain of a TCRα chain or a variant thereof, and NLB4 from the C-terminus to the N-terminus; h) The β chain comprises OX40, a linker, a constant domain of a TCRβ chain or a variant thereof, and NLB4 from C-terminus to N-terminus; the α chain comprises OX40, a linker, a constant domain of a TCRα chain or a variant thereof, and NLB14 from C-terminus to N-terminus.

9. The use according to any one of claims 1 to 8, characterized in that: The STAR complex comprises STAR and CD3 molecules, and the CD3 molecules preferably comprise one or more of CD3ε, CD3γ, CD3δ or CD3ζ.

10. The use according to claims 1-9, characterized in that: The immune cells include one or more of T cells, NK cells, NKT cells, TIL cells, mast cells, macrophages, dendritic cells, CIK cells or stem cell-derived immune cells.

11. The use according to any one of claims 1 to 10, characterized in that: The method for preparing the immune cells expressing STAR or STAR complex comprises introducing a nucleic acid encoding STAR or STAR complex or a vector expressing STAR or STAR complex into the immune cells.

12. The use according to any one of claims 1 to 11, characterized in that: The tumor includes a blood tumor or a solid tumor; Preferably, the tumor includes lymphoma (preferably including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, T-cell lymphoma or Waldenstrom's macroglobulinemia), leukemia (preferably including acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia (such as M4 or M5 type), chronic myeloid leukemia (such as chronic myelomonocytic leukemia) or plasma cell leukemia), multiple myeloma, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer , colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer (e.g., non-small cell 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, mesothelioma, B-cell malignancies, myelodysplastic syndrome, or sarcoma (e.g., osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma), one or more of the following:

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