Specific TIGIT peptide fragment

Abstract

The present application relates to a TIGIT-derived polypeptide or a mutant thereof, and a fusion protein, an engineered receptor or an engineered cell comprising the polypeptide or the mutant, wherein the binding capacity of the TIGIT-derived polypeptide or the mutant thereof with CD112 is reduced.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to the modification and application of TIGIT extracellular domain polypeptides. Background Art

[0002] CAR-T is one of the most promising immunotherapy methods, especially in the treatment of B-ALL and lymphoma, but its application effect on solid tumors is poor, mainly due to the antigenic heterogeneity of solid tumors and immunosuppressive microenvironment.

[0003] CD155 is the fifth member of the Nectin-like molecule family and acts as a poliovirus receptor, so CD155 is also called necl-5 or PVR (poliovirus receptor). As an immunoglobulin-like adhesion molecule, CD155 is involved in cell motility, natural killer cells, and T cell-mediated immunity. It is barely expressed or weakly expressed in various normal human tissues, but is often overexpressed in human malignant tumors. CD155 overexpression promotes tumor cell invasion and migration, and is associated with tumor progression and poor prognosis. Therefore, it can be used as an advantageous target for solid tumor cell therapy such as CAR-T and CAR-NK.

[0004] CD155 is a ligand for the co-stimulatory receptor CD226 and the co-inhibitory receptors TIGIT and CD96 on natural killer cells and T cells. The common design idea in this field is to select the anti-CD155 antibody ScFv segment as the extracellular antigen binding domain, or to select the CD155 receptor such as TIGIT or CD96 as the extracellular antigen binding domain of the CAR structure. TIGIT, namely T cell immunoglobulin and ITIM domain protein, is a T cell and NK cell co-inhibitory receptor, which is mainly expressed in activated T cells, NK cells, Treg cells, and helper T cells. In addition to CD155, the ligand of TIGIT also includes CD112; CD112 is also called connexin-2 or PVR-related protein 2 (PVRL2), which is a member of the connexin family and is mainly located in the adhesion junctions of epithelial cells. Although CD112 is also expressed in tumors, it is widely expressed in various cells, including epithelial cells, endothelial cells, neurons and fibroblasts. It is used in CAR structure design, and there is a safety risk caused by off-target. Therefore, if TIGIT is to be used in the field of cell therapy, it is necessary to improve the antigen specificity of TIGIT. Summary of the invention

[0005] The present application relates to a TIGIT extracellular region polypeptide, which is derived from the extracellular segment of the TIGIT protein, and whose ability to bind to CD155 is significantly higher than its ability to bind to CD112, and the difference between its ability to bind to CD155 and its ability to bind to CD112 is significantly greater than the difference between the extracellular segment of the wild-type TIGIT protein (amino acid sequence as shown in SEQ ID NO: 1). The present application also relates to a fusion protein and an engineered receptor comprising the TIGIT extracellular region polypeptide, an engineered cell comprising the engineered receptor, and the use of the engineered TIGIT extracellular region polypeptide, fusion protein, engineered receptor or engineered cell.

[0006] Specifically, this application relates to:

[0007] 1. A TIGIT extracellular domain polypeptide comprising a mutation at position 48 relative to a reference sequence, wherein the reference sequence is an amino acid sequence as shown in SEQ ID NO: 1, and wherein the numbering of amino acid positions is defined by the reference sequence.

[0008] 2. The TIGIT extracellular region polypeptide according to item 1, which comprises at least the amino acids corresponding to positions 33 to 93 of the reference sequence. In some embodiments, the polypeptide comprises a deletion of one or more amino acids at the amino acid sites corresponding to positions 33 to 93 of the reference sequence. In some embodiments, the polypeptide comprises one or more additional amino acids at any two amino acid sites corresponding to the amino acid sites at positions 33 to 93 of the reference sequence. In some embodiments, the polypeptide does not comprise other amino acids at the C-terminus and / or N-side corresponding to the amino acids at positions 33 to 93 of the reference sequence, such as amino acids corresponding to amino acids with reference sequence numbers less than 33 and / or amino acids with reference sequence numbers greater than 93. In some embodiments, the polypeptide also comprises other amino acids at the C-terminus and / or N-side corresponding to amino acids at positions 33 to 93 of the reference sequence, such as amino acids corresponding to amino acids with reference sequence numbers less than 33 and / or amino acids with reference sequence numbers greater than 93.

[0009] 3. The TIGIT extracellular domain polypeptide according to item 1 or 2, wherein the mutation is selected from any one of the following:

[0010] C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H and C48 deletion.

[0011] 4. The TIGIT extracellular region polypeptide according to any one of items 1 to 3, further comprising one or more amino acid sites selected from the following:

[0012] 2M, 2W, 2T, 9T, 9S, 12I, 12N, 14A, 14V, 20I, 20T, 21I, 21F, 22L, 22F, 34T , 34S, 37N, 37D, 37E, 39E, 39G, 39K, 39V, 42D, 42G, 44L, 44F, 61K, 61R, 7 0L, 70Q, 70P, 71G, 71D, 76S, 76P, 79V, 79E, 80N, 80Y, 86F, 86S, 86L, ​​101 I, 101T, 102S, 102F, 106L, 106Q, 110V, 110Y, 110E, 110A, 113H, and 113Y.

[0013] 5. The TIGIT extracellular region polypeptide according to item 4, further comprising a combination of amino acid sites of any one of the following 1)-12):

[0014] 1) 22F, 37D, 39G;

[0015] 2) 37D;

[0016] 3)20T, 37D, 39K, 44F;

[0017] 4)22L, 37E, 71D, 102S;

[0018] 5)20T, 37D, 42G, 70Q;

[0019] 6)21F, 34S, 37E, 39K, 70P, 80Y, 101T, 110A;

[0020] 7)9S, 37D, 39V, 61R, 101T;

[0021] 8) 20T, 37D, 39K;

[0022] 9) 37D, 86S, 113Y;

[0023] 10)2W, 37D, 56F;

[0024] 11) 12N, 14V, 21F, 37D, 39K, 70Q; or

[0025] 12)2T, 37D, 39K, 86L.

[0026] 6. The TIGIT extracellular region polypeptide according to item 1, comprising any one of the following amino acid sequences or a conservatively substituted variant of any one of the following amino acid sequences, or in some embodiments, the polypeptide comprises an amino acid sequence having 80% (e.g., 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%) or more identity with any one of the following amino acids:

[0027] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0028] 7. A fusion protein comprising the TIGIT extracellular domain polypeptide described in any one of items 1-6.

[0029] 8. The fusion protein according to item 7 further comprises one or more polypeptides that bind to tumor antigens and / or immune checkpoint proteins. Optionally, the polypeptides that bind to tumor antigens and / or immune checkpoint proteins are antibodies, ligands or receptors of the tumor antigens and / or immune checkpoint proteins. Optionally, the antibodies of the tumor antigens and / or immune checkpoint proteins are single-chain antibodies (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or diabodies.

[0030] 9. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the TIGIT extracellular region polypeptide described in any one of items 1-6, or the fusion protein described in item 7 or 8.

[0031] 10. An engineered receptor according to item 9, which further comprises a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

[0032] 11. The engineered receptor according to item 9 or 10, which is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell antigen coupling agent (TAC) or a fusion protein.

[0033] 12. An engineered receptor according to any one of items 9 to 11, wherein the co-stimulatory domain comprises a signal transduction domain selected from one or more of the following molecules:

[0034] CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83.

[0035] 13. An engineered receptor according to any one of items 9 to 12, wherein the primary signal transduction domain comprises a signal transduction domain of one or more molecules selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 and DAP12.

[0036] 14. An engineered receptor according to item 13, wherein a transmembrane domain is further included between the antigen binding domain and the signal transduction domain, and the transmembrane domain includes a transmembrane domain selected from any one or more of the following molecules: ICOS, CD4, CD8α, CD28, CD3ζ and TIGIT.

[0037] 15. An engineered receptor according to item 14, wherein the antigen binding domain is connected to the transmembrane domain via a hinge region, preferably the hinge region is the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28 or a combination thereof.

[0038] 16. The engineered receptor according to any one of items 9 to 15, which comprises or is, from N-terminus to C-terminus, the TIGIT extracellular region polypeptide, the transmembrane domain and the co-stimulatory domain, wherein the TIGIT extracellular region polypeptide and the transmembrane domain further comprise or do not comprise a hinge region, wherein:

[0039] The co-stimulatory domain comprises or is a signal transduction domain selected from one or more of the following molecules:

[0040] CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83;

[0041] The transmembrane domain comprises or is a transmembrane domain selected from any one or more of the following molecules: ICOS, CD4, CD8α, CD28, CD3ζ and TIGIT; and

[0042] The hinge region comprises or is the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28, or a combination thereof.

[0043] In some embodiments, the engineered receptor comprises from N-terminus to C-terminus: the TIGIT extracellular region polypeptide, the transmembrane domain, and the co-stimulatory domain; or the engineered receptor comprises from N-terminus to C-terminus: the TIGIT extracellular region polypeptide, the hinge region, the transmembrane domain, and the co-stimulatory domain.

[0044] 17. The engineered receptor according to item 16, which comprises or is, from N-terminus to C-terminus: TIGIT extracellular domain polypeptide, CD28 transmembrane domain and CD28 signal transduction domain.

[0045] 18. The engineered receptor according to any one of items 9 to 15, which comprises or is any one of 1) to 10) from N-terminus to C-terminus:

[0046] 1) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signal transduction domain and CD3ζ signal transduction domain;

[0047] 2) TIGIT extracellular domain polypeptide, CD8 hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0048] 3) TIGIT extracellular domain polypeptide, CD8 hinge region, C28 transmembrane domain, 4-1BB signaling domain, and CD3ζ signaling domain;

[0049] 4) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0050] 5) TIGIT extracellular domain polypeptide, ICOS transmembrane domain, ICOS signal transduction domain and CD3ζ signal transduction domain;

[0051] 6) TIGIT extracellular domain polypeptide, CD8 transmembrane domain, CD134 signaling domain, and CD3ζ signaling domain;

[0052] 7) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain;

[0053] 8) TIGIT extracellular domain polypeptide, CD7 hinge region, C28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0054] 9) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain; and

[0055] 10) TIGIT extracellular domain polypeptide, CD8 hinge region, CD8 transmembrane domain, 4-1BB signaling domain and CD3ζ signaling domain.

[0056] 19. An engineered nucleic acid molecule comprising a protein encoding a TIGIT extracellular domain polypeptide according to any one of items 1 to 6, a fusion protein according to item 7 or 8, or an engineered receptor according to any one of items 9 to 18. In some embodiments, the engineered nucleic acid molecule is DNA, RNA (eg, mRNA), or a hybrid molecule of RNA and DNA.

[0057] In some embodiments, the engineered nucleic acid molecules are chemically modified. For example, in some embodiments, one or more thymidines of the engineered nucleic acid molecules are replaced by uridines. In some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by thymidines. In some embodiments, one or more guanosines of the engineered nucleic acid molecules are replaced by creatinine. In some embodiments, the chemical modification is to replace one or more nucleotides of the nucleic acid molecules with their corresponding nucleotide derivatives. For example, in some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by one or more selected from the following: 5-(carboxyhydroxymethyl)uridine (CHM5U), 5-carboxymethylaminomethyluridine (CMNM5U), 5-carboxymethylaminomethyl-2-thiouridine (CMNM5S2U), dihydrouridine (DHU), 2'-O-methylpseudouridine (FM), 1-methylpseudouridine (M1F), 3-(3-amino-3-carboxy-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl uridine (M5U), 5-methoxycarbonylmethyl-2-thiouracil (MCM5S2U), 5-methoxyuridine (MO5U), 5-methyl-2-thiouridine (S2T), 2-thiouridine (S2U), 4-thiouridine (S4U), 5-methyluridine (M5U), 2'-O-methyl-5-methyluridine (TM), 2'-O-methyluridine (UM), 5-methylaminomethyluridine (MAM5U), 5-methylaminomethyl-2-thiouridine (MAM5S2U), pseudouridine (P), and 5-methoxycarbonylmethyl-2-thioguanosine (MCM5S2U). In some embodiments, one or more guanosines of the engineered nucleic acid molecule are replaced by one or more selected from the following: wybutoxosine (osyw), ybutoxosine (yw), 1-methylinosine (m1i), 2'-O-methylguanosine (gm), 1-methylguanosine (m1g), 2,2-dimethylguanosine (m22g), 2-methylguanosine (m2g), 7-methylguanosine (m7g) and β, D-galactose Q nucleoside (gal q), Q nucleoside (q) and β, D-mannose Q nucleoside (man q).In some embodiments, one or more adenosines of the engineered nucleic acid molecule are replaced by one or more selected from the following: N6-isopentenyl adenosine (i6a), 1-methyladenosine (m1a), 2-methyladenosine (m2a), N6-methyladenosine (m6a), 2-methylthio-N6-isopentenyl adenosine (ms2i6a), N-((9-β-D-ribofuranosyl-2-thiomethylpurin-6-yl)carbamoyl)threonine (ms2t6a), N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine (mt6a), N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine (t6a) and β, Q nucleoside (q) and D-mannose Q nucleoside (man q). In some embodiments, one or more cytidines of the engineered nucleic acid molecule are replaced by one or more selected from the following: 4-acetylcytidine (ac4c), 2'-O-methylcytidine (cm), 3-methylcytosine (m3c), N4-methylcytidine (m4c), 5-methylcytidine (m5c), β, and 2-thiocytidine (s2c). In some embodiments, the chemical modification includes 2'-O-methylation modification on the ribose of the nucleotide or 3'thiophosphate bond modification between nucleotides or both. In some embodiments, the modification is 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, 3'thiophosphate modification between the first three nucleotides at the 5' end, and 3'thiophosphate modification between the last three nucleotides at the 3' end.

[0058] 20. An engineered cell comprising the TIGIT extracellular domain polypeptide described in any one of items 1 to 6, the fusion protein described in item 7 or 8, the engineered receptor described in any one of items 9 to 18 and / or the engineered nucleic acid molecule described in item 19.

[0059] 21. The engineered cell according to item 20, which comprises two or more engineered receptors that bind to the same target molecule or different target molecules.

[0060] 22. An engineered cell according to item 21, wherein one of the target molecules is CD155, and the other target molecules are selected from one, two or three of PSCA, CD123 and CEA. In some embodiments, the target molecules are CD155 and PSCA. In some embodiments, the target molecules are CD155 and CD123. In some embodiments, the target molecules are CD155 and CEA. In some embodiments, the engineered receptor that binds to the target molecule CD155 is an engineered receptor of any one of claims 16-18, and the engineered receptor that binds to one, two or three target molecules selected from PSCA, CD123 and CEA is a CAR that targets PSCA, CD123 or CEA, respectively.

[0061] 23. An engineered cell according to item 22, wherein in some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a target molecule selected from one, two or three CARs of PSCA, CD123 and CEA. In some embodiments, the engineered receptor targeting CD155 comprises: an antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the intracellular signaling domain has only a co-stimulatory domain. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting PSCA, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a transmembrane domain and a co-stimulatory domain from the N-terminus to the C-terminus, and the antigen binding domain comprises a TIGIT extracellular region polypeptide described in any one of items 1 to 6. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting PSCA, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a hinge region, a transmembrane domain, and a co-stimulatory domain from N-terminus to C-terminus, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of Items 1 to Item 6. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting CD123, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a transmembrane domain, and a co-stimulatory domain from N-terminus to C-terminus, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of Items 1 to Item 6. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting CD123, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a hinge region, a transmembrane domain, and a co-stimulatory domain from N-terminus to C-terminus, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 6. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting CEA, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a transmembrane domain, and a co-stimulatory domain from N-terminus to C-terminus, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 6. In some embodiments, the engineered cells comprise: an engineered receptor targeting CD155 and a CAR targeting CEA, wherein the engineered receptor targeting CD155 comprises an antigen binding domain, a hinge region, a transmembrane domain and a co-stimulatory domain from N-terminus to C-terminus, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 6.

[0062] According to the engineered cells of item 22, in some embodiments, the engineered cells comprise: an engineered receptor targeting CD155 and a target molecule selected from one, two or three CARs of PSCA, CD123 and CEA, wherein the engineered receptor targeting CD155 may be a structure comprising: an antigen binding domain, a hinge structure, a transmembrane domain and an intracellular signaling domain or an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the intracellular signaling domain may be a CAR structure comprising only a primary signal transduction domain such as a CD3ζ signaling domain, or may be a structure comprising: at least one co-stimulatory domain and a primary signal transduction domain.

[0063] In some embodiments, the antigen binding domain of the engineered receptor that binds CD155 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-26 or a conservatively substituted variant thereof;

[0064] The antigen binding domain of the CAR that binds to the target molecule PSCA comprises an amino acid sequence as shown in SEQ ID NO: 27 or 28 or a conservatively substituted variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0065] The antigen binding domain of the CAR that binds to the target molecule CEA comprises an amino acid sequence as shown in SEQ ID NO: 29 or a conservatively substituted variant thereof or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and,

[0066] The antigen binding domain of the CAR that binds to the target molecule CD123 comprises an amino acid sequence as shown in SEQ ID NO: 30 or a conservatively substituted variant thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0067] 24. The engineered cell according to any one of items 20-23, which is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof. In some embodiments, the engineered cell is a CAR-T, a CAR-NK, a CAR-macrophage, or a CAR-DC. In some embodiments, the engineered cell is a TCR-T cell. In some embodiments, the engineered cell is a TAC-T cell.

[0068] 25. Use of the TIGIT extracellular domain polypeptide described in any one of items 1 to 6, the fusion protein described in item 7 or 8, the engineered receptor described in any one of items 9 to 18, the nucleic acid molecule described in item 19, or the engineered cell described in any one of items 20 to 24 for preparing a drug for treating cancer.

[0069] 26. The method according to claim 25, wherein the cancer is selected from one or more of the following:

[0070] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer. In some embodiments, the cancer is: breast cancer, pancreatic cancer, bladder cancer and / or human acute myeloid leukemia.

[0071] In addition, the present application also provides a method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the TIGIT extracellular domain polypeptide of any one of items 1-6, the fusion protein of item 7 or 8, or the engineered receptor of any one of items 9-18, the nucleic acid molecule of item 19, or the engineered cell of any one of items 20-25. In some embodiments of the method for treating cancer, the cancer is selected from one or more of the following:

[0072] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer. In some embodiments, the cancer is: breast cancer, pancreatic cancer, bladder cancer and / or human acute myeloid leukemia. In some embodiments, the subject or patient is a human patient.

[0073] 28. A method for prolonging the in vivo persistence of CAR-T cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of items 1 to 6, the fusion protein described in item 7 or 8, or the engineered receptor described in any one of items 9 to 18 on the membrane of the CAR-T cells.

[0074] 29. A method for improving the in vivo proliferation ability of CAR-T cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of items 1 to 6, the fusion protein described in item 7 or 8, or the engineered receptor described in any one of items 9 to 18 on the membrane of the CAR-T cells.

[0075] 30. A method for enhancing the in vivo killing ability of CAR-T against target cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of items 1-6, the fusion protein described in item 7 or 8, or the engineered receptor described in any one of items 9-18 on the CAR-T cell membrane.

[0076] In some embodiments, the aforementioned in vivo refers to a cancer patient or subject in vivo. In some embodiments, the in vivo refers to a patient or subject in which CD155 is abnormally expressed in certain tissues and organs. In some embodiments, the cancer is selected from one or more of the following:

[0077] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer.

[0078] 2A self-cleaving polypeptide is a common multi-gene expression scheme for realizing multiple gene co-expression at the translation level. The polypeptide was first found in foot-and-mouth disease virus (FMDV) in 1991, with an average length of 18-22 amino acids. Multiple different 2A peptides have been found, respectively derived from foot-and-mouth disease virus 2A (F2A), type 1 porcine Teschovirus 2A (P2A), bright vein flat moth virus 2A (T2A), and vest rhinitis virus 2A (E2A). Its main working principle is that when the ribosome recognizes the 2A peptide end during translation, it will skip the glycyl-prolyl peptide bond synthesis, and slide, thereby directly generating 2 independent proteins. The purpose of the application using 2A peptides is to obtain the engineered immune cells expressing the engineered receptors of the application, and finally verify the function of the engineered immune cells by the test of the embodiment, and further verify the unpredictable effect of the engineered receptors of the application for the engineered immune cells. In addition to the above-mentioned 2A self-cleaving peptide, IRES (internal ribosome entry site sequence (Internal ribosome entry site, IRES)) can also be used. IRES can recruit ribosomes to translate mRNA, and multiple proteins can be selected for independent expression. The internal ribosome entry site (IRES) is used to separate the coding genes (also referred to as ORF) of the target molecules such as the engineered receptor of the present application, at least one chimeric antigen receptor (CAR), and other fusion proteins, and a single mRNA transcript will produce multiple proteins. The above-mentioned 2A peptide and IRES, as well as other small molecule sequences with similar functions, can be referred to as linkers. In addition to using the above-mentioned linkers to achieve multi-gene expression, the above-mentioned engineered cells can also be achieved by transducing different genes into target cells separately, or by transducing the target genes simultaneously with the constructed vectors expressing different genes; whether it is using linkers or constructing expression vectors of multiple genes separately to transduce target cells, the purpose of obtaining engineered cells expressing TIGIT extracellular region polypeptides can be achieved in the end, and the functions and unpredictable effects of engineered cells are mainly based on the protein molecules expressed and the final engineered cells themselves.

[0079] The polypeptides, fusion proteins, CARs and engineered cells provided in this application can more specifically recognize CD155 in the presence of CD112 and are less interfered by CD112. Since CD112 is widely expressed in normal mammalian cells, the CARs and engineered cells provided in this application can have relatively higher in vivo safety. In the functional verification of the CAR-T and CAR-NK fields, the CAR provided in this application has indeed achieved a more specific and high-proportion killing effect on target cells expressing CD155. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1The flow cytometry staining of CD112 in MDA-MB-231-Luc-GFP is shown, where the horizontal axis represents the experimental group and the control group, and the vertical axis represents the mean fluorescence intensity (MFI) of the experimental group and the control group after cells were stained with CD112 antibody. The results show that the MFI of CD112 in MDA-MB-231-Luc-GFP is about 4 times higher than that in the control group.

[0081] Figure 2A Peptide-specific detection of five TIGIT mutants is shown.

[0082] Figure 2B Specific detection of 2 TIGIT mutant peptides is shown.

[0083] Figure 2C Peptide-specific detection of seven TIGIT mutants is shown.

[0084] Figure 2D Peptide-specific detection of nine TIGIT mutants is shown.

[0085] Figure 2E Peptide-specific detection of 3 TIGIT mutants is shown.

[0086] Figure 3 The screened TIGIT mutant peptides are shown to bind to CD112.

[0087] Figure 4 The screened TIGIT mutant peptides are shown to bind to CD155.

[0088] Figure 5 Functional validation of the screened TIGIT mutant peptides against CD155-negative cells was shown.

[0089] Figure 6 Shows in vivo efficacy evaluation in the MDA-MB-231 tumor model.

[0090] Fig. 7A The killing effect of 7 CARs with different structures on MDA-MB-231-Luc-GFP is shown.

[0091] Figure 7B The killing effect of 5 CARs with different structures on HPAC-Luc-GFP is shown.

[0092] Figure 7C The killing effect of 2 CARs with different structures on HT1376-Luc-GFP is shown.

[0093] Fig. 8A The expression of CEA in DLD1-CEA-Luc-GFP is shown.

[0094] Figure 8BThe expression of CEA in DLD1-Luc-GFP is shown.

[0095] Figure 8C The expression of CD155 in DLD1-CEA-Luc-GFP is shown.

[0096] Fig. 9A The killing effect of CEA and CD155 dual-targeting CAR37 on DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP cells is shown.

[0097] Fig. 9B The in vitro killing of Molm-13-Luc-GFP by CAR38 dual-targeting CD123 and CD155 is shown.

[0098] Fig. 9C The in vitro killing effects of CAR-T cells with four different CAR structures dual-targeting PSCA and CD155 in three cell models are shown.

[0099] Fig.10 The killing effects of CAR42 and CAR45 in two cell models are shown.

[0100] Fig.11 The IFN-γ cytokine secretion of CAR42 and CAR45 during the in vitro killing process of two cell models is shown.

[0101] Fig.12 The in vivo efficacy of CAR42 and CAR45 in the DLD-1-CEA-Luc-GFP peritoneal tumor model is shown.

[0102] Fig.13 Shows the blood copy number status in the peritoneal tumor evaluation model.

[0103] Fig.14 Verification of the effect of TIGIT mutant peptide on the in vivo efficacy of CAR.

[0104] Fig.15A Verification of the effectiveness of multi-target CAR-T targeting CD155 against acute myeloid leukemia.

[0105] Fig. 15B Verification of the effectiveness of multi-target CAR-T targeting CD155 against pancreatic cancer and bladder cancer. Specific implementation plan

[0106] The present application relates to a TIGIT mutant peptide, which is derived from the extracellular segment of the TIGIT protein, and its ability to bind to CD155 is significantly higher than its ability to bind to CD112, and the difference between its ability to bind to CD155 and its ability to bind to CD112 is significantly greater than the difference between the extracellular segment of the wild-type TIGIT protein (amino acid sequence as shown in SEQ ID NO: 1). For example, in some embodiments, the TIGIT mutant peptide has a CD155 binding ability comparable to or stronger than the extracellular segment of the wild-type TIGIT protein, but a significantly reduced binding ability to CD112. In some embodiments, the TIGIT mutant peptide has a stronger CD155 binding ability and a stronger CD112 binding ability than the extracellular segment of the wild-type TIGIT protein, but relative to the extracellular segment of the wild-type TIGIT protein, the improvement of the TIGIT mutant peptide's ability to bind to CD155 is much greater than the improvement of its ability to bind to CD112. In some embodiments, the TIGIT mutant peptide has a weaker CD155 binding ability and a weaker CD112 binding ability than the extracellular segment of the wild-type TIGIT protein, but relative to the extracellular segment of the wild-type TIGIT protein, the reduction in the TIGIT mutant peptide's ability to bind to CD155 is much less than the reduction in its ability to bind to CD112. In some embodiments, the TIGIT mutant peptide has a stronger CD155 binding ability and a relatively unchanged CD112 binding ability than the extracellular segment of the wild-type TIGIT protein. In some embodiments, the TIGIT mutant peptide has a stronger CD155 binding ability and a weaker CD112 binding ability than the extracellular segment of the wild-type TIGIT protein. In addition, the present application also relates to a fusion protein or engineered receptor comprising the TIGIT mutant peptide, such as a fusion protein comprising an antibody or an antigen recognition fragment of the antibody, a CAR, an engineered TCR, a TAC, etc.; and an engineered cell comprising the fusion protein or engineered receptor, such as a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof; and the uses of the aforementioned TIGIT mutant peptide, engineered receptor, fusion protein or engineered cell.

[0107] definition

[0108] It should be understood that the present disclosure is not limited to the aspects described herein, which may of course vary in nature. It should also be understood that the terminology used herein is only used to describe particular aspects and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0109] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the technology belongs. All techniques and patent disclosures cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, those skilled in the art will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art.

[0110] The term "TIGIT" is an abbreviation for a T cell immunoreceptor with Ig and ITIM domains, also known as WUCAM, Vstm3 or VSIG9. It consists of an extracellular region of an extracellular immunoglobulin variable domain (IgV), a type 1 transmembrane domain, and an intracellular domain with a typical immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoglobulin tyrosine tail (ITT) motif. TIGIT is a member of the poliovirus receptor / nectin family, a subset of the immunoglobulin superfamily. TIGIT is an immunoreceptor inhibitory checkpoint involved in tumor immune surveillance. TIGIT competes with the immune activator receptor CD226 (DNAM-1) for the same group of ligands: CD155 (PVR or poliovirus receptor) and CD112 (nectin-2 or PVRL2). An exemplary TIGIT is human TIGIT, which is encoded by the gene with gene ID 201633 in the NCBI database. The term "TIGIT extracellular region" refers to the amino acid sequence of positions 1 to 120 of the TIGIT protein or its variant corresponding to the reference sequence SEQ ID NO: 1, which can be determined by sequence alignment with the reference sequence, for example, by introducing gaps, etc., so that any TIGIT protein or its variant can have the same residues as the reference sequence at as many positions as possible. After alignment, the amino acids at the same positions as positions 1 and 120 in the amino acid sequence shown in SEQ ID NO: 1 and all the amino acid positions in between in the TIGIT protein or its variant can be connected in the order of their positions in the TIGIT protein or its variant and can be called the "TIGIT extracellular region". For example, the TIGIT protein with GenBank accession number ACD74757.1 has an extracellular region from positions 22 to 141.

[0111] As used herein, "TIGIT extracellular region polypeptide" may be used to refer to any peptide fragment in the TIGIT extracellular region or any truncated form of the TIGIT extracellular region, or the full-length TIGIT extracellular region.

[0112] As used herein, "reference sequence" refers specifically to the amino acid sequence shown in SEQ ID NO: 1, which is used to define the positions of amino acids in this application. Unless otherwise specified, the amino acid position numbers of this application are defined by the reference sequence. As used in this application, a mutation "relative to a reference sequence" refers to a position of an amino acid defined by sequence alignment, at which position there is an amino acid different from the amino acid at the reference sequence at the position (replacement), more than one amino acid at the amino acid position (addition), and / or there is a vacancy (deletion) at the amino acid position. For example, a TIGIT extracellular region polypeptide "comprising a mutation relative to a reference sequence at position 48" means that the amino acid at position 48 of the TIGIT extracellular region polypeptide corresponding to SEQ ID NO: 1 is not 48C of SEQ ID NO: 1 (but, for example, 48W), or has two or more amino acids at position 48, or does not contain any amino acid at position 48. In the present application, "the numbering of amino acid sites in a polypeptide, protein or amino acid sequence is defined by the reference sequence" means that after the polypeptide, protein or amino acid sequence has the same residues as the reference sequence at as many positions as possible by introducing vacancies or deleting amino acids into the polypeptide, protein or amino acid sequence, the amino acids in the reference sequence are numbered sequentially starting from 1 in sequence order, and the positions of amino acids in the polypeptide, protein or amino acid sequence and the reference sequence are defined by the same numbering by aligning the corresponding amino acids.

[0113] "CD155" is also known as "PVR", which stands for poliovirus receptor, also known as Necl5 and Tage4. CD155 is a cell surface adhesion molecule that is dramatically overexpressed in several human malignancies, while its expression is low or absent in most healthy tissues. Consistent with the biological properties of PVR, its overexpression promotes tumor cell invasion, migration and proliferation, and is associated with poor prognosis and enhanced tumor progression.

[0114] "CD112" is also known as "PVRL2", which is adhesion protein-2. It is a single-channel type I membrane protein with two Ig-like C2-type domains and one Ig-like V-type domain. It is one of the plasma membrane components of adhesion junctions.

[0115] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0116] As used herein, a "variant" has at least one amino acid difference relative to a reference amino acid sequence, for example, at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" may be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Gln); u); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met). In some other embodiments, the variant may also include non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared to the amino acid sequence, the "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range consisting of any two of the foregoing values. As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one or more amino acid residues in which the overall conformation and function of the protein or enzyme are changed by amino acid substitution, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90% is optimal, and have the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0117] As used herein, "amino acid" refers to any monomer unit that can be incorporated into a peptide, polypeptide or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). In some embodiments, the application also includes non-natural amino acids or amino acid analogs derived or modified on the basis of any of the above natural amino acids. As used herein, "amino acid" also includes non-natural amino acids, modified amino acids (e.g., with modified side chains and / or backbones) and amino acid analogs. To further illustrate, amino acids are generally organic acids comprising substituted or unsubstituted amino, substituted or unsubstituted carboxyl and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photosensitive crosslinker, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing metals, amino acids containing novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photolabile (photocaged) and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.The amino acids described in the present application include, but are not limited to, 20 natural amino acids and 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminoalanine (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or pipecolic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptaneic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisomethacrylic acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), deoxyephedrine (Des), 2,2'-diaminopimelic acid (Dp m), 2,3-diaminopropanesulfonic acid (Dpr), ethylglycine (EtGly), N-ethylaspartic acid (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine or sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orm). Therefore, in some embodiments, after the mutation, the amino acid mutation at the site comprises a substitution mutation converted to any one of the above 20 natural amino acids and the above non-natural amino acids. In some embodiments, the amino acid mutation comprises a substitution mutation to any one of the amino acids selected from the group consisting of G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H, Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Hyl, aHyl, 3Hyp, 4Hyp, Ide, aIle, MeGly, MeIle, MeLys, MeVal, Nva, Nle, and Orm.

[0118] In the context of the present invention, the terms "DNA" and "RNA" refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms "DNA" and "DNA molecule" refer to double-stranded DNA molecules consisting of A, C, G and / or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules consisting of A, C, G and / or U nucleotides. In this article, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0119] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0120] As used herein, the "DNA and RNA hybrid molecule" is a molecule comprising a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. The DNA and RNA hybrid molecule can be obtained by:

[0121] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0122] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0123] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials by biological or chemical synthesis. It should be noted that the method of obtaining a hybrid molecule of DNA and RNA is not limited to the above method, and the hybrid molecule of DNA and RNA obtained by any method belongs to the category of "hybrid molecule of DNA and RNA" defined in this application.

[0124] As used herein, if two nucleic acid molecules are described as having "the same genetic information", it means that the two nucleic acid molecules are complementary, or contain exactly the same base sequence, or one or more thymines in the base sequence of one of the nucleic acid molecules are converted to uracil to obtain a nucleic acid molecule with exactly the same base sequence as another nucleic acid molecule. Therefore, any two of DNA, RNA, and hybrid molecules of DNA and RNA can have the same genetic information. Among them, the term "base sequence" refers to the order of arrangement of bases in a polynucleotide molecule. Those skilled in the art should know that, unless otherwise specified, the base sequence or polynucleotide sequence described in this application can be used to describe DNA sequences. "T" refers to thymine, but when the base sequence or polynucleotide sequence is used to describe RNA (such as mRNA), "T" will be replaced by "U" (uracil). Therefore, any DNA disclosed by a specific sequence number (SEQ ID NO) herein also discloses an RNA (such as mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is replaced by "U".

[0125] In this article, "coding" refers to i) genetic information contained in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) genetic information contained in an RNA molecule that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof that can be translated into a protein in an mRNA precursor or mature mRNA, and can also refer to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that is used as a template to transcribe the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application may further include polynucleotide sequences encoding proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, Poly (A) tail, 5'UTR, 3'UTR, etc. Among them, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is called the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is called the "coding nucleic acid" of the amino acid sequence.

[0126] Unless otherwise specified, "peptide", "polypeptide" and "protein" are used interchangeably in this application and can be used to refer to any natural active substance formed by covalently linking two or more amino acids through peptide bonds, which may or may not contain the secondary or tertiary structure of the protein molecule.

[0127] As used herein, the term "contacting" is used according to its simple common meaning, referring to the process of allowing at least two different substances to become close enough to react, interact or physically contact. It should be understood that the resulting reaction product can be directly produced by the reaction between the added reagents, or produced by the intermediate of one or more added reagents, which can be produced in the reaction mixture. The term "contacting" can include allowing two substances to react, interact or physically contact, wherein the two substances can be, for example, engineered receptors (or engineered nucleic acid molecules) and cells provided herein. In an embodiment, contacting includes, for example, allowing engineered nucleic acid molecules or engineered peptides described in the present application to enter cells.

[0128] As used herein, "CAR-T cell in vivo persistence" refers to the duration of CAR-T cells in the patient or subject's body, that is, the period from the in vivo administration of CAR-T cells to their final disappearance from the body. It can be calculated or measured by comparing the copy number of CAR. For example, after a specific period of time of use of CAR-T in vivo, an increase in its copy number can be used to indicate an extension of the in vivo persistence of CAR-T cells.

[0129] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0130] TIGIT extracellular domain polypeptide

[0131] On the one hand, the present application relates to a TIGIT extracellular domain polypeptide (unless otherwise specified, it may also be referred to as a TIGIT mutant peptide), which comprises a mutation at position 48 relative to a reference sequence, wherein the reference sequence is an amino acid sequence as shown in SEQ ID NO: 1, and wherein the numbering of amino acid sites is defined by the reference sequence.

[0132] In some embodiments, the TIGIT extracellular region polypeptide comprises amino acids corresponding to positions 33 to 93 of the reference sequence, that is, it comprises amino acids corresponding to positions 33 and 93 of the reference sequence in any TIGIT extracellular region amino acid sequence, and all amino acids between positions 33 to 93 in any TIGIT extracellular region amino acid sequence. It should be understood that it does not require that all amino acids between positions 33 to 93 correspond to all amino acids at positions 33 to 93 of the reference sequence, but allows it to include the deletion or addition of amino acids at any position corresponding to the amino acid positions 33 to 93 of the reference sequence. Furthermore, it should be understood that when the amino acid sequence of any TIGIT extracellular region does not have an amino acid at position 33 relative to the reference sequence, the amino acids corresponding to positions 33 to 93 of the reference sequence start from an amino acid site greater than position 33 of the reference sequence and closest to position 33 of the reference sequence; and when the amino acid sequence of any TIGIT extracellular region does not have an amino acid at position 93 relative to the reference sequence, the amino acids corresponding to positions 33 to 93 of the reference sequence end at an amino acid site less than position 93 of the reference sequence and closest to position 93 of the reference sequence. In some embodiments, the engineered TIGIT extracellular region polypeptide may further comprise one or more continuous amino acid sequences extending from position 33 of any TIGIT extracellular region amino acid sequence to the carbon terminus (C terminus), and / or one or more continuous amino acid sequences extending from position 93 of any TIGIT extracellular region amino acid sequence to the nitrogen terminus (N terminus). In some embodiments, the engineered TIGIT extracellular region polypeptide may further comprise one or more continuous amino acid sequences extending from position 33 of any TIGIT extracellular region amino acid sequence to the nitrogen terminus (N terminus), and / or one or more continuous amino acid sequences extending from position 93 of any TIGIT extracellular region amino acid sequence to the carbon terminus (C terminus). In some embodiments, the TIGIT extracellular region polypeptide comprises only amino acids corresponding to positions 33 to 93 of the reference sequence.

[0133] In some embodiments, the TIGIT extracellular region polypeptide comprises a mutation at position 48 relative to the reference sequence selected from any one of the following:

[0134] C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H and deletion at position 48. Wherein the deletion at position 48 means that the TIGIT extracellular domain polypeptide does not have an amino acid at position 48 relative to the reference sequence. In some embodiments, the TIGIT extracellular region polypeptide comprises a substitution of a non-natural amino acid at position 48, thus in some embodiments, the TIGIT extracellular region is Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Hyl, aHyl, 3Hyp, 4Hyp, Ide, aIle, MeGly, MeIle, MeLys, MeVal, Nva, Nle, or Orm at position 48 relative to the reference sequence.

[0135] In some embodiments, the TIGIT extracellular region polypeptide comprises: amino acids corresponding to positions 33 to 93 of the reference sequence, the mutation at position 48 relative to the reference sequence, and one or more amino acid positions selected from the following:

[0136] 2M, 2W, 2T, 9T, 9S, 12I, 12N, 14A, 14V, 20I, 20T, 21I, 21F, 22L, 22F, 34T , 34S, 37N, 37D, 37E, 39E, 39G, 39K, 39V, 42D, 42G, 44L, 44F, 61K, 61R, 7 0L, 70Q, 70P, 71G, 71D, 76S, 76P, 79V, 79E, 80N, 80Y, 86F, 86S, 86L, ​​101 I, 101T, 102S, 102F, 106L, 106Q, 110V, 110Y, 110E, 110A, 113H, and 113Y.

[0137] In some embodiments, the TIGIT extracellular region polypeptide comprises: amino acids corresponding to positions 33 to 93 of the reference sequence, the above-mentioned mutation at position 48 relative to the reference sequence, and the following amino acid positions:

[0138] 1) 22F, 37D, 39G;

[0139] 2) 37D;

[0140] 3)20T, 37D, 39K, 44F;

[0141] 4)22L, 37E, 71D, 102S;

[0142] 5)20T, 37D, 42G, 70Q;

[0143] 6)21F, 34S, 37E, 39K, 70P, 80Y, 101T, 110A;

[0144] 7)9S, 37D, 39V, 61R, 101T;

[0145] 8) 20T, 37D, 39K;

[0146] 9) 37D, 86S, 113Y;

[0147] 10)2W, 37D, 56F;

[0148] 11) 12N, 14V, 21F, 37D, 39K, 70Q; or

[0149] 12)2T, 37D, 39K, 86L.

[0150] In some embodiments, any of the aforementioned letters referring to natural amino acids can also be used to refer to non-natural amino acids or amino acid analogs formed by modification or derivatization of the natural amino acids.

[0151] In some embodiments, the TIGIT extracellular region polypeptide comprises any one of the following amino acid sequences or a conservatively substituted variant of any one of the following amino acid sequences:

[0152] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0153] In some embodiments, the amino acid sequence of the TIGIT extracellular region polypeptide is selected from any of the following amino acid sequences or comprises a conservative substitution variant of any of the following amino acid sequences:

[0154] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0155] In some embodiments, the TIGIT extracellular domain polypeptide is chemically modified, for example, one or more amino acids therein are replaced by non-natural amino acids or amino acid analogs.

[0156] Fusion Protein

[0157] On the one hand, the present application also provides a fusion protein comprising the aforementioned TIGIT extracellular region polypeptide, and the fusion protein can be a protein complex formed by combining the aforementioned TIGIT extracellular region polypeptide with another one or more proteins, polypeptides or protein functional domains in any manner. In some embodiments, the binding is that the TIGIT extracellular region polypeptide is directly and / or indirectly connected to the other one or more proteins, polypeptides or protein functional domains. In some embodiments, the carbon (C) end of the TIGIT extracellular region polypeptide in the fusion protein is connected to the nitrogen (N) end of the other protein, polypeptide or protein functional domain. In some embodiments, the nitrogen (N) end of the TIGIT extracellular region polypeptide in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the TIGIT extracellular region polypeptide in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the TIGIT extracellular region polypeptide in the fusion protein is connected to the multiple proteins, polypeptides or protein functional domains in series. In some embodiments, the C-terminus and / or N-terminus of the TIGIT extracellular region polypeptide in the fusion protein is connected to at least two or more additional proteins, polypeptides or protein domains, and the two or more additional proteins, polypeptides or protein domains are not connected in series. In some embodiments, the one or more proteins are homologous proteins of the TIGIT extracellular region polypeptide or parts of the homologous proteins. In some embodiments, the one or more proteins are heterologous proteins of the TIGIT extracellular region polypeptide, or parts of the heterologous proteins. The term "direct" connection or "direct connection" refers to a connection achieved only by a chemical bond, that is, the two proteins or polypeptides are not connected by other molecules, and the chemical bond can be a non-covalent bond (such as an ionic bond, a hydrogen bond, a hydrophobic bond or a van der Waals bond), or a covalent bond (such as a peptide bond). The term "indirect" connection or "indirect connection" refers to a connection through a linker, and the two proteins or polypeptides connected to each other using a linker are respectively connected to one end of the linker by a covalent or non-covalent bond. The "linker" can be a peptide linker (i.e., a peptide chain, such as a peptide chain consisting of 1 to 50 amino acids or their derivatives) or a non-peptide linker, and the linker can be cleavable (i.e., hydrolyzed by an enzyme in the body, such as a mammal) or non-cleavable. Exemplary non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, polymerized lipids, chitin and hyaluronic acid, or derivatives thereof, or combinations thereof.

[0158] In some embodiments, the other one or more proteins, polypeptides or protein domains comprise a recognition polypeptide that specifically binds to another one or more proteins, and the other one or more proteins may be tumor antigens, for example, one or more selected from the following: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD3 3; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor alpha (IL-11Rα); prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor PDGFR-β; stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bD Galp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa lightchain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variant of tumor necrosis zone; G protein-coupled receptor class C group 5 - member D (GPRC5D);X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding cell surface receptor 2 ( Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic disease viral oncogene neuroblastoma-derived homolog MYCN; Ras homolog family member C (RhoC); cytochrome P4501B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin leukocyte immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155.

[0159] In some embodiments, the one or more other proteins, polypeptides or protein domains comprise a recognition polypeptide that specifically binds to one or more other proteins, and the one or more other proteins may be immune checkpoint proteins, such as one or more selected from the following:

[0160] 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0161] In some embodiments, the other one or more proteins, polypeptides or protein functional domains contain a recognition polypeptide that specifically binds to another one or more proteins, and the other one or more proteins include any one or more immune checkpoint proteins selected from the above-mentioned, and any one or more tumor antigens mentioned above.

[0162] In some embodiments, the recognition polypeptide that specifically binds to another one or more proteins is an antibody or an antigen-binding fragment of the antibody or a ligand or receptor or a fragment thereof that binds to the tumor antigen and / or immune checkpoint protein.

[0163] In some embodiments, the one or more other proteins, polypeptides or protein functional domains may be a hinge structure connecting the TIGIT extracellular region polypeptide and the transmembrane structure, for example, one or more selected from the following: the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28 or a combination thereof; in some embodiments, the one or more other proteins, polypeptides or protein functional domains may be a transmembrane structure connecting the TIGIT extracellular region polypeptide and the intracellular signaling structure, for example, one or more selected from the following: ICOS, CD4, CD8α, CD28, CD3ζ and TIGIT; in some embodiments, the one or more other proteins, polypeptides or protein functional domains may also include an intracellular signaling domain, which is a co-stimulatory signal molecule, comprising a signal transduction domain selected from one or more of the following molecules:

[0164] CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD 160. CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83.

[0165] Further, in some embodiments, the fusion protein may be a TIGIT fusion protein comprising a TIGIT extracellular domain polypeptide, a transmembrane structure and an intracellular co-stimulatory domain.

[0166] Engineered receptors

[0167] The present application also provides an engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide or the aforementioned fusion protein. In some embodiments, the engineered receptor further comprises a signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a costimulatory domain or a secondary signal transduction domain, but does not comprise a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain and a costimulatory domain.

[0168] In some embodiments, the engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-transmembrane domain-costimulatory domain (or secondary signal transduction domain). In some embodiments, the engineered receptor comprises or does not comprise a hinge region, and the hinge region connects the antigen binding domain and the transmembrane domain or the signal transduction domain. In some embodiments, the hinge is derived from 7h (CD7 hinge region) / G4h / 8h (CD8 hinge region). In some embodiments, the transmembrane domain is from the transmembrane domain of ICOS, CD4, CD8α, CD28, CD3ζ or TIGIT, or a combination thereof. In some embodiments, the co-stimulatory domain or secondary signaling domain comprises one or more of the following, or consists of one or more of the following: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a signal transduction domain of a ligand that specifically binds to CD83.

[0169] In some embodiments, the engineered receptor may comprise an extracellular antigen binding domain, may comprise an additional hinge structure or may not comprise a transmembrane domain and an intracellular signal transduction domain, wherein the extracellular antigen binding domain is selected from any of the following amino acid sequences or comprises a conservative substitution variant of any of the following amino acid sequences: SEQ ID NO: 5-17, 19, 22, and SEQ ID NO: 24-26. Specifically, in some embodiments, the structure of the engineered receptor is TIGIT extracellular region polypeptide-28TM-28. In some embodiments, the TIGIT extracellular region polypeptide is the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the TIGIT extracellular region polypeptide may be any of the following amino acid sequences or comprises a conservative substitution variant of any of the following amino acid sequences: SEQ ID NO: 5-17, 19, 22, and SEQ ID NO: 24-26, 28TM is a transmembrane domain derived from human CD28, and 28 is an intracellular signal transduction domain derived from human CD28. In some embodiments, the structure of the engineered receptor is TIGIT extracellular region polypeptide-28TM-28, wherein the TIGIT extracellular region polypeptide can be the polypeptide described in SEQ ID NO: 7, 28TM is the transmembrane domain derived from human CD28, and 28 is the intracellular signal transduction domain derived from human CD28.

[0170] In some embodiments, the engineered receptor is: a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell antigen coupling agent (TAC) or the aforementioned fusion protein. In some embodiments, the fusion protein comprises an extracellular antigen binding domain and an intracellular domain, wherein the extracellular antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide, and the intracellular domain is selected from the intracellular domain of any one or more membrane proteins. In some embodiments, the fusion protein comprises an extracellular antigen binding domain and an intracellular domain, wherein the extracellular antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide, and the intracellular domain comprises a T cell receptor costimulatory domain. In some embodiments, the fusion protein comprises an extracellular antigen binding domain and an intracellular domain, wherein the extracellular antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide, and the intracellular domain comprises one or more T cell receptor co-stimulatory domains, and the T cell receptor co-stimulatory domains are derived from the intracellular domains of the following proteins: CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, ICOS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, and ligands that specifically bind to CD83.

[0171] As used herein, the term "CAR", i.e., a chimeric antigen receptor, comprises: i) an antigen binding domain that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); ii) a transmembrane domain; and iii) an intracellular signal transduction domain. Among them, the "intracellular signal transduction domain" comprises a primary signal transduction domain and / or a co-stimulatory domain. In some embodiments, the antigen binding domain is selected from one or more of the following groups: an extracellular antigen binding domain of a receptor or ligand, a single domain antibody (sdAb), a single-chain Fv (scFv), and a Fab. In some embodiments, the transmembrane domain is from any one molecule selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD5, CD6, CD7, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1. In some embodiments, the transmembrane domain is from CD8α. In some embodiments, the intracellular signal transduction domain comprises a primary signal transduction domain, which is from a molecule selected from any one of the following groups: CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10 and DAP12. In some embodiments, the primary signal transduction domain is from CD3ζ. As used herein, the "primary signal transduction domain" generally comprises an immune receptor tyrosine activation motif (ITAM), the basic composition of which is: YXXL / V. Wherein Y is tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA connected thereto can be phosphorylated under the action of a class of protein tyrosine kinase PTK connected to the cell membrane, thereby recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell.

[0172] In some embodiments, the intracellular signaling domain further comprises a co-stimulatory domain from one or more co-stimulatory receptor molecules selected from the group consisting of CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, ICOS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the costimulatory domain is from 4-1BB. In some embodiments, CAR further comprises a hinge domain between the C-terminus of the antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is from CD8α or CD28. In some embodiments, the antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the antigen binding domain is the aforementioned engineered TIGIT extracellular region polypeptide. As used herein, a "costimulatory domain" is generally derived from a costimulatory receptor of an immune cell, providing a second signal or secondary intracellular signal for activated immune cells (eg, T cells).

[0173] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an antigen binding domain (as used in this application, an antigen binding domain comprises a domain that binds to an antigen, a ligand domain that binds to a receptor, or a receptor domain that binds to a ligand), which comprises an antigen binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) optionally a first linker; (c) optionally The extracellular antigen binding domain or part thereof of the first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) the transmembrane domain of the second TCR subunit (e.g., TCRα, TCRβ); and (e) the intracellular signal transduction domain comprising the third TCR subunit (e.g., TCRα, TCRβ); wherein the first, second and third TCR subunits are independently selected from any one of the following groups: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ and CD3ζ. In some embodiments, the first, second and third TCR subunits are the same (e.g., all CD3ε, all TCRα or all TCRβ). In some embodiments, the first, second and third TCR subunits are different. In some embodiments, the engineered TCR further comprises a hinge domain between the C-terminus of the antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is from CD8α. In some embodiments, the antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the antigen binding domain is the aforementioned engineered TIGIT extracellular region polypeptide.

[0174] As used herein, "TAC" is a T cell antigen conjugate comprising (i) an antigen binding domain, (ii) a TCR binding domain (e.g., scFv), and (iii) a co-receptor domain (e.g., hinge, transmembrane and / or cytosolic region). See, for example, Helsen et al. Nat Commun. 2018; 9(1): 3049. In some embodiments, the antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the antigen binding domain is the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the TAC comprises: (a) an antigen binding domain (as used herein, the antigen binding domain comprises a domain that binds to an antigen, a ligand domain that binds to a receptor, or a receptor domain that binds to a ligand), which comprises an antigen binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) optionally a first linker; (c) an extracellular TCR binding domain (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes an extracellular antigen binding domain of a TCR subunit (e.g., CD3ε); (d) optionally A second connector; (e) optionally an extracellular antigen binding domain or a portion thereof of a first TCR co-receptor (e.g., CD4, CD8); (f) a transmembrane domain comprising a transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) optionally an intracellular signal transduction domain comprising an intracellular signal transduction domain of a third TCR co-receptor (e.g., CD4, CD8); wherein the TCR subunit is selected from any one or more of the following groups: CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ, and TCRδ; and wherein the first, second, and third TCR co-receptors are each independently selected from any one of the following groups: CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further comprises a hinge domain (e.g., from CD8α) located between the C-terminus of the antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the antigen binding domain comprises the aforementioned engineered TIGIT extracellular region polypeptide. In some embodiments, the antigen binding domain is the aforementioned engineered TIGIT extracellular region polypeptide.

[0175] As used herein, the term "antigen binding domain" covers the concepts of "ligand binding domain" and "receptor binding domain", which are usually located in the extracellular segment of a cell (especially an immune cell) receptor and can specifically bind to a certain protein, and the scope of the certain protein is not subject to any restrictions. Therefore, in some embodiments, the certain protein is a certain receptor, and the "antigen binding domain" is the part of the ligand of the certain receptor that specifically recognizes the certain receptor; in some embodiments, the protein is a certain ligand, and the "antigen binding domain" is the part of the receptor of the certain ligand that specifically recognizes the ligand; in some embodiments, the protein is an antibody or an antigen binding domain of the antibody, such as a single-chain antibody (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or a diabody.

[0176] Chimeric Antigen Receptor (CAR)

[0177] The present application also provides a chimeric antigen receptor (CAR) that binds to CD155, which comprises one or more antigen binding domains, a transmembrane domain and an intracellular signal transduction domain, wherein the one or more antigen binding domains comprise the aforementioned TIGIT extracellular region polypeptide.

[0178] In some embodiments, the one or more antigens further include one or more tumor antigens and / or immune checkpoint proteins in addition to CD155 that binds to the aforementioned TIGIT extracellular region polypeptide.

[0179] Wherein, in some embodiments, the one or more tumor antigens are selected from: PSCA, CEA, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD22, CD30, CD70, CD123, CD138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCA M, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSEA-4, MUC1, MUC6, EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, sLe, ganglioside GM3 (aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer, TGS5, HMWMAA, OAcGD2, folate receptor, CD248, TEM7R, Claudin6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, kappa light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, carcinoembryonic variant of tumor necrosis area, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K , OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155.

[0180] Wherein, in some embodiments, the one or more immune checkpoint proteins are selected from:

[0181] 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0182] In some embodiments, the CAR comprises multiple antigen binding domains, and the multiple antigen binding domains are connected in series with each other. In some embodiments, the CAR comprises multiple antigen binding domains, and at least two of the multiple antigen binding domains are connected to the transmembrane domain of the CAR in parallel with each other. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD4, CD8α, CD28 or CD3ζ.

[0183] In some embodiments, the CAR is directly connected to the transmembrane domain at one or more antigen binding domains, and the one or more antigen binding domains comprise the aforementioned TIGIT extracellular region polypeptide that binds to CD155.

[0184] In some embodiments, the CAR further comprises a hinge region between the one or more antigen binding domains and the transmembrane domain. In some embodiments, the CAR comprises multiple antigen binding domains, and at least two of the multiple antigen binding domains are respectively connected to the same hinge region. In some embodiments, the CAR comprises multiple antigen binding domains, and the multiple antigen binding domains are connected to at least two hinge regions. In some embodiments, the CAR comprises multiple antigen binding domains in series, and the multiple antigen binding domains in series are directly or indirectly connected to a hinge region. In some embodiments, the hinge region is selected from the hinge region of IgG, IgD, CD7, CD8α or CD28. In some embodiments, the intracellular signal transduction domain comprises a primary signal transduction domain, and the primary signal transduction domain is a signal transduction domain of CD3ζ, CD3γ, CD3δ, CD3ε, FcεRIγ, FcεR1β, CD79α, CD79β, FcγRIIa, DAP10 or DAP12 molecules. In some embodiments, the signal transduction domain further comprises a costimulatory domain, and the costimulatory domain is derived from the signal transduction domain of one or more of the following molecules: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83. In some embodiments, the signal transduction domain is the signal transduction domain of the CD3ζ molecule, and the co-stimulatory domain is from the signal transduction domain of 4-1BB, CD134, ICOS or CD28.In some embodiments, the signal transduction domain is the signal transduction domain of the CD3ζ molecule, and the co-stimulatory domain is from the signal transduction domains of 4-1BB and CD28.

[0185] In addition, the present application also provides a protein combination comprising any one or more of the above-mentioned fusion proteins, engineered receptors or CARs, such as a combination of the above-mentioned fusion proteins and CARs targeting any antigen. In some embodiments, the fusion protein comprises an extracellular region and an intracellular region, wherein the extracellular region comprises the aforementioned TIGIT extracellular region polypeptide, and the intracellular region comprises a co-stimulatory domain. In some embodiments, the fusion protein comprises an extracellular region and an intracellular region, wherein the extracellular region comprises the aforementioned TIGIT extracellular region polypeptide, and the intracellular region comprises a co-stimulatory domain, and the co-stimulatory domain is derived from the signal transduction domain of one or more co-stimulatory receptor molecules selected from the group consisting of: CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, I COS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the fusion protein comprises an extracellular region and an intracellular region, wherein the extracellular region comprises the aforementioned TIGIT extracellular region polypeptide, and the intracellular region comprises the signal transduction domain of CD28. In some embodiments, the fusion protein comprises an extracellular region, an intracellular region, and a transmembrane domain connecting the extracellular region and the intracellular region, wherein the extracellular region comprises the aforementioned TIGIT extracellular region polypeptide, the intracellular region comprises a costimulatory domain, and the transmembrane domain comprises a transmembrane domain selected from any one or more of the following molecules: TIGIT, ICOS, CD4, CD8α, CD28, and CD3ζ. In some embodiments, the fusion protein comprises an extracellular region, an intracellular region, and a transmembrane domain connecting the extracellular region and the intracellular region, wherein the extracellular region comprises the aforementioned TIGIT extracellular region polypeptide, the intracellular region comprises a signal transduction domain of CD28, and the transmembrane domain comprises the transmembrane domain of CD28.

[0186] In some embodiments, the protein combination comprises any one structure selected from the following:

[0187] 1) PSCA-8h-8TM-BBZ and TIGIT-28TM-28Z (1)

[0188] 2) CEA-8h-8TM-BBZ and TIGIT-28TM-28Z (1)

[0189] 3) CD123-8h-8TM-2B4Z and TIGIT-28TM-28Z (1)

[0190] 4) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28Z (1)

[0191] 5) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28Z (1)

[0192] 6) PSCA-7h-28TM-28Z and TIGIT-28TM-28Z (1)

[0193] 7) PSCA-8h-8TM-BBZ and TIGIT-28TM-28

[0194] 8) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28

[0195] 9) CEA-8h-8TM-BBZ and TIGIT-28TM-28

[0196] 10) CD123-8h-8TM-2B4Z and TIGIT-28TM-28

[0197] 11) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28; or

[0198] 12) PSCA-7h-28TM-28Z (1) and TIGIT-28TM-28;

[0199] Wherein, CEA is an antibody or an antigen-binding fragment thereof (e.g., scFv) that binds to CEA, CD123 is an antibody or an antigen-binding fragment thereof (e.g., scFv) that binds to CD123, PSCA is an antibody or an antigen-binding fragment thereof (e.g., scFv) that binds to PSCA, TIGIT is the aforementioned engineered receptor, 28TM is the transmembrane domain of CD28, 28 is the intracellular signal transduction domain of CD28, BB is the intracellular signal transduction domain of 4-1BB, Z(1) or Z(3) is the intracellular region of CD3ζ, 2B4 is the intracellular signal transduction domain of 2B4, 8h is the hinge region of CD8, 8TM is the transmembrane domain of CD8, G4h is the hinge region of Igg4, 7h is the hinge region of CD7, and "-" represents a peptide bond or a connecting peptide.

[0200] Engineered Nucleic Acid Molecules

[0201] The present application also provides an engineered nucleic acid molecule encoding the aforementioned engineered receptor, fusion protein or chimeric antigen receptor, the nucleic acid molecule comprising a target protein coding sequence, the target protein coding sequence can be expressed as the engineered receptor, fusion protein or chimeric antigen receptor. The term "engineered nucleic acid molecule" is used to distinguish it from a "natural nucleic acid molecule". "Natural nucleic acid molecule" refers to a nucleic acid molecule that exists in a natural form in nature. "Engineered nucleic acid molecule" is a restriction on the source or preparation method of the nucleic acid molecule, and does not constitute any restriction on its function or structure. Therefore, an engineered nucleic acid molecule can be used to refer to any nucleic acid molecule that can be obtained by any or multiple bioengineering means, which can have a polynucleotide sequence that is exactly the same as a natural nucleic acid molecule, have a modification that is exactly the same as a natural nucleic acid molecule, and even form a structure that is exactly the same as a natural nucleic acid molecule, but the difference between an engineered nucleic acid molecule and its corresponding natural nucleic acid molecule, or a natural nucleic acid molecule with the same polynucleotide sequence as it at least includes: the engineered nucleic acid molecule is not directly purified or extracted from an animal or plant that exists naturally in nature in its natural form.

[0202] In some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Therefore, the DNA molecule, in addition to comprising the target protein coding sequence, also comprises a gene manipulation or regulatory element for replication and / or expression in a prokaryotic and / or eukaryotic cell.

[0203] The necessary structural elements that make the engineered DNA molecule replicate or efficiently replicate in cells are known in the art, including, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further comprises a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of DNA elements, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing a plasmid comprising the marker gene, and can be selected from, for example, an antibiotic resistance gene. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0204] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double-stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in cells independently of the genomic genetic information stored in the nucleoid or nucleoid of prokaryotes, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are such vectors: they are based on pUC, pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) etc. that contain an ORI that supports high copies of plasmids.

[0205] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a prokaryotic nucleoid or nucleoid, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the target protein coding sequence or its complementary sequence can be replicated along with the prokaryotic genome.

[0206] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule also includes a coding sequence of an element that can be used to start or regulate the expression of the protein, polypeptide or its fragment after transcription, and the element includes but is not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal), etc. In some embodiments, the engineered DNA molecule includes a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR and the target protein coding sequence. In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR, the target protein coding sequence, the coding sequence of 3'UTR, the tailing signal (or the DNA sequence corresponding to the Ploy (A) tail sequence) from 5' to 3', and the start codon (5' end) and the stop codon (3' end) at both ends of the target protein coding sequence may be included, respectively, which are the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule, respectively. The 5'UTR usually contains at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the start of translation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, the 5'UTR includes an internal ribosome entry site or IRES. The 3'UTR may contain one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0207] On the basis of the aforementioned embodiments, in some embodiments, the target gene fragment further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for amino acid sequences that enhance the translation of mRNA molecules, regulatory elements (such as riboswitches), and / or nucleotide sequences that have a positive impact on translation initiation. In addition, within the 5'UTR, preferably there is no functional upstream open reading frame, an out-of-frame upstream translation initiation site, an out-of-frame upstream start codon, and / or a nucleotide sequence that produces a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0208] The target protein coding sequence comprises codons that can be translated into an amino acid sequence. All the codons contained in the target protein coding sequence may be naturally occurring codons encoding amino acids, or may be partially or entirely composed of artificially synthesized codons. In some embodiments, some or all of the codons have been codon-optimized. In some embodiments, some or all of the codons encode non-natural amino acids.

[0209] In some embodiments, the engineered DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end side of the target gene fragment, and the structural element is known in the art. In some embodiments, the structural element at least comprises a promoter. Promoters and their sequences are known in the art, including weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, etc. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in prokaryotes. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in eukaryotic cells. The "promoter" comprises at least one transcription recognition site and a subsequent transcription factor binding site. The recognition and binding site can interact with an amino acid sequence that mediates or regulates transcription. Compared with the recognition site, the binding site is closer to the aforementioned target gene fragment. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element of the enhancing promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence that promotes the transcription of the aforementioned target protein coding sequence. Strong promoters are known to those skilled in the art, such as OXB18, OXB19 and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises other regulatory elements before it, such as an enhancer that can promote the transcription of the aforementioned target protein coding sequence in a DNA plasmid.

[0210] In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the DNA molecule is a yeast display vector.

[0211] In addition, the present application also provides an engineered RNA molecule comprising an engineered receptor, fusion protein, engineered receptor or chimeric antigen receptor. In some embodiments, the engineered RNA molecule is obtained by transcription of the engineered DNA molecule. In some embodiments, the engineered RNA molecule has the same sequence as the RNA molecule obtained by transcription of the engineered DNA molecule. In some embodiments, the engineered RNA is mRNA. As used herein, "mRNA" (messenger RNA) is any RNA that encodes at least one protein, polypeptide or fragment thereof, naturally occurring, non-naturally occurring or modified, and the mRNA has the ability to be translated to produce the encoded protein, polypeptide or fragment thereof in vitro, in vivo, in situ or in vitro. Therefore, the mRNA can be a mature mRNA or a pre-mature mRNA, and the elements or structures that must be included or selectively included are known in the art. In some embodiments, the mRNA includes a plurality of necessary functional component coding sequences to express, regulate, or enhance the expression level of the protein, polypeptide or fragment thereof. The functional components include but are not limited to 5' cap, 5' UTR, 3' UTR, etc. Both the 5'UTR and the 3'UTR are usually transcribed from genomic DNA and are elements present in the pre-mature mRNA.

[0212] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate connection with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II respectively. Type O refers to the ribose of the terminal nucleotide is not methylated, type I refers to the ribose of one terminal nucleotide is methylated, and type II refers to the ribose of both terminal nucleotides is methylated. In some embodiments, the 5' cap can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG) according to the manufacturer's protocol. For example, in some embodiments, 5' capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from Type I cap structures followed by 2'-O-methylation of the 5'-third to last nucleotide using 2'-O methyl-transferase. Type III cap structures can be generated from Type II cap structures followed by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0213] In some embodiments, the mRNA further comprises a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop and optionally a poly(A) sequence or a polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop, although both have alternative mechanisms in nature, their synergistic effect can increase protein expression to a level that exceeds that observed for any single element. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is generally derived from a histone gene and comprises two adjacent partial or complete reverse complementary sequences separated by a spacer (composed of a short sequence) and the intramolecular base pairing forms a loop. Unpaired loop regions are generally unable to base pair with any of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0214] In some embodiments, one or more AU-rich sequences of the mRNA can be removed. These sequences are sometimes referred to as AURES, which are destabilizing sequences found in the 3'UTR. AURES can be removed from the mRNA. Alternatively, AURES can be retained in the mRNA.

[0215] In some embodiments, the mRNA is configured in a lipid nanoparticle (LNP). In some embodiments, lipids are mixed with the mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed into empty lipid nanoparticles and are combined or wrapped with the mRNA of the vaccine before being administered (e.g., within a few minutes to an hour).

[0216] The lipid nanoparticles generally include ionizable lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acids, such as the above-mentioned mRNA. Lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 037551, PCT / US2017 / 037552, PCT / US2017 / 037551 ... S2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575 and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[0217] In some embodiments, the engineered nucleic acid molecule may also be a hybrid molecule of DNA and RNA, wherein the hybrid molecule of DNA and RNA has the same genetic information as the engineered DNA molecule or the engineered RNA molecule.

[0218] Engineered cells

[0219] The present application also provides engineered cells, which express or contain on their cell membranes the aforementioned TIGIT extracellular domain polypeptide, fusion protein, engineered receptor, chimeric antigen receptor, or engineered nucleic acid molecule.

[0220] In some embodiments, the engineered cells are engineered immune cells. In some embodiments, the engineered cells are T cells, NK cells, macrophages, DC cells, B cells, or precursor cells thereof. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting one or more epitopes of CD155. In some embodiments, the engineered cells are TCR-T cells targeting one or more epitopes of CD155. In some embodiments, the engineered cells are TAC-T cells targeting one or more epitopes of CD155.

[0221] In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises a TIGIT extracellular region, a fusion protein, an engineered receptor or a chimeric antigen receptor as described above, and one or more CARs targeting other tumor antigens. In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises a TIGIT extracellular region, a fusion protein, an engineered receptor or a chimeric antigen receptor as described above, and one or more CARs targeting immune checkpoint proteins. In some embodiments, the engineered cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises a TIGIT extracellular region, a fusion protein, an engineered receptor or a chimeric antigen receptor as described above, one or more CARs targeting other tumor antigens, and one or more CARs targeting immune checkpoint proteins. In some embodiments, the engineered cell comprises the aforementioned engineered receptor, and the engineered cell comprises one or more CARs targeting other tumor antigens and / or one or more CARs targeting immune checkpoint proteins; wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain) or wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain).

[0222] In some embodiments, the engineered cell is a TCR-T cell targeting multiple different antigens, comprising a TIGIT extracellular region, a fusion protein, or an engineered receptor as described above, the antigen binding domain of the TCR comprising the aforementioned TIGIT extracellular region polypeptide, and the TCR-T further comprises one or more TCRs targeting other tumor antigens. In some embodiments, the engineered cell is a TCR-T cell targeting multiple different antigens, comprising a TIGIT extracellular region, a fusion protein, or an engineered receptor as described above, the antigen binding domain of the TCR comprising the aforementioned TIGIT extracellular region polypeptide, and the TCR-T further comprises a TCR targeting one or more immune checkpoint proteins. In some embodiments, the engineered cell is a TCR-T cell targeting multiple different antigens, comprising a TIGIT extracellular region, a fusion protein, or an engineered receptor as described above, the antigen binding domain of the TCR comprising the aforementioned TIGIT extracellular region polypeptide, and the TCR-T further comprises one or more TCRs targeting other tumor antigens, and one or more TCRs targeting immune checkpoint proteins. In some embodiments, the engineered cell comprises the aforementioned engineered receptor, and the engineered cell comprises one or more TCRs targeting other tumor antigens and / or one or more TCRs targeting immune checkpoint proteins; wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain) or wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain).

[0223] In some embodiments, the engineered cell is a TAC-T cell targeting multiple different antigens, which comprises the TIGIT extracellular region, fusion protein, or engineered receptor as described above, the antigen binding domain of the TAC comprises the aforementioned TIGIT extracellular region polypeptide, and the TAC-T further comprises one or more TACs targeting other tumor antigens. In some embodiments, the engineered cell is a TAC-T cell targeting multiple different antigens, which comprises the TIGIT extracellular region, fusion protein, or engineered receptor as described above, the antigen binding domain of the TAC comprises the aforementioned TIGIT extracellular region polypeptide, and the TAC-T further comprises a TAC targeting one or more immune checkpoint proteins. In some embodiments, the engineered cell is a TAC-T cell targeting multiple different antigens, which comprises the TIGIT extracellular region, fusion protein, or engineered receptor as described above, the antigen binding domain of the TAC comprises the aforementioned TIGIT extracellular region polypeptide, and the TAC-T further comprises one or more TACs targeting other tumor antigens, and one or more TACs targeting immune checkpoint proteins. In some embodiments, the engineered cell comprises the aforementioned engineered receptor, and the engineered cell comprises one or more TACs targeting other tumor antigens and / or one or more TACs targeting immune checkpoint proteins; wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain) or wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain).

[0224] In some embodiments, the engineered cell expresses or contains one or more engineered receptors on its cell membrane, and at least one of the engineered receptors contains the aforementioned TIGIT extracellular region polypeptide, and the engineered receptor binds to CD155 and activates or inhibits the downstream signaling pathway of the engineered receptor. In some embodiments, the engineered receptor is selected from one or more of CAR, TCR, TAC, and fusion protein. In some embodiments, the engineered cell expresses or contains one or more engineered receptors on its cell membrane, wherein the engineered receptor is selected from one or more of CAR, TCR, TAC, or fusion protein, and at least one of the engineered receptors contains the aforementioned TIGIT extracellular region polypeptide or fusion protein, and the engineered receptor binds to CD155 and activates or inhibits the downstream signaling pathway of the engineered receptor. In some embodiments, the engineered cell comprises the aforementioned engineered receptor, and the engineered cell comprises one or more TACs, CARs or TCRs targeting other tumor antigens and / or comprises one or more TACs, TCRs or CARs targeting immune checkpoint proteins; wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain) or wherein the aforementioned engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane domain-co-stimulatory domain (or secondary signal transduction domain).

[0225] In some embodiments, the engineered cells are selected from: T cells, NK cells, macrophages, DC cells, B cells, or precursor cells thereof.

[0226] In some embodiments, the tumor antigens described herein are selected from one or more of the following:

[0227] PSCA, CEA, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD22, CD30, CD70, CD123, CD138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDG FR-β, SSEA-4, MUC1, MUC6, EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII, NCAM, CAIX, LMP2, EphA2, fucosyl GM1, sLe, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer, TGS5, HMWMAA, OAcGD2, folate receptor, CD248, TEM7R, Claudin6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, κ light chain, CSPG 4. EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, carcinoembryonic variant of tumor necrosis, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AM L, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155.

[0228] In some embodiments, the immune checkpoint proteins described herein are selected from one or more of the following: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0229] use

[0230] The present application also provides the use of the aforementioned engineered TIGIT extracellular domain polypeptide, fusion protein, engineered receptor, chimeric antigen receptor, engineered nucleic acid molecule and engineered cell in the preparation of a drug for treating cancer. In some embodiments, the cancer is selected from one or more of the following:

[0231] Bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, and uterine cancer.

[0232] In some embodiments, the cancer involves tumor cells that highly express CD155.

[0233] In addition, the present application also provides the use of the aforementioned engineered TIGIT extracellular domain polypeptide, fusion protein, engineered receptor, chimeric antigen receptor or engineered cell as a TIGIT antagonist, which prevents TIGIT on the surface of immune cells from binding to CD155, thereby reversing tumor immunosuppression caused by the binding of TIGIT to CD155. Therefore, in some embodiments, the engineered TIGIT extracellular domain polypeptide, fusion protein, engineered receptor, chimeric antigen receptor or engineered cell can be administered in combination with other anticancer agents.

[0234] Example

[0235] Example 1: Screening of TIGIT mutant peptides

[0236] After random mutation of the TIGIT extracellular region, TIGIT mutant peptides were specifically screened by in vitro killing based on the principle of molecular recognition.

[0237] 1) Random mutation of TIGIT extracellular domain:

[0238] Method 1 was used to perform random mutations on the TIGIT extracellular region (SEQ ID NO: 1) to obtain 24 mutants Mut1 to Mut24 as shown in the sequence at the end of the article.

[0239] The mutation sites in mutants Mut1 to Mut24 relative to wild-type TIGIT are marked in bold.

[0240] A chimeric antigen receptor (CAR) is constructed using TIGIT as an antigen binding domain (or extracellular antigen binding domain). Table 1 below shows the correspondence between the CAR and the TIGIT mutants contained therein.

[0241] Table 1: CAR and its antigen binding domain

[0242]

[0243]

[0244] 2) Screening of mutant peptides: The mutant peptides obtained in 1) are constructed as extracellular antigen binding domains on the classic CD28TM-28Z (the amino acid sequence of CD28TM is shown in SEQ ID NO: 36, the amino acid sequence of 28 is shown in SEQ ID NO: 37, the amino acid sequence of Z is shown in SEQ ID NO: 41, and the amino acid sequences corresponding to Mut1 to Mut24 are shown in SEQ ID NO: 2 to SEQ ID NO: 25). Specifically, the CAR structure is: TIGIT mutant peptide-CD28TM-28Z, using common methods in the field such as enzyme cleavage and ligation. For specific steps, refer to Molecular Cloning Experiment Guide (3rd Edition, J. Sambrook et al.), 21st Century College Textbook "Gene Engineering" edited by Lou Shilin, Yang Shengchang et al., etc. Construct a vector target plasmid containing the gene encoding the CAR (CAR gene). The above target plasmid is transfected into a 293T derivative cell line by calcium transfection method for virus preparation. The titer calculation method is: Titer (TU / ml) = 1×10 5× positive rate × dilution factor ÷ virus volume × 1000. PBMCs or T cells obtained by Ficoll separation or apheresis, or cells frozen and revived from the above-obtained PBMCs or T cells, are used for CAR-T cell preparation. The obtained PBMCs or T cells are activated by anti-CD3 and anti-CD28 monoclonal antibodies or activated magnetic beads (CD3 / CD28 Dynabead (40203D; Gibco)) coated with anti-CD3 and anti-CD28 monoclonal antibodies, and virally transduced with CAR viruses prepared with the corresponding vectors in Table 2 to obtain CAR-T cells. The above-mentioned CAR-T cells are used to recognize CD155-positive and CD155-negative but CD112-positive target cells, respectively. Among them: the cell line used in MDA-MD-231-Luc-GFP is the MDA-MD-231 cell line, which is a CD155-positive and CD112-positive human breast cancer cell line; MDA-MD-231-CD155(-)-Luc-GFP is a CD155-negative but CD112-positive human breast cancer cell line. Figure 1 The expression of CD112 in the MDA-MB-231-Luc-GFP group is shown. The control (CT) group refers to the result of incubation of the MDA-MB-231 cell line with PBS, and the MDA-MB-231-Luc-GFP group refers to the result of co-incubation of MDA-MB-231-Luc-GFP with anti-CD112 antibody (CD112 (Nectin2) Monoclonal Antibody (R2.525) PE Lot: WC3208861A). Figure 2A-2E By screening mutant peptides that kill CD155-positive cells but have significantly reduced or no killing ability on CD155-negative cells, mutant peptides that can specifically recognize CD155 but do not recognize or have weakened recognition ability on CD112 were screened out. The effector-target ratio used was 1:1.

[0245] like Figures 2A to 2EAs shown: the horizontal axis represents CAR-T containing different TIGIT mutant peptides, and the vertical axis represents the in vitro killing of CD155 positive cells and negative cells by CAR-T cells. The above are the specific screening killing results of 24 CAR-Ts. According to the specific screening criteria, the Mut6 mutant peptide corresponding to CAR6 is determined, that is, relative to the wild type, the mutant peptide with a mutation from C to W at position 48 can specifically recognize CD155 in biological activity and significantly reduce the recognition of CD112. The ratio of the binding activity of the TIGIT mutant peptide sequences corresponding to CAR4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 21, 23, and 24 to CD155 is also significantly higher than that of the wild type, so the corresponding TIGIT mutant peptide sequences SEQ ID NO: 5-17, SEQ ID NO: 19 and SEQ ID NO: 22, 24, 25 are also optional sequences of the present application.

[0246] The sequences used for each TIGIT mutant peptide and each element in the CAR structure used in the examples of this application are shown in the sequence table at the end of the article.

[0247] CAR structural elements: The CAR structural elements described in this article are structural regions that constitute the chimeric antigen receptor, such as the extracellular recognition region (such as the TIGIT sequence described in this application, the extracellular recognition sequence that recognizes PSCA, CD123, and CEA), the hinge region (sometimes also called the spacer region, a sequence extending from the transmembrane domain to the binding region, such as 8h, 7h, G4h, etc. described in this application), the transmembrane region, and the intracellular signaling region (which may include activation signals and / or co-stimulatory signals). The molecules that constitute the above structure are structural elements.

[0248] Example 2: Verification of specific recognition sites

[0249] Analysis of the Mut6 sequence revealed that the amino acid at position 48 mutated from C to W. In order to determine whether the mutation of amino acid 48 to a key amino acid can improve specificity, this mutant peptide and wild-type TIGIT (WT) were used as extracellular antigen binding domains to construct the classic CD28TM-28Z CAR structure, which is: TIGIT peptide-CD28TM-28Z, forming CAR6 and CAR30, wherein CAR30 is a control CAR structure (WT) containing a wild TIGIT polypeptide.

[0250] 1) CAR6 and CAR30 were packaged into viruses and infected into CHO cells. First, TIGIT antibody was labeled by flow cytometry to confirm the successful preparation of CAR-CHO. Then CD155 and CD112 molecules were labeled and the binding of different TIGIT mutant peptides to CD155 and CD112 was detected by flow cytometry to determine the binding specificity of different TIGIT mutants to them.

[0251] Data analysis method: The flow cytometry data generated by method 1) were analyzed. The analysis process was to first calculate the MFI (mean fluorescence intensity) value of the positive cell population in which TIGIT antibody binds to TIGIT mutants, and then calculate the MFI value of the positive cell population in which TIGIT mutants bind to CD155 antigen and CD112 antigen.

[0252] CD155 relative MFI = CD155 positive cell population MFI / TIGIT positive cell population MFI

[0253] CD112 relative MFI = CD112 positive cell population MFI / TIGIT positive cell population MFI

[0254] The relative MFI values ​​of CD155 and CD112 between groups were statistically analyzed with the values ​​of CAR30 by T Test, and P < 0.05 indicated a significant difference.

[0255] like Figure 3 The horizontal axis represents different CAR-CHO, and the vertical axis represents the relative MFI value of CD112. The results show that CAR6 and CD112 have a weaker binding.

[0256] Table 2 shows the statistical analysis results of the binding of CAR6 and CAR30 to CD112

[0257] Table 2

[0258]

[0259] As shown in Table 2 above, the first column represents the CAR molecules, the second column represents the relative MFI value of CD112, and the numerical meaning is that the lower the value compared with CAR30, the weaker the binding to CD112, and the third column represents the significance analysis results through T Test.

[0260] Conclusion: According to Figure 3 As shown in Table 2, the binding ability of CAR6 to CD112 was significantly reduced compared with the wild type.

[0261] Figure 4 The binding status of CAR6 and CAR30 with CD155 is shown. Figure 4The horizontal axis represents different CAR-CHO molecules, and the vertical axis represents the relative MFI value of CD155.

[0262] Table 3 shows the analysis of the binding ability of four TIGIT mutant peptides to CD155.

[0263] Table 3

[0264]

[0265] As described in Table 3, the first column represents the CAR molecules, the second column represents the relative MFI value of CD155, and the numerical meaning is that the higher the value compared with CAR30, the stronger the binding to CD155 is, and the third column represents the significance analysis results through T Test.

[0266] Conclusion: According to Figure 4 As described in Table 3, the results showed that the binding ability of CAR6 to CD155 was significantly increased compared with the wild type.

[0267] Summary: Comprehensive analysis determined that CAR6 had significantly higher binding to CD155 and significantly lower binding to CD112 compared to the wild type.

[0268] In summary, the mutant TIGIT in which the amino acid at position 48 can mutate to W (mut6) is a more specific peptide.

[0269] 2) Further, the functional performance of the above mutant peptides on T cells was verified by in vitro killing.

[0270] CAR-T cells were prepared using the scheme of Example 1, and viruses containing CAR6 and CAR30 vector genes were prepared for viral transduction. MDA-MB-231-CD155(-)-Luc-GFP was used as target cells, which did not express CD155 but expressed CD112. The above-prepared cells containing CAR6 and CAR30 were killed at a 1:1 effector-target ratio. Three batches (3 types of white membranes) were used to prepare CAR-T cells to detect the positive rate of CAR-T, and three in vitro functional evaluations were performed: the mutant peptide was verified as an extracellular recognition domain to kill CD155-positive cells and CD155-negative but CD112-positive cells in vitro, and the killing results were statistically analyzed using T TEST.

[0271] Figure 5The killing of MDA-MB-231-CD155(-)-Luc-GFP by CAR6 and CAR30 is shown. The results are the statistical results of in vitro killing of 3 batches of CAR-T preparations. The horizontal axis represents different CAR molecules, and the vertical axis represents the in vitro killing ratio (%) of MDA-MB-231-CD155(-)-Luc-GFP. The results show that CAR6 significantly reduces the killing of MDA-MB-231-CD155(-)-Luc-GFP. * represents P < 0.05, and ** represents P < 0.01.

[0272] Conclusion: In summary, CAR6 significantly reduced the killing effect of MDA-MB-231-CD155(-)-Luc-GFP, among which the TIGIT peptide corresponding to CAR6 is the preferred peptide segment of this application, that is, amino acid 48 can be mutated to W (mut6).

[0273] 3) In vivo effectiveness of TIGIT mutant peptide as the extracellular recognition domain of CAR structure

[0274] The expression of CD155 on target cells was detected by flow cytometry. As shown in Table 4, the expression of CD155 in HT1376-Luc-GFP and HPAC-Luc-GFP cells was shown.

[0275] Table 4

[0276] cell CD155 expression positive rate MDA-MB-231-Luc-GFP 100％ HT-1376-Luc-GFP 100％ HPAC-Luc-GFP 100％

[0277] Female NCG mice aged 6-8 weeks were selected, and MDA-MB-231-Luc-GFP cells were intravenously injected into the tail of the mice at a volume of 3×10^5 / mouse to construct a tumor-bearing model. On the 3rd day of tumor formation, the above-mentioned CAR30 and CAR6 T cells were intraperitoneally injected at a volume of 5×10^5CAR-T cells / mouse; the Control T group was infused with the same total number of T lymphocytes on the 3rd day. The results are shown in Figure 6 As shown, the in vivo efficacy evaluation of the CAR structure with the above mutant peptide as the extracellular recognition domain of the CAR structure is shown in the MDA-MB-231 tumor model.

[0278] The results showed that there was no significant difference in the efficacy of CAR30 and CAR6 in the MDA-MB-231-Luc-GFP tumor model, proving that the TIGIT mutant peptide of the present invention, as the extracellular recognition domain of the CAR structure, can achieve effective killing of CD155 target cells in vivo, and its effectiveness is better than that of the wild-type TIGIT peptide.

[0279] Example 3: Verification of the applicability of mutant peptides as the extracellular antigen binding domain of CAR structures in different CAR structures

[0280] Use Mut6 to design different CAR structures, select TIGIT's own hinge or 7h (CD7 hinge region) / G4h / 8h (CD8 hinge region), select different transmembrane CD28TM (CD28 transmembrane domain), ICOSTM (ICOS transmembrane domain) or CD8TM (CD8 transmembrane domain), select costimulatory domains from different costimulatory receptor molecules, the costimulatory receptor molecules include CD28, ICOS, CD134, 4-1BB, select different CD3 including Z or Z (1) or Z (3) and select the structural design of second-generation CAR and third-generation CAR, forming the CAR structure design as shown in Table 6 below, wherein Z or Z (1) or Z (3) is a different mutant peptide of CD3ζ, which is used as an intracellular signal sequence of the CAR structure in different public texts. In Table 5, except for the "-" of 4-1BB, which is the name of the molecule itself, the remaining "-" all indicate that they are connected by peptide bonds or linkers (such as short peptides). Structures without "-" can be connected directly or through a linker (such as a short peptide).

[0281] Table 5

[0282]

[0283] The exemplary amino acid sequences used for each element in Table 5 are shown in the sequence listing at the end of the text. Among them, the amino acid sequence of Mut6 is shown in SEQ ID NO:7, the amino acid sequence of 28TM (i.e., the transmembrane sequence derived from human CD28, also represented by CD28TM, which is a transmembrane domain) is shown in SEQ ID NO:36, the amino acid sequence of 28 (i.e., the intracellular signal sequence derived from human CD28, also represented by CD28 in this application) is shown in SEQ ID NO:37, the amino acid sequence of Z(1) (which is the intracellular signal sequence derived from human CD3ζ) is shown in SEQ ID NO:42, the amino acid sequence of ICOSTM (i.e., the transmembrane sequence derived from human ICOS) is shown in SEQ ID NO:44, the amino acid sequence of ICOS (i.e., the intracellular signal sequence derived from human ICOS) is shown in SEQ ID NO:46, the amino acid sequence of Z (which is the intracellular signal sequence derived from human CD3ζ) is shown in SEQ ID NO:41, and the amino acid sequence of CD28-4-1BB (a costimulatory signal combination composed of the CD28 intracellular signal sequence and the 4-1BB intracellular signal sequence) is shown in SEQ ID NO:47. NO:40, the amino acid sequence of Z(3) (i.e., the intracellular signal sequence derived from human CD3ζ) is shown in SEQ ID NO:43, the amino acid sequence of 7h (i.e., the hinge sequence derived from human CD7) is shown in SEQ ID NO:33, the amino acid sequence of 8h (i.e., the hinge sequence derived from human CD8) is shown in SEQ ID NO:32, the amino acid sequence of G4h (i.e., the hinge sequence derived from IgG4) is shown in SEQ ID NO:34, the amino acid sequence of 8TM (i.e., the transmembrane sequence derived from human CD8, also represented by CD8TM) is shown in SEQ ID NO:35, the amino acid sequence of BB (i.e., the intracellular signal sequence derived from human 4-1BB (also known as CD137)) is shown in SEQ ID NO:38, and the amino acid sequence of 134 (i.e., the intracellular signal sequence derived from human CD134) is shown in SEQ ID NO:45.

[0284] MDA-MB-231-Luc-GFP, HPAC-Luc-GFP, and HT-1376-Luc-GFP cells expressing CD155 with different indications were used as positive target cells, among which MDA-MB-231-Luc-GFP was for human breast cancer indication, HPAC-Luc-GFP was for human pancreatic cancer indication, and HT-1376-Luc-GFP was for human bladder cancer indication; CAR-T cells were plated in the target cells at a ratio of 1:1, and the ACEA xCELLigence RTCA MP instrument was used to detect the killing ability of different CAR-T on target cells. The experimental steps were carried out according to the instrument manual. The principle of ACEA xCELLigence RTCA MP is to record the resistance index of tumor cells attached to the bottom of the well every 15 minutes, and judge the proliferation or death of the attached target cells by the resistance index. The formula for analyzing the results using the resistance index is: cell killing rate (%) = (Cell Index value of the control group - Cell Index value of the experimental group) / (Cell Index value of the control group) × 100%.

[0285] The results are as follows Figures 7A-7C As shown, the horizontal axis represents different CARs, and the vertical axis represents the killing effects of different CARs on MDA-MB-231-Luc-GFP, HPAC-Luc-GFP, and HT1376-Luc-GFP. The results showed that the mutated TIGIT, as the extracellular recognition domain of various CAR structures, has a significant killing effect on various malignant tumors expressing CD155, such as breast cancer, pancreatic cancer, and bladder cancer.

[0286] Furthermore, the above CAR-T was tested for cytokines after being activated by target cells. The cytokine IFN-γ detection was performed using the Elisa method and a BD kit. The test kit item number is 555142, the production batch number is 6266958, and the specific steps are shown in the kit instructions. The cytokine secretion results are shown in Table 6 below, which shows the secretion of IFN-γ factors by the above 7 CARs with different structures in the process of killing 3 target cells.

[0287] Table 6

[0288] Factor secretion MDA-MB-231-Luc-GFP HT-1376-Luc-GFP HPAC-Luc-GFP CAR31 23371.516 / / CAR32 45592.297 / / CAR33 / 2582.139 1239.714 CAR6 / 16583.42 9826.8 CAR36 23333.294 / 6779.085 CAR34 75127.335 / 14634.484 CAR35 47706.754 / 16733.595 Control-T 0 0 0

[0289] As shown in Table 6 above, in the experiments targeting different target cells, while there was no factor secretion in the Control-T group, there was obvious cytokine secretion in the CAR-T cell group.

[0290] The above Figure 7 and Table 6 analyzed the in vitro killing and factor secretion of CAR-T cells for 7 different CAR structures with mutant TIGIT peptide as extracellular recognition domain and multiple different malignant tumor indications, proving that TIGIT as an extracellular recognition domain can adapt to all different CAR structures, and has significant recognition and functional killing effects on different solid tumor cells, including but not limited to CD155-expressing malignant tumors such as breast cancer, pancreatic cancer and bladder cancer.

[0291] Example 4: Application verification of mutant peptide as the extracellular antigen binding domain of CAR structure in dual CAR

[0292] Different dual CAR structures were designed based on CAR6, targeting the targets PSCA, CD123, CEA and TIGIT mutant peptides respectively. Different hinges, i.e. 8h, G4h and 7h, different transmembrane CD8TM and CD28TM, different co-stimulatory signals, i.e. 4-1BB, 2B4, CD28, CD28-4-1BB and different CD3Z, i.e. Z, Z(3), and different signal combinations were verified during the CAR structure design. The CAR structures are shown in Table 7.

[0293] Table 7

[0294]

[0295]

[0296] The above CAR-T cells were prepared using the method of Example 1. The sequence information of each element is summarized as shown in the sequence table at the end of the article. Among them, the amino acid sequence of PSCA (1) (recognizing the extracellular recognition sequence of PSCA) is shown in SEQ ID NO: 27, the amino acid sequence of 8h is shown in SEQ ID NO: 32, the amino acid sequence of 8TM is shown in SEQ ID NO: 35, the amino acid sequence of BB is shown in SEQ ID NO: 38, the amino acid sequence of Z is shown in SEQ ID NO: 41, the amino acid sequence of P2A is shown in SEQ ID NO: 31, the amino acid sequence of mut6 is shown in SEQ ID NO: 7, the amino acid sequence of 28TM is shown in SEQ ID NO: 36, the amino acid sequence of 28 is shown in SEQ ID NO: 37, the amino acid sequence of Z (1) is shown in SEQ ID NO: 42, the amino acid sequence of CEA (i.e., the antigen binding domain that binds to CEA) is shown in SEQ ID NO: 29, the amino acid sequence of CD123 (i.e., the antigen binding domain that binds to CD123) is shown in SEQ ID NO: 30, the amino acid sequence of 2B4 is shown in SEQ ID NO: 39, and the amino acid sequence of PSCA (9) (recognizing the extracellular recognition sequence of PSCA) is shown in SEQ ID NO: NO:28, the amino acid sequence of G4h is shown in SEQ ID NO:34, the amino acid sequence of 28-4-1BB is shown in SEQ ID NO:40, the amino acid sequence of Z(3) is shown in SEQ ID NO:43, and the amino acid sequence of 7h is shown in SEQ ID NO:33.

[0297] 1) Detection of antigen expression in target cells

[0298] Flow cytometry was used to detect target cell antigen expression. As shown in Figure 8, the horizontal axis is the antigen fluorescence intensity. The positive expression rate of CEA in DLD1-CEA-Luc-GFP is 100%, and CEA is very weakly expressed in DLD1-Luc-GFP. The positive expression rate of CD155 in DLD1-CEA-Luc-GFP is 100%; the difference between DLD1-CEA-Luc-GFP and DLD1-Luc-GFP is only the exogenously expressed CEA molecule, so the CD155 of DLD1-Luc-GFP is also 100% expressed.

[0299] The dual-target CAR-T designed in Table 8 containing mutant TIGIT as the extracellular domain was used to verify the dual-target effectiveness. The specific CAR-T preparation and killing process are shown in Examples 1 and 3. The following verifications were performed on the dual-target CAR-T targeting CEA×CD155, CD123×CD155, and PSCA×CD155, respectively.

[0300] Table 8: Antigen expression of different target cells

[0301] cell CD155 expression Second antigen expression Molm13-Luc-GFP 100％ 100%(CD123) PC3-PSCA-Luc-GFP 100％ 100% (PSCA) HT-1376-Luc-GFP 100％ 80% (PSCA) HPAC-Luc-GFP 100％ 60% (PSCA)

[0302] As shown in Figure 9 above, CAR37 has significant killing effects on both DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP, and the killing effect on DLD-1-CEA-Luc-GFP is significantly higher than that on DLD-1-Lcu-GFP. The main reason is that the dual CAR recognizes the CEA and CD155 of DLD-1-CEA-Luc-GFP cells and kills them more than the one target antigen of DLD-1-Luc-GFP, namely CD155, which shows higher killing effect, proving that the dual CAR can exert better in vitro pharmacodynamic effects; CAR38 has significant killing effect on Molm13-Luc-GFP, proving that CAR38 can exert significant in vitro pharmacodynamic effects in hematological tumors, such as human acute myeloid leukemia tumor cell models; as shown above Fig. 9C As shown, the horizontal axis represents different CARs, and the vertical axis represents the in vitro killing percentage in different cells. The results show that CAR39, CAR36, CAR40 and CAR41 have significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, and their killing percentages are all higher than 50%, proving that different CARs can exert obvious in vitro pharmacodynamic effects in various solid tumor cell models.

[0303] Furthermore, the above CAR-T cells were tested for cytokines after being activated by target cells. The cytokine IFN-γ was tested using the Elisa method and a BD kit. The kit number is 555142, the production batch number is 6266958, and the specific steps are shown in the kit manual. The cytokine secretion results are shown in Table 9 below.

[0304] Table 9: Summary of IFN-γ secretion by five CARs during in vitro cell killing

[0305]

[0306] By analyzing the in vitro killing and factor secretion of five types of CAR-T cells, it was demonstrated that the mutant TIGIT peptide can be used as one of the extracellular recognition domains of dual-CAR and multi-target CAR structures, and is applicable to multiple indications for hematological tumors and different solid tumors.

[0307] Example 5. Verification of the combined application of fusion protein constructed by mutant peptide and CAR targeting non-CD155 targets

[0308] In addition to the above-mentioned extracellular recognition domain as a single CAR or dual CAR structure for direct tumor antigen recognition and initiation of T cell killing, the TIGIT mutant peptide described in the present invention can also be designed to form a TIGIT mutant peptide fusion protein (in the embodiments and drawings of the present application, it specifically refers to an engineered receptor that does not contain a primary signal transduction domain in the signal transduction domain, such as an engineered receptor composed of an antigen binding domain, a transmembrane domain and a co-stimulatory domain from the N-terminus to the C-terminus, or an engineered receptor composed of an antigen binding domain, a hinge region, a transmembrane domain and a co-stimulatory domain from the N-terminus to the C-terminus), which is designed in combination with the CAR structure to transform the immune microenvironment and promote the effectiveness of CAR-T cells.

[0309] 1) The inventors designed mut6-28TM-28 and combined it with different CARs to verify the adaptability of TIGIT mutant peptides to different transmembrane and intracellular signals. TIGIT mutations are adaptable to a variety of peptide transmembrane and intracellular signals.

[0310] 2) The inventors also made different designs for the CAR structure and considered the compatibility of different targets such as PSCA, CD123 and CEA with TIGIT mutant peptide fusion proteins. The structural design is shown in Table 10 below.

[0311] Table 10: CAR structure design

[0312] CAR structure CAR43 PSCA(1)-8h-8TM-BBZ-P2A-mut6-28TM-28 CAR44 PSCA(9)-G4h-28TM-CD28-4-1BBZ(3)-P2A-mut6-28TM-28 CAR45 CEA-8h-8TM-BBZ-P2A-mut6-28TM-28 CAR42 CEA-8h-8TM-BBZ CAR46 CD123-8h-8TM-2B4Z-P2A-mut6-28TM-28 CAR47 PSCA(9)-G4h-28TM-CD28-4-1BBZ(3)-P2A-mut6-28TM-28 CAR48 PSCA(9)-7h-28TM-28Z(1)-P2 A-mut6-28TM-28

[0313] The sequences of the components in the above table are shown in the sequence table at the end of the article. Among them, the amino acid sequence of PSCA (1) is shown in SEQ ID NO: 27, the amino acid sequence of 8h is shown in SEQ ID NO: 32, the amino acid sequence of 8TM is shown in SEQ ID NO: 35, the amino acid sequence of BB is shown in SEQ ID NO: 35, the amino acid sequence of Z is shown in SEQ ID NO: 41, the amino acid sequence of P2A is shown in SEQ ID NO: 31, the amino acid sequence of mut6 is shown in SEQ ID NO: 7, the amino acid sequence of 28TM is shown in SEQ ID NO: 36, the amino acid sequence of 28 is shown in SEQ ID NO: 37, the amino acid sequence of PSCA (9) is shown in SEQ ID NO: 28, the amino acid sequence of G4h is shown in SEQ ID NO: 34, the amino acid sequence of CD28-4-1BB is shown in SEQ ID NO: 40, the amino acid sequence of Z (3) is shown in SEQ ID NO: 43, the amino acid sequence of CEA (i.e., the antigen binding domain that binds to CEA) is shown in SEQ ID NO: 29, and the amino acid sequence of CD123 (i.e., the antigen binding domain that binds to CD123) is shown in SEQ ID NO: The amino acid sequence of 2B4 is shown in SEQ ID NO:39, the amino acid sequence of 7h is shown in SEQ ID NO:33, and the amino acid sequence of Z(1) is shown in SEQ ID NO:42.

[0314] CAR-T cells were prepared using the scheme of Example 1, CAR (CAR42) targeting the CEA target was selected, CAR45 with a TIGIT mutant peptide fusion protein was designed, DLD-1-CEA-Luc-GFP and DLD1-Luc-GFP were used as target cells, the effector-target ratio was selected as 1:1, and the method of Example 3 was used to verify the in vitro killing ability of CAR42 and CAR45 to evaluate the effectiveness of CAR-T cells expressing the fusion protein.

[0315] like Fig.10 As shown, the horizontal axis is CAR42 and CAR45, and the vertical axis is the killing percentage of the two cells to DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP. The results showed that CAR42 and CAR45 had obvious killing effects on DLD-1-CEA-Luc-GFP, and the killing effect of CAR45 on DLD-1-Luc-GFP was significantly higher than that of the CAR42 group. It can be seen that CAR-T expressing fusion protein is superior to CAR-T without fusion protein in terms of effective killing ability.

[0316] like Fig.11As shown, the horizontal axis represents CAR42 and CAR45, and the vertical axis represents their IFN-γ cytokine secretion. The results showed that the factor secretion of CAR45 was significantly higher than that of CAR42, proving that CAR combined with TIGIT mutant peptide to form a fusion protein can enhance cytokine secretion during the killing process.

[0317] Female NCG mice aged 6-8 weeks were selected, and DLD-1-CEA-Luc-GFP cells were injected intraperitoneally at a volume of 3×10^5 / mouse at the tail of the mouse to construct a tumor-bearing model. Seven days after tumor formation, the above-mentioned CAR42 and CAR45T cells were injected into the tail vein at a volume of 5×10^5CAR-T cells / mouse; the Control T group was re-infused with the same total number of T lymphocytes on the 7th day. In vivo imaging of mice was performed every 7 days after administration to detect the retention of tumor cells in the mouse body. The imaging fluorescence values ​​were statistically analyzed using T Test for significance. The results are shown in Figure 2. Fig.12 As shown. In the peritoneal tumor efficacy evaluation model, blood was collected from the midpoint of the mice's eye sockets, and the number of CAR-T copies in the blood was detected by fluorescent quantitative PCR. The results showed that the number of CAR-T copies in the blood of CAR45 on the 7th, 20th and 27th days after CAR-T administration was significantly higher than that of CAR42, proving that CAR combined with TIGIT mutant peptide to form a fusion protein, namely CAR45, can significantly improve the persistence of CAR in the in vivo model. Fig.13 It shows the blood copy number in the peritoneal tumor evaluation model.

[0318] DLD-1-CEA-Luc-GFP was used to form tumors subcutaneously. CAR-T cells were reinfused into the tail vein 12 days after tumor formation. Live imaging of mice was performed every 7 days after administration to detect the retention of tumor cells in the mice. The fluorescence values ​​of the imaging were statistically analyzed using T Test for significance. Fig.14 shown.

[0319] The results showed that the in vivo efficacy of CAR45 was significantly better than that of CAR42, proving that the CAR45 CAR-T cells formed by CAR combined with TIGIT mutant peptide fusion protein can significantly improve the in vivo efficacy. And the effectiveness comes from the recognition of the CD155 target by the mutant TIGIT of CAR45, further proving the feasibility of TIGIT mutant peptide as one of the extracellular recognition domains of multi-target CAR structure.

[0320] Furthermore, the inventors have also verified the applicability of TIGIT mutant peptides as one of the extracellular recognition domains of multi-target CAR products targeting CD155 targets in various indications such as acute myeloid leukemia, pancreatic cancer, bladder cancer and other blood system tumors and solid tumors. The horizontal axis represents different CARs, and the vertical axis represents the in vitro killing percentage in different cells. Fig.15A As shown above, CAR46 has a significant killing effect on Molm13-Luc-GFP. Fig. 15B As shown, the results showed that CAR44, CAR43, CAR47 and CAR48 all had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, proving that dual-target and multi-target CAR-Ts using TIGIT mutant peptides as extracellular recognition domains targeting CD155 targets and any other malignant tumor targets can exert significant in vitro pharmacodynamic effects in various solid tumor cell models.

[0321] The above CAR-T cells were activated by target cells to detect cytokines. The cytokine IFN-γ was detected by Elisa method using a BD kit. The kit number is 555142, the production batch number is 6266958, and the specific steps are shown in the kit instructions. The cytokine secretion results are shown in Table 11 below.

[0322] Table 11: Summary of IFN-γ secretion by five CARs during in vitro cell killing

[0323]

[0324]

[0325] By analyzing the in vitro killing and factor secretion of five types of CAR-T cells, it was demonstrated that different CARs had obvious recognition and functional killing effects on hematological tumor cell lines and different solid tumor cell lines, namely Molm13-Luc-GFP, HT1376-Luc-GFP, HPAC-Luc-GFP, and PC3-PSCA-Luc-GFP. The TIGIT mutant peptide, as one of the extracellular recognition domains of the multi-target CAR structure containing the CD155 target, is applicable to a variety of hematological tumors and solid tumors.

[0326] It should be noted that although the present invention takes CAR-T cells as an example, based on the contents disclosed in the present invention, technicians in this field can obviously infer that other immune cells such as NK cells, macrophages, DC cells and related prerequisite cells can also become single-target CARs, dual-target or multi-target CARs designed to express mutant TIGIT, as well as fusion proteins expressing mutant TIGIT.

[0327] The inventors constructed TIGIT single CARs with different CAR structures to prepare CAR-NK cells and verified the expression. The results are shown in Table 12.

[0328] Table 12

[0329] CAR structure NK cell source Virus titer CAR expression positive rate mut6-28TM-28Z B-756 4.57E+07 76.15％ mut6-8h-28TM-28Z B-771 5.88E+08 69.74％ mut6-8h-8TM-BBZ B-758 7.88E+08 70.94％ mut6-G4h-28TM-28Z B-759 3.68E+08 52.53％

[0330] Example 6: Specificity and functional verification of truncated TIGIT containing the mutation site described in the present invention

[0331] Based on Mut6, we truncated it and obtained the following truncated sequence (the sequence contains the polypeptide fragment of positions 33-93 of the amino acid sequence shown in the reference sequence SEQ ID NO: 1):

[0332] VTQVNWEQQDQLLAIWNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYF CIYHTYP (SEQ ID NO: 26, the mutation site at position 48 relative to the reference sequence is located at position 16 in this sequence).

[0333] The scheme of Example 2 was used to verify the specificity of the truncated TIGIT mutant peptide. The results showed that the truncated TIGIT peptide containing the mutation site we described (reference SEQ ID NO: 1, position 48 C mutated to W) also had weak recognition for CD112 and better specific recognition ability for CD155.

[0334] The schemes of Example 3 and Example 5 were used to verify the application of truncated TIGIT in constructing CAR structures and fusion proteins, and in engineered cells, proving that the truncated TIGIT mutant peptide is applicable as one of the extracellular recognition domains or domains of single-target or multi-target CAR structures containing the CD155 target in a variety of hematological tumors and solid tumors; and that the truncated TIGIT constructed fusion protein can be expressed in engineered cells alone or in combination with CAR to play the role of transforming the tumor microenvironment.

[0335] Based on the results of the above-mentioned truncated TIGIT mutant peptides and non-truncated TIGIT mutations, we can prove that as long as the TIGIT peptides contain the mutation site we discovered (reference SEQ ID NO: 1, position 48 C mutated to W) and the polypeptide fragment corresponding to positions 33-93 of the reference sequence in the TIGIT extracellular segment, they have better specific recognition ability for CD155.

[0336] The corresponding sequences of the components used in the examples of the present application are shown in the following sequence table.

[0337] Sequence Listing

[0338]

[0339]

[0340]

Claims

1. A TIGIT extracellular domain polypeptide comprising a mutation at position 48 relative to a reference sequence, wherein the reference sequence is an amino acid sequence as shown in SEQ ID NO: 1, and wherein the numbering of amino acid positions is defined by the reference sequence.

2. The TIGIT extracellular domain polypeptide according to claim 1, which comprises at least the amino acids corresponding to positions 33 to 93 of the reference sequence.

3. The TIGIT extracellular domain polypeptide according to claim 1 or 2, wherein the mutation is selected from any one of the following: C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H and C48 deletion.

4. The TIGIT extracellular region polypeptide according to any one of claims 1 to 3, further comprising one or more amino acid sites selected from the following: 2M, 2W, 2T, 9T, 9S, 12I, 12N, 14A, 14V, 20I, 20T, 21I, 21F, 22L, 22F, 34T , 34S, 37N, 37D, 37E, 39E, 39G, 39K, 39V, 42D, 42G, 44L, 44F, 61K, 61R, 7 0L, 70Q, 70P, 71G, 71D, 76S, 76P, 79V, 79E, 80N, 80Y, 86F, 86S, 86L, ​​101 I, 101T, 102S, 102F, 106L, 106Q, 110V, 110Y, 110E, 110A, 113H, and 113Y.

5. The TIGIT extracellular region polypeptide according to claim 4, further comprising a combination of amino acid sites of any one of the following 1)-12): 1) 22F, 37D, 39G; 2)37D； 3)20T, 37D, 39K, 44F; 4)22L, 37E, 71D, 102S; 5)20T, 37D, 42G, 70Q; 6)21F, 34S, 37E, 39K, 70P, 80Y, 101T, 110A; 7)9S, 37D, 39V, 61R, 101T; 8) 20T, 37D, 39K; 9) 37D, 86S, 113Y; 10)2W, 37D, 56F; 11) 12N, 14V, 21F, 37D, 39K, 70Q; or 12)2T, 37D, 39K, 86L.

6. The TIGIT extracellular region polypeptide according to claim 1, comprising any one of the following amino acid sequences or a conservatively substituted variant of any one of the following amino acid sequences: SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

7. A fusion protein comprising the TIGIT extracellular domain polypeptide according to any one of claims 1 to 6.

8. The fusion protein according to claim 7, further comprising one or more polypeptides that bind to tumor antigens and / or immune checkpoint proteins, optionally, the polypeptides that bind to tumor antigens and / or immune checkpoint proteins are antibodies, ligands or receptors of the tumor antigens and / or immune checkpoint proteins, optionally, the antibodies of the tumor antigens and / or immune checkpoint proteins are single-chain antibodies (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or diabodies.

9. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the TIGIT extracellular region polypeptide according to any one of claims 1-6, or the fusion protein of claim 7 or 8.

10. An engineered receptor according to claim 9, further comprising a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

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