Specific tigit peptide fragment

Abstract

Provided are a TIGIT-derived polypeptide or a mutant thereof, and a fusion protein, engineered receptor or engineered cell containing the polypeptide or the mutant, wherein the binding capacity of the TIGIT-derived polypeptide or mutant thereof to CD112 is reduced.

Description

Specific TIGIT peptide 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, and has achieved great results. However, its application effect on solid tumors is poor, mainly due to the antigenic heterogeneity and immunosuppressive microenvironment of solid tumors.

[0003] CD155 is the fifth member of the nectin-like molecule family and functions as a poliovirus receptor. Therefore, CD155 is also known as necl-5 or PVR (poliovirus receptor). As an immunoglobulin-like adhesion molecule, CD155 participates in cell motility, natural killer cell-mediated immunity, 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 serve as an advantageous target for solid tumor cell therapies 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 and T cells. A common design approach in this field is to select an anti-CD155 antibody ScFv fragment as the extracellular antigen-binding domain, or to select a CD155 receptor such as TIGIT or CD96 as the extracellular antigen-binding domain of the CAR structure. TIGIT, or T cell immunoglobulin and ITIM domain protein, is a co-inhibitory receptor for T cells and NK cells, primarily expressed on activated T cells, NK cells, Treg cells, and helper T cells. In addition to CD155, TIGIT's ligands also include CD112; CD112, also known as connexin-2 or PVR-related protein 2 (PVRL2), is a member of the connexin family and is primarily localized at the adherens 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. Its use in CAR structure design presents safety risks due to off-target effects. Therefore, if TIGIT is to be used in the field of cell therapy, it is necessary to improve the antigen specificity of TIGIT.

[0005] Summary of the Invention

[0006] The present application relates to a TIGIT extracellular region polypeptide, which is derived from the extracellular segment of the TIGIT protein, and has a significantly higher ability to bind to CD155 than to CD112. The difference in its ability to bind to CD155 and CD112 is significantly greater than that of 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 fusion proteins and engineered receptors comprising the TIGIT extracellular region polypeptide, engineered cells comprising the engineered receptor, and uses of the engineered TIGIT extracellular region polypeptide, fusion proteins, engineered receptors, or engineered cells.

[0007] Specifically, this application relates to:

[0008] 1. A TIGIT extracellular domain polypeptide comprising a mutation at position 48 relative to a reference sequence, wherein the reference sequence is the amino acid sequence shown in SEQ ID NO: 1, and wherein the numbering of amino acid positions is defined by the reference sequence.

[0009] 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 corresponding to the amino acid sites at positions 33 to 93 of the reference sequence. In some embodiments, the polypeptide comprises one or more additional amino acids in any two of the 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 numbered less than 33 of the reference sequence and / or amino acids numbered greater than 93 of the reference sequence. In some embodiments, the polypeptide further 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 numbered less than 33 of the reference sequence and / or amino acids numbered greater than 93 of the reference sequence.

[0010] 3. The TIGIT extracellular domain polypeptide according to item 1 or 2, wherein the mutation is selected from any one of the following:

[0011] C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H, and deletion of position C48.

[0012] 4. The TIGIT extracellular domain polypeptide according to any one of items 1 to 3, further comprising one or more amino acid sites selected from the following:

[0013] 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, 70L, 70Q, 70P, 71G, 71D, 76S, 76P, 79V, 79E, 80N, 80Y, 86F, 86S, 86L, ​​101I, 101T, 102S, 102F, 106L, 106Q, 110V, 110Y, 110E, 110A, 113H, and 113Y.

[0014] 5. The TIGIT extracellular domain polypeptide according to item 4, further comprising a combination of amino acid sites of any one of the following 1) to 12):

[0015] 1) 22F, 37D, 39G;

[0016] 2) 37D;

[0017] 3)20T, 37D, 39K, 44F;

[0018] 4)22L, 37E, 71D, 102S;

[0019] 5)20T, 37D, 42G, 70Q;

[0020] 6)21F, 34S, 37E, 39K, 70P, 80Y, 101T, 110A;

[0021] 7)9S, 37D, 39V, 61R, 101T;

[0022] 8)20T, 37D, 39K;

[0023] 9)37D, 86S, 113Y;

[0024] 10) 2W, 37D, 56F;

[0025] 11) 12N, 14V, 21F, 37D, 39K, 70Q; or

[0026] 12)2T, 37D, 39K, 86L.

[0027] 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:

[0028] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0029] 7. A fusion protein comprising the TIGIT extracellular domain polypeptide described in any one of items 1-6.

[0030] 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 to the tumor antigens and / or immune checkpoint proteins are single-chain antibodies (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or diabodies.

[0031] 9. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the TIGIT extracellular domain polypeptide described in any one of items 1-6, or the fusion protein described in item 7 or 8.

[0032] 10. The engineered receptor according to item 9, further comprising a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

[0033] 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.

[0034] 12. The engineered receptor of any one of items 9 to 11, wherein the costimulatory domain comprises a signal transduction domain selected from one or more of the following molecules:

[0035] 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.

[0036] 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.

[0037] 14. An engineered receptor according to claim 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.

[0038] 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.

[0039] 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 domain polypeptide, the transmembrane domain, and the co-stimulatory domain, wherein the TIGIT extracellular domain polypeptide and the transmembrane domain further comprise or do not comprise a hinge region, wherein:

[0040] The costimulatory domain comprises or is a signal transduction domain selected from one or more of the following molecules:

[0041] 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, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB 2. 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;

[0042] 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

[0043] The hinge region comprises or is the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28, or a combination thereof.

[0044] 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.

[0045] 17. The engineered receptor according to item 16, which comprises or is, from N-terminus to C-terminus, the following: TIGIT extracellular domain polypeptide, CD28 transmembrane domain and CD28 signal transduction domain.

[0046] 18. The engineered receptor according to any one of items 9 to 15, which comprises or is any one of 1) to 10) in sequence from N-terminus to C-terminus:

[0047] 1) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0048] 2) TIGIT extracellular domain polypeptide, CD8 hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0049] 3) TIGIT extracellular domain polypeptide, CD8 hinge region, C28 transmembrane domain, 4-1BB signaling domain, and CD3ζ signaling domain;

[0050] 4) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0051] 5) TIGIT extracellular domain polypeptide, ICOS transmembrane domain, ICOS signaling domain, and CD3ζ signaling domain;

[0052] 6) TIGIT extracellular domain polypeptide, CD8 transmembrane domain, CD134 signaling domain, and CD3ζ signaling domain;

[0053] 7) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain;

[0054] 8) TIGIT extracellular domain polypeptide, CD7 hinge region, C28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0055] 9) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain; and

[0056] 10) TIGIT extracellular domain polypeptide, CD8 hinge region, CD8 transmembrane domain, 4-1BB signaling domain and CD3ζ signaling domain.

[0057] 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 (e.g., mRNA), or a hybrid molecule of RNA and DNA.

[0058] 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 creatinines. 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 group consisting of 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-carboxyl-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl The uridine residues are 5-methyluridine (MCM5U), 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 group consisting of wybutoxosine (osyw), whibutoxosine (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 group consisting of 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), 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 of 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 a 2'-O-methylation modification on the ribose of the nucleotide or a 3'thiophosphate bond modification between nucleotides or both. In some embodiments, the modification is a 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, a 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, a 3'thiophosphate modification between the nucleotides of the first three nucleotides at the 5' end, and a 3'thiophosphate modification between the nucleotides of the last three nucleotides at the 3' end.

[0059] 20. An engineered cell comprising the TIGIT extracellular domain polypeptide of any one of items 1-6, the fusion protein of item 7 or 8, the engineered receptor of any one of items 9-18 and / or the engineered nucleic acid molecule of item 19.

[0060] 21. The engineered cell according to item 20, comprising two or more engineered receptors that bind to the same target molecule or different target molecules.

[0061] 22. The engineered cell of claim 21, wherein one of the target molecules is CD155 and the additional 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 according to any one of claims 16 to 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.

[0062] 23. The 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, wherein the intracellular signaling domain has only a costimulatory 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, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, wherein the antigen binding domain comprises the TIGIT extracellular region polypeptide of 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, from N-terminus to C-terminus, an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, 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 CD123, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, 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 CD123, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, 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, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, 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.

[0063] 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 among 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 a structure comprising: at least one co-stimulatory domain and a primary signal transduction domain.

[0064] 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 conservative substitution variant thereof;

[0065] The antigen binding domain of the CAR that binds to the target molecule PSCA comprises the 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;

[0066] The antigen binding domain of the CAR that binds to the target molecule CEA comprises the 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

[0067] 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 conservative substitution 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.

[0068] 24. The engineered cell of any one of items 20-23, which is a T cell, NK cell, macrophage, DC cell, B cell, or a precursor thereof. In some embodiments, the engineered cell is a CAR-T, CAR-NK, CAR-macrophage, or CAR-DC. In some embodiments, the engineered cell is a TCR-T cell. In some embodiments, the engineered cell is a TAC-T cell.

[0069] 25. Use of the TIGIT extracellular domain polypeptide described in any one of items 1-6, the fusion protein described in item 7 or 8, the engineered receptor described in any one of items 9-18, the nucleic acid molecule described in item 19, or the engineered cell described in any one of items 20-24 for preparing a drug for treating cancer.

[0070] 26. The method according to claim 25, wherein the cancer is selected from one or more of the following:

[0071] 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.

[0072] In addition, the present application also provides a method for treating cancer, 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:

[0073] 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.

[0074] 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 1-6, 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.

[0075] 29. A method for enhancing the in vivo expansion ability of CAR-T 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] 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.

[0077] In some embodiments, the aforementioned in vivo refers to a cancer patient or subject. 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:

[0078] 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.

[0079] Specifically, this application relates to:

[0080] 1. A TIGIT extracellular domain polypeptide comprising a mutation at the following position relative to a reference sequence:

[0081] 1) No. 48, No. 57 and No. 86; or

[0082] 2) No. 48, No. 71 and No. 88;

[0083] The reference sequence is the amino acid sequence shown in SEQ ID NO: 1, and the numbering of amino acid positions is defined by the reference sequence.

[0084] 2. The TIGIT extracellular domain polypeptide according to item 1, comprising the following mutation combinations relative to the reference sequence:

[0085] 1) C48W, S57P and F86S; or

[0086] 2)C48W, G71D and I88V.

[0087] In some embodiments, the TIGIT extracellular domain polypeptide comprises only the following mutations relative to amino acids 33 to 93 of the reference sequence:

[0088] 1) C48W, S57P and F86S; or

[0089] 2)C48W, G71D and I88V.

[0090] 3. The TIGIT extracellular domain polypeptide according to claim 2, comprising a combination of amino acid sites according to any one of 1) to 4):

[0091] 1) 34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S and 88I;

[0092] 2) 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F and 88V;

[0093] 3) 9T, 20I, 21I, 34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S, 88I, 101I and 110V; or

[0094] 4)9T, 20I, 21I, 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F, 88V, 101I and 110V.

[0095] 4. The TIGIT extracellular region polypeptide according to any one of items 1 to 3, 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 corresponding to the amino acid sites at positions 33 to 93 of the reference sequence. In some embodiments, the polypeptide comprises one or more additional amino acids in any two of the 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 of the amino acids corresponding to positions 33 to 93 of the reference sequence, such as amino acids corresponding to amino acids numbered less than 33 of the reference sequence and / or amino acids numbered greater than 93 of the reference sequence. In some embodiments, the polypeptide further comprises other amino acids at the C-terminus and / or N-side of the amino acids corresponding to positions 33 to 93 of the reference sequence, such as amino acids corresponding to amino acids numbered less than 33 of the reference sequence and / or amino acids numbered greater than 93 of the reference sequence.

[0096] 5. The TIGIT extracellular region polypeptide according to any one of items 1 to 5, 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:

[0097] SEQ ID NO: 2-8.

[0098] 6. A fusion protein comprising the TIGIT extracellular domain polypeptide according to any one of items 1 to 5.

[0099] 7. The fusion protein according to item 6, further comprising one or more polypeptides that bind to tumor antigens and / or immune checkpoint proteins,

[0100] Optionally, the polypeptide that binds to a tumor antigen and / or immune checkpoint protein is an antibody, ligand or receptor for the tumor antigen and / or immune checkpoint protein;

[0101] Optionally, the antibody to the tumor antigen and / or immune checkpoint protein is a single-chain antibody (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb or diabody.

[0102] 8. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the TIGIT extracellular domain polypeptide described in any one of items 1-5, or the fusion protein described in item 6 or 7.

[0103] 9. The engineered receptor according to item 8, further comprising a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

[0104] 10. The engineered receptor according to item 8 or 9, which is a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a T cell antigen coupling agent (TAC).

[0105] 11. The engineered receptor of any one of items 9 or 10, wherein the costimulatory domain comprises a signal transduction domain selected from one or more of the following molecules:

[0106] 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.

[0107] 12. An engineered receptor according to any one of items 9 to 11, 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.

[0108] 13. The engineered receptor according to item 12, further comprising a transmembrane domain between the antigen binding domain and the signal transduction domain, wherein the transmembrane domain comprises a transmembrane domain selected from any one or more of the following molecules: ICOS, CD4, CD8α, CD28, CD3ζ and TIGIT.

[0109] 14. An engineered receptor according to item 13, 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.

[0110] 15. The engineered receptor according to any one of claims 9 to 14, which comprises or is, from N-terminus to C-terminus, the TIGIT extracellular domain polypeptide, the transmembrane domain, and the co-stimulatory domain, wherein the TIGIT extracellular domain polypeptide and the transmembrane domain further comprise or do not comprise a hinge region, wherein:

[0111] The costimulatory domain comprises or is a signal transduction domain selected from one or more of the following molecules:

[0112] 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;

[0113] 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

[0114] The hinge region comprises or is the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28, or a combination thereof.

[0115] 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.

[0116] 16. The engineered receptor according to item 15, which comprises or is, from N-terminus to C-terminus, the following: TIGIT extracellular domain polypeptide, CD28 transmembrane domain and CD28 signal transduction domain.

[0117] 17. The engineered receptor according to any one of items 8 to 14, which comprises or is any one of 1) to 4) from N-terminus to C-terminus:

[0118] 1) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain;

[0119] 2) TIGIT extracellular domain polypeptide, CD8 hinge region, CD8 transmembrane domain, 4-1BB signaling domain, and CD3ζ signaling domain;

[0120] 3) TIGIT extracellular domain polypeptide, CD7 hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain; and

[0121] 4) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain-4-1BB signaling domain and CD3ζ signaling domain.

[0122] 18. An engineered nucleic acid molecule encoding a TIGIT extracellular domain polypeptide according to any one of items 1 to 5, a fusion protein according to item 6 or 7, or an engineered receptor according to any one of items 8 to 17. In some embodiments, the engineered nucleic acid molecule is DNA, RNA (e.g., mRNA), or a hybrid molecule of RNA and DNA.

[0123] 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 creatinines. 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 group consisting of 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-carboxyl-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl The uridine residues are 5-methyluridine (MCM5U), 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 group consisting of wybutoxosine (osyw), whibutoxosine (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 group consisting of 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), 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 of 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 a 2'-O-methylation modification on the ribose of the nucleotide or a 3'thiophosphate bond modification between nucleotides or both. In some embodiments, the modification is a 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, a 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, a 3'thiophosphate modification between the nucleotides of the first three nucleotides at the 5' end, and a 3'thiophosphate modification between the nucleotides of the last three nucleotides at the 3' end.

[0124] 19. An engineered cell comprising the TIGIT extracellular domain polypeptide described in any one of items 1-5, the fusion protein described in item 6 or 7, the engineered receptor described in any one of items 8-17 and / or the engineered nucleic acid molecule described in item 18.

[0125] 20. The engineered cell according to item 19, comprising two or more engineered receptors that bind to the same target molecule or different target molecules.

[0126] 21. The engineered cell of claim 20, wherein one of the target molecules is CD155 and the additional 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 according to any one of claims 15 to 17, 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.

[0127] 22. The engineered cell according to item 21, 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, wherein the intracellular signaling domain has only a costimulatory 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, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, wherein the antigen binding domain comprises the TIGIT extracellular region polypeptide of any one of items 1 to 5. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting PSCA, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 5. In some embodiments, the engineered cell comprises: an engineered receptor targeting CD155 and a CAR targeting CD123, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 5. In some embodiments, the engineered cells comprise: an engineered receptor targeting CD155 and a CAR targeting CD123, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a hinge region, a transmembrane domain, and a costimulatory domain, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 5. In some embodiments, the engineered cells comprise: an engineered receptor targeting CD155 and a CAR targeting CEA, wherein the engineered receptor targeting CD155 comprises, from N-terminus to C-terminus, an antigen binding domain, a transmembrane domain, and a costimulatory domain, and the antigen binding domain is a TIGIT extracellular region polypeptide according to any one of items 1 to 5. 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 5.

[0128] According to the engineered immune cell of item 21, 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, wherein the engineered receptor targeting CD155 comprises: 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 can be a CAR structure comprising only a primary signal transduction domain such as a CD3ζ signal transduction domain, or it can be a CAR structure comprising: at least one co-stimulatory domain and a primary signal transduction domain.

[0129] 23. The engineered cell according to item 22, wherein the antigen binding domain of the engineered receptor that binds CD155 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 2-8 or a conservatively substituted variant thereof;

[0130] 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: 9 or 10, 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;

[0131] The antigen binding domain of the CAR that binds to the target molecule CEA comprises the amino acid sequence as shown in SEQ ID NO: 11 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

[0132] The antigen binding domain of the CAR that binds to the target molecule CD123 comprises the amino acid sequence shown in SEQ ID NO: 12 or a conservative substitution 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.

[0133] 24. The engineered cell of any one of items 19-23, which is a T cell, NK cell, macrophage, DC cell, B cell, or a precursor thereof. In some embodiments, the engineered cell is a CAR-T, CAR-NK, CAR-macrophage, or CAR-DC. In some embodiments, the engineered cell is a TCR-T cell. In some embodiments, the engineered cell is a TAC-T cell.

[0134] 25. Use of the TIGIT extracellular domain polypeptide described in any one of items 1 to 5, the fusion protein described in item 6 or 7, or the engineered receptor described in item 8 to item 17, the nucleic acid molecule described in item 18, or the engineered cell described in any one of items 19 to 24 for preparing a drug for treating cancer.

[0135] 26. The method according to claim 25, wherein the cancer is selected from one or more of the following:

[0136] 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.

[0137] In addition, the present application also provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the TIGIT extracellular domain polypeptide of any one of Items 1-5, the fusion protein of Item 6 or 7, or the engineered receptor of any one of Items 8-17, the nucleic acid molecule of Item 18, or the engineered immune cell of any one of Items 19-20. In some embodiments of the method for treating cancer, the cancer is selected from one or more of the following:

[0138] 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.

[0139] 27. 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 1-5, the fusion protein described in item 6 or 7, or the engineered receptor described in any one of items 8-17 on the CAR-T cell membrane.

[0140] 28. A method for improving the in vivo expansion ability of CAR-T cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of items 1-5, the fusion protein described in item 6 or 7, or the engineered receptor described in any one of items 8-17 on the CAR-T cell membrane.

[0141] 29. 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 1-5, the fusion protein described in item 6 or 7, or the engineered receptor described in any one of items 8-17 on the CAR-T cell membrane.

[0142] In some embodiments, the aforementioned in vivo refers to a cancer patient or subject. 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:

[0143] 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.

[0144] 2A self-cleavage polypeptide is a kind of commonly used multi-gene expression scheme that translation level realizes multiple gene co-expression.The polypeptide was first found in foot-and-mouth disease virus (FMDV) in 1991, with an average length of 18-22 amino acids. It has been found that multiple different 2A peptides are derived from foot-and-mouth disease virus 2A (F2A), type 1 porcine teschovirus 2A (P2A), bright vein flat moth virus 2A (T2A), and horse-type rhinitis virus 2A (E2A). Its main working principle is that ribosomes are in the translation process. When identifying 2A peptide ends, they can skip glycyl-prolyl peptide bond synthesis, and sliding occurs, 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 the function of the engineered immune cells is verified by the test of embodiment, and further verify the unpredictable effect of the engineered receptors of the application for engineered immune cells. In addition to the above-mentioned 2A self-cleavage peptide, IRES (internal ribosome entry site sequence (Internal ribosome entry site, IRES)) can also be used. IRES can recruit ribosomes to translate mRNA, and can choose to express multiple proteins independently. The internal ribosome entry site (IRES) is used to separate the coding genes (also referred to as ORFs) 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 separately transducing different genes into target cells, or by simultaneously transducing target genes with constructed vectors expressing different genes; whether using linkers or separately constructing expression vectors of multiple genes to transduce target cells, the purpose of obtaining engineered cells expressing TIGIT extracellular domain polypeptides can be achieved in the end, and the functions and unpredictable effects of engineered cells are mainly based on the protein molecules expressed therein and the final engineered cells themselves.

[0145] 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 with 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

[0146] Figure 1 shows the flow cytometry staining of CD112 in MDA-MB-231-Luc-GFP, 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 showed that the MFI of CD112 in MDA-MB-231-Luc-GFP was approximately 4 times higher than that in the control group.

[0147] Figure 2A shows peptide-specific detection of five TIGIT mutants.

[0148] Figure 2B shows the specific detection of two TIGIT mutant peptides.

[0149] Figure 2C shows peptide-specific detection of seven TIGIT mutants.

[0150] Figure 2D shows peptide-specific detection of nine TIGIT mutants.

[0151] Figure 2E shows peptide-specific detection of three TIGIT mutants.

[0152] FIG3 shows the binding assay of the screened TIGIT mutant peptides to CD112.

[0153] FIG4 shows the binding assay of the screened TIGIT mutant peptides to CD155.

[0154] FIG5 shows the functional validation of the screened TIGIT mutant peptides against CD155-negative cells.

[0155] FIG6 shows the in vivo drug efficacy evaluation in the MDA-MB-231 tumor model.

[0156] FIG7A shows the killing effect of CARs with seven different structures on MDA-MB-231-Luc-GFP.

[0157] FIG7B shows the killing effect of CARs with five different structures on HPAC-Luc-GFP.

[0158] FIG7C shows the killing effect of CARs with two different structures on HT1376-Luc-GFP.

[0159] FIG8A shows the expression of CEA in DLD1-CEA-Luc-GFP.

[0160] FIG8B shows the expression of CEA in DLD1-Luc-GFP.

[0161] FIG8C shows the expression of CD155 in DLD1-CEA-Luc-GFP.

[0162] Figure 9A shows the killing effect of CEA and CD155 dual-targeting CAR37 on DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP cells.

[0163] Figure 9B shows the in vitro killing of Molm-13-Luc-GFP by CD123 and CD155 dual-targeting CAR38.

[0164] Figure 9C shows the in vitro killing effects of CAR-T cells with four different CAR structures dual-targeting PSCA and CD155 in three cell models.

[0165] FIG10 shows the killing effect of CAR42 and CAR45 in two cell models.

[0166] Figure 11 shows the IFN-γ cytokine secretion of CAR42 and CAR45 during the in vitro killing process of two cell models.

[0167] Figure 12 shows the in vivo efficacy of CAR42 and CAR45 in the DLD-1-CEA-Luc-GFP peritoneal tumor model.

[0168] FIG13 shows the blood copy number in the peritoneal tumorigenesis evaluation model.

[0169] Figure 14 Verification of the effect of TIGIT mutant peptide on the in vivo effectiveness of CAR.

[0170] Figure 15A Verification of the effectiveness of multi-target CAR-T targeting CD155 against acute myeloid leukemia.

[0171] Figure 15B Verification of the effectiveness of multi-target CAR-T targeting CD155 against pancreatic cancer and bladder cancer.

[0172] Figures 16A and 16B show the in vivo validation of CAR-T cells constructed with different TIGIT mutant peptides. Figure 16A is a visual graph of CAR-T cells expressing CARs with different TIGIT mutant peptides as the extracellular recognition region, demonstrating tumor killing in an immunodeficient mouse model of acute lymphoblastic leukemia; Figure 16B is a statistical curve of fluorescence values.

[0173] FIG17 shows the in vitro killing of three target cells by CAR53.

[0174] Figure 18 Verification of the effectiveness of CAR-T using mutant TIGIT peptide as the extracellular recognition domain against pancreatic cancer.

[0175] Figure 19 Verification of the effectiveness of CAR-T using mutant TIGIT peptide as the extracellular recognition domain against bladder cancer.

[0176] Figure 20 CAR-T cells with different CAR structures kill malignant tumors with different indications.

[0177] Figure 21 In vitro killing of target cells by CAR53 and CAR61 in three different indications.

[0178] Figure 22 In vitro efficacy data of CAR-T cells expressing engineered receptors with TIGIT mutant peptides as antigen binding domains.

[0179] Figure 23 shows the in vivo effectiveness of CAR-T cells expressing an engineered receptor with TIGIT mutant peptide as the antigen binding domain against colorectal cancer tumors in colorectal cancer-bearing mice.

[0180] Figure 24 is a graph of in vivo tumor fluorescence curves of CAR-T cells expressing an engineered receptor with TIGIT mutant peptide as the antigen binding domain against colorectal cancer tumors in colorectal cancer-bearing mice.

[0181] Figure 25 shows the copy number in the mice after CAR-T cells expressing an engineered receptor with TIGIT mutant peptide as the antigen binding domain killed the tumor in colorectal cancer-bearing mice.

[0182] FIG26 shows the in vitro killing effect of CAR60 on Molm-13-Luc-GFP. Specific implementation plan

[0183] The present application relates to a TIGIT mutant peptide, which is derived from the extracellular segment of the TIGIT protein, for example, it contains a mutation at position 48 relative to the reference sequence, and its ability to bind to CD155 is significantly higher than its ability to bind to CD112, and the difference in its ability to bind to CD155 and its ability to bind to CD112 is significantly greater than the difference in 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 that is comparable to or stronger than the extracellular segment of the wild-type TIGIT protein, but its binding ability to CD112 is significantly reduced. 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 in the binding ability of the TIGIT mutant peptide to CD155 is much greater than the improvement in the binding ability 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. The present application relates to an engineered polypeptide derived from the extracellular segment of the TIGIT protein, for example, it comprises mutations at positions 48, 57 and 86 relative to the reference sequence, or comprises mutations at positions 48, 71 and 88; relative to the extracellular segment of the wild-type TIGIT protein (amino acid sequence as shown in SEQ ID NO: 1), its ability to bind to CD155 is significantly improved. And the engineered receptor with the engineered polypeptide as the antigen binding domain (or ligand binding domain) can result in a stronger target cell killing ability of the immune effector cells described therein compared to the engineered receptor with the extracellular segment of the wild-type TIGIT protein as the antigen binding domain (or ligand binding domain). Wherein, the target cell is a cell that highly expresses CD155. In some embodiments, the target cell is a tumor cell, such as a bladder cancer cell, a prostate cancer cell, a pancreatic cancer cell, etc.In addition, the present application also relates to fusion proteins or engineered receptors comprising the TIGIT mutant peptide, such as fusion proteins comprising antibodies or antigen recognition fragments of the antibodies, CARs, engineered TCRs, TACs, etc.; and engineered cells comprising the fusion proteins or engineered receptors, such as T cells, NK cells, macrophages, DC cells, B cells, or their precursor cells; and the uses of the aforementioned TIGIT mutant peptides, engineered receptors, fusion proteins or engineered cells.

[0184] definition

[0185] It should be understood that the present disclosure is not limited to the aspects described herein, which can of course vary. It should also be understood that the terminology used herein is used to describe particular aspects only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0186] 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 technical and patent disclosures cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA within the skill of the art will be employed by those skilled in the art.

[0187] The term "TIGIT" is an abbreviation for the 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-based inhibitory motif (ITIM) and immunoglobulin tyrosine-based 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 that is 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 the TIGIT protein or its variant corresponding to positions 1 to 120 of the reference sequence SEQ ID NO: 1. This can be determined by sequence alignment with the reference sequence. For example, by introducing gaps, any TIGIT protein or its variant can have identical residues at as many positions as possible with the reference sequence. After alignment, the amino acids at positions 1 and 120 in the amino acid sequence of SEQ ID NO: 1 in the TIGIT protein or its variant, as well as all amino acid positions therebetween, connected in the order of their positions in the TIGIT protein or its variant, can be referred to as the "TIGIT extracellular region." For example, the extracellular region of the TIGIT protein with GenBank accession number ACD74757.1 is from positions 22 to 141.

[0188] As used herein, "TIGIT extracellular region polypeptide" can be used to refer to any peptide segment in the TIGIT extracellular region or any truncated form of the TIGIT extracellular region, or the full-length TIGIT extracellular region.

[0189] 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 in this application are defined by the reference sequence. As used herein, a mutation "relative to a reference sequence" refers to a position of an amino acid defined by sequence alignment, where the position of the amino acid has an amino acid different from the amino acid at the reference sequence at the position (substitution), more than one amino acid at the amino acid position (addition), and / or there is a vacancy at the amino acid position (deletion). For example, a TIGIT extracellular domain polypeptide "comprising a mutation at position 48 relative to the reference sequence" means that the amino acid at position 48 of the TIGIT extracellular domain 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 gaps 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, 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.

[0190] "CD155," also known as "PVR," 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 expression is low or absent in most healthy tissues. Consistent with PVR's biological properties, its overexpression promotes tumor cell invasion, migration, and proliferation, and is associated with poor prognosis and enhanced tumor progression.

[0191] "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.

[0192] As used herein, percentages of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refer to a degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, the percentage of positions with identical bases or amino acid residues is determined as a ratio of the total number of positions after two sequences have been aligned to have identical residues at as many positions as possible, such as by introducing gaps. Percentages of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at the following address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0193] As used herein, a "variant" has at least one amino acid difference relative to a reference amino acid sequence, e.g., 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 ... Lu); 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 comprise non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%, 95%, 96%, 97%, 98%, or 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 aforementioned 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%, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0194] As used herein, "amino acid" refers to any monomeric 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, when referring to any of the above natural amino acids, also includes non-natural amino acids or amino acid analogs derived or modified therefrom. 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 photosensitive crosslinkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising 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), methamphetamine (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 into 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 following amino acids: 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.

[0195] 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 composed of A, C, G and / or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules composed of A, C, G and / or U nucleotides. As used herein, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0196] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0197] 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:

[0198] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0199] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0200] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials through biological or chemical synthesis. It should be noted that the methods for obtaining DNA / RNA hybrid molecules are not limited to the above methods; DNA / RNA hybrid molecules obtained by any method fall within the scope of "DNA / RNA hybrid molecules" as defined in this application.

[0201] As used herein, when 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 that after one or more thymines in the base sequence of one nucleic acid molecule are converted to uracil, a nucleic acid molecule with exactly the same base sequence as the other nucleic acid molecule can be obtained. 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 in which bases are arranged in a polynucleotide molecule. It should be understood by those skilled in the art that, unless otherwise specified, the base sequence or polynucleotide sequence described in this application can be used to represent thymine when used to describe a DNA sequence, but when the base sequence or polynucleotide sequence is used to describe RNA (e.g., 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 (e.g., mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" in the DNA sequence is replaced by a "U".

[0202] In this article, "coding" refers to i) the genetic information contained in the DNA sequence that can be transcribed into an RNA molecule, and / or ii) the genetic information contained in the 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 in an mRNA precursor or mature mRNA that can be translated into a protein, and can also refer to the complementary sequence of a deoxyribonucleotide (DNA) sequence or a fragment thereof that is used as a template for transcribing the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application can further include polynucleotide sequences that encode 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.

[0203] Unless otherwise specified, "peptide", "polypeptide" and "protein" are used interchangeably in this application to refer to any natural active substance formed by two or more amino acids covalently linked by peptide bonds, which may or may not contain the secondary or tertiary structure of the protein molecule.

[0204] As used herein, the term "contacting" is used in accordance with its simple common meaning and refers to a process in which at least two different substances are allowed to become sufficiently close to react, interact or physically contact. It should be understood that the resulting reaction product can be produced directly by the reaction between the added reagents, or by an intermediate of one or more added reagents, which can be produced in a reaction mixture. The term "contacting" can include allowing two substances to react, interact or physically contact, wherein the two substances can be, for example, an engineered receptor (or engineered nucleic acid molecule) and a cell as provided herein. In an embodiment, contacting includes, for example, allowing an engineered nucleic acid molecule or an engineered peptide as described herein to enter a cell.

[0205] As used herein, "CAR-T cell persistence" refers to the duration of CAR-T cell persistence in a patient or subject, i.e., the period from in vivo administration of CAR-T cells to their eventual disappearance from the body. This can be calculated or measured by comparing CAR copy numbers. For example, an increase in CAR-T copy number after a specific period of in vivo use can be used to indicate an increase in CAR-T cell persistence.

[0206] Unless defined otherwise 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.

[0207] TIGIT extracellular domain polypeptide

[0208] On the one hand, the present application relates to a TIGIT extracellular region polypeptide (which may also be referred to as a TIGIT mutant peptide unless otherwise specified), 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.

[0209] 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 one-to-one 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 the 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 position 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 the 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 position 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 consecutive amino acid sequences extending from position 33 of the amino acid sequence of any TIGIT extracellular region to the carbon terminus (C-terminus), and / or one or more consecutive amino acid sequences extending from position 93 of the amino acid sequence of any TIGIT extracellular region to the nitrogen terminus (N-terminus). In some embodiments, the engineered TIGIT extracellular region polypeptide may further comprise one or more consecutive 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 consecutive 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.

[0210] In some embodiments, the TIGIT extracellular domain polypeptide comprises a mutation at position 48 relative to the reference sequence selected from any one of the following:

[0211] C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H, and deletion at position 48. 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.

[0212] In some embodiments, the TIGIT extracellular domain 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:

[0213] 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.

[0214] In some embodiments, the TIGIT extracellular domain 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 the following amino acid positions:

[0215] 1) 22F, 37D, 39G;

[0216] 2) 37D;

[0217] 3)20T, 37D, 39K, 44F;

[0218] 4)22L, 37E, 71D, 102S;

[0219] 5)20T, 37D, 42G, 70Q;

[0220] 6)21F, 34S, 37E, 39K, 70P, 80Y, 101T, 110A;

[0221] 7)9S, 37D, 39V, 61R, 101T;

[0222] 8)20T, 37D, 39K;

[0223] 9)37D, 86S, 113Y;

[0224] 10) 2W, 37D, 56F;

[0225] 11) 12N, 14V, 21F, 37D, 39K, 70Q; or

[0226] 12)2T, 37D, 39K, 86L.

[0227] 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.

[0228] 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:

[0229] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0230] In some embodiments, the amino acid sequence of the TIGIT extracellular region polypeptide is selected from any one of the following amino acid sequences or a conservative substitution variant comprising any one of the following amino acid sequences:

[0231] SEQ ID NOs: 5-17, 19, 22, and SEQ ID NOs: 24-26.

[0232] In some embodiments, the TIGIT extracellular region polypeptide comprises a mutation relative to a reference sequence at one or more sites selected from:

[0233] 9th, 20th, 21st, 34th, 39th, 48th, 57th, 61st, 70th, 71st, 80th, 86th, 88th, 101st and 110th.

[0234] In some embodiments, the TIGIT extracellular domain polypeptide comprises a mutation at the following positions relative to the reference sequence:

[0235] 1) No. 48, No. 57 and No. 86; or

[0236] 2) No. 48, No. 71 and No. 88.

[0237] In some embodiments:

[0238] The mutation at position 48 is selected from any one of the following:

[0239] C48G, C48A, C48V, C48L, C48I, C48P, C48F, C48W, C48M, C48Y, C48S, C48T, C48N, C48Q, C48D, C48E, C48K, C48R, C48H, and deletion at position 48;

[0240] The mutation at position 57 is selected from any one of the following: S57G, S57A, S57V, S57L, S57I, S57P, S57F, S57W, S57M, S57Y, S57C, S57T, S57N, S57Q, S57D, S57E, S57K, S57R, S57H, and deletion at position 57;

[0241] The mutation at position 71 is selected from any one of the following: G71S, G71A, G71V, G71L, G71I, G71P, G71F, G71W, G71M, G71Y, G71C, G71T, G71N, G71Q, G71D, G71E, G71K, G71R, G71H, and deletion at position 71;

[0242] The mutation at position 86 is selected from any one of the following: F86G, F86A, F86V, F86L, F86I, F86P, F86S, F86W, F86M, F86Y, F86C, F86T, F86N, F86Q, F86D, F86E, F86K, F86R, F86H and deletion at position 86; and / or

[0243] The mutation at position 88 is selected from any one of the following: I88G, I88A, I88V, I88L, I88S, I88P, I88F, I88W, I88M, I88Y, I88C, I88T, I88N, I88Q, I88D, I88E, I88K, I88R, I88H and deletion at position 88.

[0244] Wherein the deletions at positions 48, 57, 71, 86 and 88 refer to that the TIGIT extracellular region polypeptide does not have an amino acid at positions 48, 57, 71, 86 and 88 relative to the reference sequence, respectively. In some embodiments, the TIGIT extracellular region polypeptide comprises a substitution of a non-natural amino acid at one or more of positions 9, 20, 21, 34, 39, 48, 57, 61, 70, 71, 80, 86, 88, 101 and 110, thus in some embodiments, the TIGIT extracellular region has a deletion at positions 9, 20, 21, 34, 39, 48, 57, 61, 70, 71, 80, 86, 88, 101 and 110 relative to the reference sequence. One or more of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 200, 211, 222, 223, 239, 240, 249, 250, 251, 261, 262, 263, 279, 280, 281, 282, 283, 284, 285

[0245] In some embodiments, the TIGIT extracellular domain polypeptide comprises at least the following mutations relative to a reference sequence:

[0246] 1) C48W, S57P and F86S; or

[0247] 2)C48W, G71D and I88V.

[0248] In some embodiments, the TIGIT extracellular domain polypeptide comprises the following amino acid sites:

[0249] 1) 34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S and 88I;

[0250] 2) 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F and 88V;

[0251] 3) 9T, 20I, 21I, 34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S, 88I, 101I and 110V; or

[0252] 4)9T, 20I, 21I, 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F, 88V, 101I and 110V.

[0253] 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.

[0254] 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:

[0255] SEQ ID NO: 47-53.

[0256] In some embodiments, the amino acid sequence of the TIGIT extracellular region polypeptide is selected from any one of the following amino acid sequences or a conservative substitution variant comprising any one of the following amino acid sequences:

[0257] SEQ ID NO: 47-53.

[0258] In some embodiments, the TIGIT extracellular domain polypeptide is chemically modified, for example, one or more amino acids therein are substituted with a non-natural amino acid or an amino acid analog.

[0259] Fusion protein

[0260] On the one hand, the present application also provides a fusion protein comprising the aforementioned TIGIT extracellular region polypeptide, which may 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 combination is that the TIGIT extracellular region polypeptide is directly and / or indirectly connected to the another one or more proteins, polypeptides, or protein functional domains. In some embodiments, the carbon (C) terminus of the TIGIT extracellular region polypeptide in the fusion protein is connected to the nitrogen (N) terminus of the another protein, polypeptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the TIGIT extracellular region polypeptide in the fusion protein is connected to the carbon (C) terminus of the another protein, polypeptide, or protein functional domain. In some embodiments, the TIGIT extracellular region polypeptide in the fusion protein and the multiple proteins, polypeptides, or protein functional domains are connected 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 functional domains, and the two or more additional proteins, polypeptides or protein functional 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, e.g., a peptide chain consisting of 1 to 50 amino acids or derivatives thereof) or a non-peptide linker, and the linker can be cleavable (i.e., hydrolyzable by enzymes in an organism, e.g., a mammalian organism) 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, polymeric lipids, chitin, and hyaluronic acid, or derivatives thereof, or combinations thereof.

[0261] In some embodiments, the one or more proteins, polypeptides or protein domains comprise a recognition polypeptide that specifically binds to the one or more proteins, and the one or more proteins can 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 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; 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); mammary 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); ET 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 mutants; 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 leukemia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (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-like receptor 1 (LAIR1); IgA receptor The Fc fragment of the human leukocyte antigen (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); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155.

[0262] 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:

[0263] 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.

[0264] In some embodiments, the one or more other proteins, polypeptides or protein functional domains comprise a recognition polypeptide that specifically binds to one or more other proteins, and the one or more other proteins include any one or more immune checkpoint proteins selected from the above-mentioned ones, and any one or more tumor antigens mentioned above.

[0265] 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.

[0266] In some embodiments, the one or more 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 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 proteins, polypeptides or protein functional domains may also include an intracellular signaling domain, which is a co-stimulatory signal molecule and includes a signal transduction domain selected from one or more of the following molecules:

[0267] 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.

[0268] Furthermore, 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.

[0269] Engineered receptors

[0270] The present application also provides an engineered receptor comprising an antigen binding domain comprising 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.

[0271] In some embodiments, the engineered receptor comprises or is from N-terminus to C-terminus: antigen binding domain-membrane spaning domain-costimulatory domain (or secondary signal transduction domain). In some embodiments, the engineered receptor comprises or does not comprise a hinge region, wherein the hinge region connects the antigen binding domain and the membrane spaning 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 membrane spaning domain is from the membrane spaning domain of ICOS, CD4, CD8α, CD28, CD3ζ or TIGIT, or a combination thereof. In some embodiments, the costimulatory 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 the signaling domain of a ligand that specifically binds to CD83.

[0272] In some embodiments, the engineered receptor may comprise an extracellular antigen-binding domain, with or without an additional hinge structure, 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 NOs: 5-17, 19, 22, SEQ ID NOs: 24-26, and SEQ ID NOs: 47-53. 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 can be any one of the following amino acid sequences or a conservative substitution variant comprising any one of the following amino acid sequences: SEQ ID NOs: 5-17, 19, 22, SEQ ID NOs: 24-26, and SEQ ID NOs: 47-53, 28TM is a transmembrane domain derived from human CD28 (e.g., the amino acid sequence set forth in SEQ ID NO: 36), and 28 is an intracellular signal transduction domain derived from human CD28 (e.g., the amino acid sequence set forth in SEQ ID NO: 37). In some embodiments, the structure of the engineered receptor is TIGIT extracellular region polypeptide-28TM-28, wherein the TIGIT extracellular region polypeptide can be a polypeptide set forth in SEQ ID NOs: 7, 48, 51 to 53, 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-28z, wherein the TIGIT extracellular region polypeptide can be any one of the following amino acid sequences or a conservative substitution variant comprising any one of the following amino acid sequences: SEQ ID NO: 5-17, 19, 22, SEQ ID NO: 24-26 and SEQ ID NO: 47-53, 28TM is a transmembrane domain derived from human CD28 (for example, the amino acid sequence shown in SEQ ID NO: 36), 28 is a signal transduction domain derived from human CD28 (for example, the amino acid sequence shown in SEQ ID NO: 37), and z is a signal transduction domain derived from human CD3ζ (for example, the amino acid sequence shown in any one of SEQ ID NO: 41 to 43).

[0273] 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 costimulatory domains, and the T cell receptor costimulatory 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.

[0274] 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. Wherein, the "intracellular signal transduction domain" comprises a primary signal transduction domain and / or a costimulatory 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 signaling domain comprises a primary signaling domain 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 signaling domain is derived from CD3ζ. As used herein, a "primary signaling domain" generally comprises an immune-receptor tyrosine-based 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 attached to it can be phosphorylated under the action of a class of protein tyrosine kinases PTKs associated with the cell membrane, thereby recruiting other free protein kinases or adaptor proteins in the cell to transmit activation signals into the cell.

[0275] 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 activating immune cells (eg, T cells).

[0276] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an antigen binding domain (as used herein, 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 identical (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.

[0277] As used herein, "TAC" refers to 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, a TAC comprises: (a) an antigen-binding domain (as used herein, 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) 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 linker; (e) optionally an extracellular antigen binding domain or 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.

[0278] As used herein, the term "antigen binding domain" encompasses the concepts of "ligand binding domain" and "receptor binding domain", which are generally 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 limited in any way. 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 diabody.

[0279] Chimeric Antigen Receptor (CAR)

[0280] 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.

[0281] In some embodiments, the one or more antigens further comprise one or more tumor antigens and / or immune checkpoint proteins in addition to CD155 that binds to the aforementioned TIGIT extracellular domain polypeptide.

[0282] 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-1 Gag, 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, tendinocytes White, carcinoembryonic variants in tumor necrosis areas, 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.

[0283] Wherein, in some embodiments, the one or more immune checkpoint proteins are selected from:

[0284] 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.

[0285] 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 the transmembrane domain of CD4, CD8α, CD28 or CD3ζ.

[0286] 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.

[0287] 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, which is a 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, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 In some embodiments, the signal transduction domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule, and the costimulatory domain is a signal transduction domain of a CD3ζ molecule,In some embodiments, the signal transduction domain is the signal transduction domain of the CD3ζ molecule, and the costimulatory domain is derived from the signal transduction domains of 4-1BB and CD28.

[0288] 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 costimulatory 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 costimulatory domain, and the costimulatory domain is derived from a signal transduction domain of one or more costimulatory 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.

[0289] In some embodiments, the protein combination comprises any one structure selected from the group consisting of:

[0290] 1) PSCA-8h-8TM-BBZ and TIGIT-28TM-28Z (1)

[0291] 2) CEA-8h-8TM-BBZ and TIGIT-28TM-28Z (1)

[0292] 3) CD123-8h-8TM-2B4Z and TIGIT-28TM-28Z (1)

[0293] 4) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28Z (1)

[0294] 5) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28Z (1)

[0295] 6) PSCA-7h-28TM-28Z and TIGIT-28TM-28Z (1)

[0296] 7) PSCA-8h-8TM-BBZ and TIGIT-28TM-28

[0297] 8) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28

[0298] 9) CEA-8h-8TM-BBZ and TIGIT-28TM-28

[0299] 10) CD123-8h-8TM-2B4Z and TIGIT-28TM-28

[0300] 11) PSCA-G4h-28TM-28-BBZ (3) and TIGIT-28TM-28;

[0301] 12) PSCA-7h-28TM-28Z (1) and TIGIT-28TM-28;

[0302] 13) CEA-8h-8TM-BBZ and TIGIT-28TM-28Z; or

[0303] 14) PSCA-8h-8TM-BBZ and TIGIT-28TM-28Z;

[0304] Wherein, CEA is an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to CEA, CD123 is an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to CD123, PSCA is an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to PSCA, TIGIT is the aforementioned TIGIT extracellular domain polypeptide, 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 or 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.

[0305] Engineered nucleic acid molecules

[0306] The present application also provides an engineered nucleic acid molecule encoding the aforementioned engineered receptor, fusion protein, or chimeric antigen receptor, wherein the nucleic acid molecule comprises a target protein coding sequence, which 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." A "natural nucleic acid molecule" refers to a nucleic acid molecule that exists in its 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 methods. It can have a polynucleotide sequence that is exactly the same as a natural nucleic acid molecule, have modifications that are exactly the same as a natural nucleic acid molecule, or even form a structure that is completely consistent with a natural nucleic acid molecule. However, 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, at least includes that the engineered nucleic acid molecule is not directly purified or extracted in its natural form from an animal or plant that naturally occurs in nature.

[0307] 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, in addition to comprising the target protein coding sequence, the DNA molecule also comprises genetic manipulations or regulatory elements for replication and / or expression in prokaryotic and / or eukaryotic cells.

[0308] The engineered DNA molecule is made to replicate or efficiently replicate the necessary structural elements in the cell, which are known in the art and include, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further includes a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of the DNA element, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing the plasmid that comprises the marker gene and can be selected from, for example, antibiotic resistance genes. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0309] 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, as well as linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify the genomic genetic information stored in the nucleoid or nucleoid of a prokaryotic cell in a cell, 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 vectors based on pUC, pTZ plasmids, or any other plasmid (e.g., pMB1, pCoIE1) containing an ORI that supports high copies of the plasmid.

[0310] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof that constitutes a nucleoid or nucleoid of a prokaryotic organism, or a DNA molecule or a fragment thereof that constitutes a eukaryotic genome, that is, the target protein coding sequence or its complementary sequence can be replicated along with the prokaryotic genome.

[0311] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule further comprises a coding sequence of an element that can be used to initiate or regulate the expression of the protein, polypeptide or fragment thereof after transcription, the element including but not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal) and the like. In some embodiments, the engineered DNA molecule comprises a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises at least the coding sequence of the 5'UTR and the target protein coding sequence. In some embodiments, the engineered DNA molecule comprises at least the coding sequence of the 5'UTR, the target protein coding sequence, the coding sequence of the 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) may also be respectively included at both ends of the target protein coding sequence, which are respectively the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule. 5'UTR generally comprises 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. RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the time of translation initiation. 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, 5'UTR includes an internal ribosome entry site or IRES. 3'UTR may comprise one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of the mRNA molecule, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0312] On the basis of the aforementioned embodiment, 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 mRNA molecule stability, binding sites for amino acid sequences that enhance mRNA molecule translation, 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, out-of-frame upstream translation initiation site, out-of-frame upstream start codon, and / or nucleotide sequences that produce secondary structures that reduce or prevent translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0313] The target protein coding sequence comprises codons that can be translated into an amino acid sequence. The target protein coding sequence may comprise all codons that encode naturally occurring amino acids, or may comprise some or all of artificially synthesized codons. In some embodiments, some or all of the codons are codon-optimized. In some embodiments, some or all of the codons encode unnatural amino acids.

[0314] In some embodiments, the engineered DNA molecule further comprises structural elements on the 5' end of the target gene segment that are necessary to initiate or regulate transcription of the RNA, and these structural elements are known in the art. In some embodiments, the structural elements comprise at least a promoter. Promoters and their sequences are known in the art and include weak promoters, medium-strength promoters, strong promoters, mini promoters, or core promoters. 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 followed by a transcription factor binding site. The recognition and binding sites can interact with amino acid sequences that mediate or regulate transcription. Compared to the recognition site, the binding site is closer to the target gene segment. 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 that enhances 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 well 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 additional regulatory elements before it, such as an enhancer that can promote the transcription of the aforementioned target protein coding sequence in a DNA plasmid.

[0315] In some embodiments, the eukaryotic cell is a yeast cell.In some embodiments, the DNA molecule is a yeast display vector.

[0316] In addition, the present application also provides engineered RNA molecules encoding the aforementioned engineered receptors, fusion proteins, engineered receptors, or chimeric antigen receptors. In some embodiments, the engineered RNA molecules are transcribed from the aforementioned engineered DNA molecules. In some embodiments, the engineered RNA molecules have the same sequence as the RNA molecules transcribed from the aforementioned engineered DNA molecules. In some embodiments, the engineered RNA is mRNA. As used herein, "mRNA" (messenger RNA) is any RNA, naturally occurring, non-naturally occurring, or modified, that encodes at least one protein, polypeptide, or fragment thereof, and that has the ability to be translated to produce the encoded protein, polypeptide, or fragment thereof in vitro, in vivo, in situ, or ex vivo. Therefore, the mRNA can be mature mRNA or pre-mature mRNA, and the elements or structures that it must contain or optionally contain are known in the art. In some embodiments, the mRNA contains coding sequences for multiple necessary functional components to express, regulate, or enhance the expression level of the protein, polypeptide, or fragment thereof. Such functional components include, but are not limited to, a 5' cap, a 5' untranslatable region (UTR), a 3' untranslatable region (UTR), and the like. Both the 5'UTR and the 3'UTR are usually transcribed from genomic DNA and are elements present in the pre-mature mRNA.

[0317] The term "5' cap" is located at the 5' end of the mRNA and contains a methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate, forming a 5',5'-triphosphate linkage with its adjacent nucleotide. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O refers to unmethylated ribose of the terminal nucleotide, type I refers to methylation of the ribose of one terminal nucleotide, and type II refers to methylation of the ribose of both terminal nucleotides. 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 produce m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from type I cap structures 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 by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0318] 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 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 polyadenylation signal and at least one histone stem-loop. Although the two have alternative mechanisms in nature, their synergistic effect can increase protein expression to levels exceeding that observed with either element alone. 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 typically derived from a histone gene and comprises two adjacent partially or completely reverse-complementary sequences separated by a spacer (composed of a short sequence) that form a loop through intramolecular base pairing. Unpaired loop regions are generally unable to base pair with either 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) can occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0319] 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.

[0320] 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 just before administration (e.g., within a few minutes to an hour).

[0321] The lipid nanoparticles generally comprise 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, PCT / US2017 / 0 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.

[0322] 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 carries the same genetic information as the engineered DNA molecule or the engineered RNA molecule.

[0323] Engineered cells

[0324] The present application also provides engineered cells that 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.

[0325] 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 their precursor cells. 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.

[0326] In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting multiple different antigens, which contain the TIGIT extracellular region, fusion protein, engineered receptor or chimeric antigen receptor as described above, and one or more CARs targeting other tumor antigens. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting multiple different antigens, which contain the TIGIT extracellular region, fusion protein, engineered receptor or chimeric antigen receptor as described above, and one or more CARs targeting immune checkpoint proteins. In some embodiments, the engineered cells are CAR-T or CAR-NK cells targeting multiple different antigens, which contain the TIGIT extracellular region, fusion protein, engineered receptor or 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).

[0327] In some embodiments, the engineered cell is a TCR-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 TCR comprises 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, which comprises the TIGIT extracellular region, fusion protein, or engineered receptor as described above, the antigen binding domain of the TCR comprises 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, which comprises the TIGIT extracellular region, fusion protein, or engineered receptor as described above, the antigen binding domain of the TCR comprises 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 cells comprise the aforementioned engineered receptors, and the engineered cells comprise 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).

[0328] 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 cells comprise the aforementioned engineered receptor, and the engineered cells comprise 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).

[0329] In some embodiments, the engineered cell expresses or comprises one or more engineered receptors on its cell membrane, and at least one of the engineered receptors comprises the aforementioned TIGIT extracellular domain polypeptide, and the engineered receptor activates or inhibits the downstream signaling pathway of the engineered receptor after binding to CD155. 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 comprises 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 comprises the aforementioned TIGIT extracellular domain polypeptide or fusion protein, and the engineered receptor activates or inhibits the downstream signaling pathway of the engineered receptor after binding to CD155. 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).

[0330] In some embodiments, the engineered cells are selected from: T cells, NK cells, macrophages, DC cells, B cells, or precursor cells thereof.

[0331] In some embodiments, the tumor antigens described herein are selected from one or more of the following:

[0332] 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-1 Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, 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, Shen 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, oncofetal 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-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.

[0333] 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.

[0334] use

[0335] This application also provides the use of the aforementioned engineered TIGIT extracellular domain polypeptides, fusion proteins, engineered receptors, chimeric antigen receptors, engineered nucleic acid molecules, and engineered cells in the preparation of drugs for treating cancer. In some embodiments, the cancer is selected from one or more of the following:

[0336] 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.

[0337] In some embodiments, the cancer involves tumor cells that highly express CD155.

[0338] 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.

[0339] Example

[0340] Example 1: Screening of TIGIT mutant peptides

[0341] 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.

[0342] 1) Random mutagenesis of the TIGIT extracellular domain:

[0343] 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.

[0344] The mutation sites in mutants Mut1 to Mut24 relative to wild-type TIGIT are marked in bold.

[0345] A chimeric antigen receptor (CAR) was constructed using TIGIT as the antigen binding domain (or extracellular antigen binding domain). Table 1 below shows the correspondence between the CAR and the TIGIT mutants contained therein.

[0346] Table 1: CAR and its antigen binding domain

[0347] 2) Screening of mutant peptides: The mutant peptides obtained in 1) were used as extracellular antigen binding domains to construct the classic CD28TM-28Z CAR structure (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. Common methods in the field, such as enzyme digestion and ligation, were used. For specific steps, reference can be made to the Molecular Cloning Laboratory Manual (3rd edition, by J. Sambrook et al.), the 21st century college textbook "Gene Engineering" edited by Lou Shilin, Yang Shengchang et al., etc. A vector target plasmid containing the gene encoding the CAR (CAR gene) was constructed. The target plasmid was transfected into a 293T derivative cell line by calcium transfection for virus preparation. The titer was calculated as follows: Titer (TU / ml) = 1×10 5= × positive rate × dilution factor ÷ virus volume × 1000. PBMCs or T cells obtained using Ficoll separation or apheresis, or cryopreserved and revived PBMCs or T cells obtained above, are used for CAR-T cell preparation. The obtained PBMCs or T cells are activated with anti-CD3 and anti-CD28 monoclonal antibodies or activated magnetic beads coated with anti-CD3 and anti-CD28 monoclonal antibodies (CD3 / CD28 Dynabeads (40203D; Gibco)) and then transduced with CAR viruses prepared using the corresponding vectors in Table 2 to obtain CAR-T cells. These CAR-T cells are used to recognize CD155-positive and CD155-negative but CD112-positive target cells, respectively. The cell line used in the MDA-MD-231-Luc-GFP assay is the MDA-MD-231 cell line, a CD155- and CD112-positive human breast cancer cell line. The MDA-MD-231-CD155(-)-Luc-GFP assay is a CD155-negative but CD112-positive human breast cancer cell line. Figure 1 shows the expression of CD112 in the MDA-MB-231-Luc-GFP group. The control (CT) group refers to the results of incubating the MDA-MB-231 cell line with PBS, while the MDA-MB-231-Luc-GFP group refers to the results of co-incubating MDA-MB-231-Luc-GFP with an anti-CD112 antibody (CD112 (Nectin2) Monoclonal Antibody (R2.525) PE Lot: WC3208861A). Figures 2A-2E screened for mutant peptides that were cytotoxic to CD155-positive cells but had significantly reduced or no cytotoxicity against CD155-negative cells. This meant that mutant peptides that specifically recognized CD155 but had no or reduced recognition of CD112 were identified. A 1:1 effector-target ratio was used.

[0348] As shown in Figures 2A to 2E: the horizontal axis represents CAR-T containing different TIGIT mutant peptides, and the vertical axis represents the in vitro killing of CD155-positive and negative cells by CAR-T cells. The above are the specificity screening killing results of 24 CAR-Ts. According to the specificity screening criteria, the Mut6 mutant peptide corresponding to CAR6 was determined, that is, the mutant peptide with a mutation from C to W at position 48 relative to the wild type can specifically recognize CD155 in terms of 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 relative to the binding activity of CD112 is also significantly higher than that of the wild type. Therefore, the corresponding TIGIT mutant peptide sequences SEQ ID NO: 5-17, SEQ ID NO: 19 and SEQ ID NO: 22, 24, and 25 are also optional sequences in this application.

[0349] The sequences of the various TIGIT mutant peptides and the various elements used in the CAR structure in the examples of this application are shown in the sequence table at the end of the article.

[0350] CAR structural elements: The CAR structural elements described herein 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.

[0351] Example 2: Verification of specific recognition sites

[0352] Analysis of the Mut6 sequence revealed a mutation of amino acid 48 from C to W. To determine whether the mutation of amino acid 48 to a key amino acid could improve specificity, this mutant peptide and wild-type TIGIT (WT) were used as extracellular antigen binding domains to construct a classic CD28TM-28Z CAR structure. The CAR structure is: TIGIT peptide-CD28TM-28Z, forming CAR6 and CAR30, where CAR30 is a control CAR structure containing a wild-type TIGIT peptide (WT).

[0353] 1) CAR6 and CAR30 were packaged into viruses and infected into CHO cells. First, TIGIT antibodies were 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 with them.

[0354] 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 antibodies bind 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.

[0355] CD155 relative MFI = CD155 positive cell population MFI / TIGIT positive cell population MFI

[0356] CD112 relative MFI = CD112 positive cell population MFI / TIGIT positive cell population MFI

[0357] The relative MFI values ​​of CD155 and CD112 between groups were statistically analyzed with the values ​​of CAR30 using T Test, and P < 0.05 indicated significant differences.

[0358] As shown in 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 weaker binding.

[0359] Table 2 shows the statistical analysis results of CAR6 and CAR30 binding to CD112.

[0360] Table 2

[0361] 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. The third column represents the significance analysis results through TTest.

[0362] Conclusion: According to Figure 3 and Table 2, the results showed that the binding ability of CAR6 to CD112 was significantly reduced compared with the wild type.

[0363] Figure 4 shows the binding of CAR6 and CAR30 to CD155. As shown in Figure 4, the horizontal axis represents different CAR-CHO molecules, and the vertical axis represents the relative MFI value of CD155.

[0364] Table 3 shows the analysis of the binding ability of four TIGIT mutant peptides to CD155.

[0365] Table 3

[0366] 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. The third column represents the significance analysis results through TTest.

[0367] Conclusion: As shown in Figure 4 and Table 3, the results show that the binding ability of CAR6 to CD155 is significantly increased compared with the wild type.

[0368] Summary: Comprehensive analysis determined that CAR6 had significantly improved binding to CD155 compared to the wild type, while its binding ability to CD112 was significantly reduced.

[0369] In summary, the mutant TIGIT in which the amino acid at position 48 can mutate to W (mut6) is a more specific peptide.

[0370] 2) Further, the functional performance of the above mutant peptides on T cells was verified by in vitro killing.

[0371] 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 the target cell. The cells 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 film) 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.

[0372] Figure 5 shows the killing of MDA-MB-231-CD155(-)-Luc-GFP by CAR6 and CAR30. The results are statistical results of in vitro killing of three 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.

[0373] 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).

[0374] 3) In vivo effectiveness of TIGIT mutant peptides as extracellular recognition domains of CAR constructs

[0375] Flow cytometry was used to detect the expression of CD155 on target cells. Table 4 shows the expression of CD155 in HT1376-Luc-GFP and HPAC-Luc-GFP cells.

[0376] Table 4

[0377] Female NCG mice aged 6-8 weeks were selected and injected intravenously into the tail of mice with MDA-MB-231-Luc-GFP cells at a dose of 3×10^5 cells / mouse to establish a tumor-bearing model. Three days after tumor formation, the aforementioned CAR30 and CAR6 T cells were injected intraperitoneally at a dose of 5×10^5 CAR-T cells / mouse. The control T group received a transfusion of the same total number of T lymphocytes on day 3. The results are shown in Figure 6, demonstrating the in vivo efficacy evaluation of the CAR construct containing the aforementioned mutant peptides as the extracellular recognition domain in the MDA-MB-231 tumor model.

[0378] 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.

[0379] Example 3: Verification of the applicability of mutant peptides as the extracellular antigen binding domain of CAR structures in different CAR structures

[0380] 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 shown in Table 6 below, wherein Z or Z (1) or Z (3) is a different mutant peptide of CD3ζ, which is used as the 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 rest of the "-" indicates 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).

[0381] Table 5

[0382] The exemplary amino acid sequences used for each element in Table 5 are shown in the sequence listing at the end of the article. 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) (i.e., 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 (i.e., 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: 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.

[0383] CD155-expressing MDA-MB-231-Luc-GFP, HPAC-Luc-GFP, and HT-1376-Luc-GFP cells, each indicated for different indications, were used as positive target cells. MDA-MB-231-Luc-GFP is indicated for human breast cancer, HPAC-Luc-GFP is indicated for human pancreatic cancer, and HT-1376-Luc-GFP is indicated for human bladder cancer. CAR-T cells were plated onto the target cells at a 1:1 ratio, and the cytotoxicity of the different CAR-T cells against target cells was assessed using the ACEA xCELLigence RTCA MP instrument. The ACEA xCELLigence RTCA MP instrument measures the electrical resistance index of tumor cells attached to the bottom of the wells every 15 minutes. This resistance index is used to determine the proliferation or death of adherent target cells. The resistance index analysis formula 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%.

[0384] The results are shown in Figures 7A-7C, where 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 show that the mutant TIGIT, as the extracellular recognition domain of various CAR structures, has a significant killing effect on various CD155-expressing malignant tumors such as breast cancer, pancreatic cancer, and bladder cancer.

[0385] Furthermore, the CAR-T cells were activated by target cells and assayed for cytokine production. IFN-γ was detected using an Elisa assay using a BD kit. Kit catalog number: 555142, batch number: 6266958. Specific steps are described in the kit instructions. Table 6 below shows IFN-γ secretion by the seven CARs with different structures in response to the three target cell lines.

[0386] Table 6

[0387] As shown in Table 6 above, in experiments targeting different target cells, while the Control-T group had no cytokine secretion, the CAR-T cell groups all had obvious cytokine secretion.

[0388] The above Figure 7 and Table 6 analyzed the in vitro killing and factor secretion of CAR-T cells with seven different CAR structures using mutant TIGIT peptides as extracellular recognition domains 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.

[0389] Example 4: Verification of the application of mutant peptides as the extracellular antigen binding domain of CAR structure in dual CAR

[0390] Different dual CAR structures were designed based on CAR6, targeting the targets PSCA, CD123 and CEA respectively, and dual CAR structures with TIGIT mutant peptides were designed. Different hinges, i.e. 8h, G4h and 7h, different transmembrane CD8TM and CD28TM, different co-stimulatory signals, 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 below.

[0391] Table 7

[0392] The above-mentioned 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.

[0393] 1) Detection of antigen expression in target cells

[0394] Target cell antigen expression was detected using flow cytometry. As shown in Figure 8, the horizontal axis represents antigen fluorescence intensity. The positive rate of CEA expression in DLD1-CEA-Luc-GFP is 100%, while CEA expression is very weak in DLD1-Luc-GFP. The positive rate of CD155 expression in DLD1-CEA-Luc-GFP is 100%. The only difference between DLD1-CEA-Luc-GFP and DLD1-Luc-GFP is the exogenously expressed CEA molecule. Therefore, CD155 expression in DLD1-Luc-GFP is also 100%.

[0395] 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.

[0396] Table 8: Antigen expression on different target cells

[0397] As shown in Figure 9 above, CAR37 showed significant killing effects on both DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP. At the same time, the killing effect on DLD-1-CEA-Luc-GFP was significantly higher than that on DLD-1-Lcu-GFP. The main reason was that the dual CAR recognized CEA and CD155 of DLD-1-CEA-Luc-GFP cells and showed higher killing effects than the recognition of one target antigen of DLD-1-Luc-GFP, namely CD155, which proved that the dual CAR could exert better in vitro pharmacodynamics. CAR38 had a better effect on Molm13-Luc-GFP. There was significant killing of P, proving that CAR38 can exert significant in vitro pharmacodynamic effects in hematological tumors, such as human acute myeloid leukemia tumor cell models; as shown in Figure 9C above, the horizontal axis represents different CARs, and the vertical axis represents the in vitro killing percentage in different cells. The results showed that CAR39, CAR36, CAR40 and CAR41 all had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, and their killing percentages were all higher than 50%, proving that different CARs can exert significant in vitro pharmacodynamic effects in various solid tumor cell models.

[0398] Furthermore, the CAR-T cells were activated by target cells and assayed for cytokine secretion. IFN-γ was detected using an Elisa assay using a BD kit. Kit catalog number: 555142, batch number: 6266958. Specific steps are described in the kit instructions. Cytokine secretion results are shown in Table 9 below.

[0399] Table 9: Summary of IFN-γ secretion by five CARs during in vitro cell killing

[0400] 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 serve 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.

[0401] Example 5. Verification of the combined application of fusion protein constructed with mutant peptides and CAR targeting non-CD155 targets

[0402] 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 this 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.

[0403] 1) The inventors designed mut6-28TM-28 and, based on this, combined it with different CARs to verify the adaptability of the TIGIT mutant peptide to different transmembrane and intracellular signals. The TIGIT mutation has adaptability to multiple peptide transmembrane and intracellular signals.

[0404] 2) The inventors also designed different CAR structures and considered the compatibility of different targets such as PSCA, CD123 and CEA with TIGIT mutant peptide fusion proteins. The structural designs are shown in Table 10 below.

[0405] Table 10: CAR structure design

[0406] 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, the amino acid sequence of CD123 (i.e., the antigen binding domain that binds to CD123) is shown in SEQ ID NO: NO:30, 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.

[0407] 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.

[0408] As shown in Figure 10, the horizontal axis is CAR42 and CAR45, and the vertical axis is the killing percentage of the two cells against 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.

[0409] As shown in Figure 11, the horizontal axis represents CAR42 and CAR45, and the vertical axis represents their IFN-γ cytokine secretion. The results show that the factor secretion of CAR45 is 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.

[0410] 6-8 week old female NCG mice were injected intraperitoneally with DLD-1-CEA-Luc-GFP cells at a dose of 3×10^5 cells / mouse to establish a tumor-bearing model. Seven days after tumor formation, the aforementioned CAR42 and CAR45 T cells were injected into the tail vein at a dose of 5×10^5 CAR-T cells / mouse. The control T group received an equal number of T lymphocytes on day 7. Live imaging of the mice was performed every 7 days after administration to assess tumor cell persistence. Fluorescence values ​​were statistically analyzed and analyzed for significance using a T-test. The results are shown in Figure 12. In the peritoneal tumor efficacy evaluation model, blood was collected from the mice at the midpoint of the eye socket, and the CAR-T cell copy number in the blood was measured by fluorescence quantitative PCR. The results showed that the CAR-T cell copy number in the blood of CAR45 was significantly higher than that of CAR42 on days 7, 20, and 27 after CAR-T administration, demonstrating that the CAR-T cell-fused protein (CAR45) can significantly improve the persistence of CAR in the in vivo model. Figure 13 shows the blood copy number in the peritoneal tumor evaluation model.

[0411] Subcutaneous tumor formation was performed using DLD-1-CEA-Luc-GFP. CAR-T cells were reinfused into the tail vein 12 days after tumor formation. In vivo imaging of mice was performed every 7 days after administration to detect the retention of tumor cells in the mice. The imaging fluorescence values ​​were statistically analyzed using T Test for significance, as shown in Figure 14.

[0412] The results showed that CAR45 had significantly better in vivo efficacy than CAR42, demonstrating that the CAR45 CAR-T cell, formed by combining CAR with a TIGIT mutant peptide fusion protein, can significantly improve in vivo efficacy. Furthermore, the effectiveness stems from the recognition of the CD155 target by the mutated TIGIT in CAR45, further demonstrating the feasibility of using the TIGIT mutant peptide as one of the extracellular recognition domains of a multi-target CAR structure.

[0413] Furthermore, the inventors have also verified the applicability of TIGIT mutant peptides as one of the extracellular recognition domains of multi-target CAR products for CD155 targets in a variety of indications, such as acute myeloid leukemia, pancreatic cancer, bladder cancer, and other hematological tumors and solid tumors. The horizontal axis represents different CARs, and the vertical axis represents the in vitro killing percentage in different cells. As shown in Figure 15A, CAR46 has a significant killing effect on Molm13-Luc-GFP. As shown in Figure 15B above, the results show that CAR44, CAR43, CAR47 and CAR48 have a significant killing effect on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP and HT1376-Luc-GFP, proving that dual-target and multi-target CAR-Ts constructed with TIGIT mutant peptides as extracellular recognition domains for CD155 targets and any other malignant tumor targets can exert significant in vitro pharmacodynamic effects in various solid tumor cell models.

[0414] After activation by target cells, the CAR-T cells were assayed for cytokine secretion. IFN-γ was detected using an Elisa assay using a BD kit. Kit catalog number: 555142, batch number: 6266958. Specific steps are described in the kit instructions. Cytokine secretion results are shown in Table 11 below.

[0415] Table 11: Summary of IFN-γ secretion by five CARs during in vitro cell killing

[0416] 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.

[0417] 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.

[0418] The inventors constructed TIGIT single CARs with different CAR structures, prepared CAR-NK cells, and verified their expression. The results are shown in Table 12.

[0419] Table 12

[0420] Example 6: Specificity and functional verification of truncated TIGIT containing the mutation site described in the present invention

[0421] Based on Mut6, we truncated it and obtained the following truncated sequence (the sequence includes the polypeptide fragment of amino acid sequence 33-93 shown in the reference sequence SEQ ID NO: 1):

[0422] VTQVNWEQQDQLLAIWNADLGWHISPSFKDRVAPGPGLGLTLQSLT VNDTGEYFCIYHTYP (SEQ ID NO: 26, the mutation site at position 48 relative to the reference sequence is located at position 16 in this sequence).

[0423] The specificity of the truncated TIGIT mutant peptide was verified using the protocol of Example 2. 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.

[0424] 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, demonstrating 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 transform the tumor microenvironment.

[0425] 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.

[0426] Example 7. Construction of TIGIT mutant peptide and CAR

[0427] 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.

[0428] 1) Random mutagenesis of the TIGIT extracellular domain:

[0429] Method 1 was used to perform random mutations on the extracellular region of TIGIT to obtain five mutants Mut25 to Mut29 (whose amino acid sequences are shown in SEQ ID NOs: 47 to 51, respectively) as shown in the sequences at the end of the article.

[0430] The mutation sites in mutants Mut25 to Mut29 relative to wild-type TIGIT are marked in bold and underlined.

[0431] A chimeric antigen receptor (CAR) was constructed using TIGIT as the antigen binding domain (or extracellular antigen binding domain). Table 13 below shows the correspondence between the CAR and the TIGIT mutants contained therein.

[0432] Table 13: CAR and its antigen binding domain

[0433] 2) Construction of CAR-T cells: The mutant peptide obtained in 1) was constructed as the extracellular antigen binding domain on the classic CD28TM-28Z CAR structure. Specifically, the CAR structure is: TIGIT mutant peptide-CD28TM-28Z, and a vector target plasmid containing the gene encoding the CAR (CAR gene) was constructed. The above target plasmid was transfected into a 293T-derived cell line by calcium transfection for lentiviral preparation. The titer calculation method is: Titer (TU / ml) = 1×10 5 × positive rate × dilution factor ÷ virus volume × 1000. The titer results of viruses containing mutant peptide coding sequences are shown in Table 13 above.

[0434] PBMCs or T cells obtained using Ficoll separation or apheresis, or cryopreserved and revived PBMCs or T cells obtained as described above, are used for CAR-T cell preparation. The obtained PBMCs or T cells are activated with anti-CD3 and anti-CD28 monoclonal antibodies or activated magnetic beads coated with anti-CD3 and anti-CD28 monoclonal antibodies (CD3 / CD28 Dynabeads (40203D; Gibco)) and then transduced with a lentivirus encoding the CAR prepared from the aforementioned target plasmid to obtain CAR-T cells.

[0435] The sequences used in each TIGIT mutant peptide and each element in the CAR structure of this embodiment are shown in the sequence table at the end of the article. Among them, the amino acid sequence of CD28TM in the classic CD28TM-28Z is shown in SEQ ID NO:36, the amino acid sequence of 28 is shown in SEQ ID NO:37, and the amino acid sequence of Z is shown in SEQ ID NO:41; the sequence information of each TIGIT mutant peptide involved is as follows: the amino acid sequence of the wild-type TIGIT extracellular region is shown in SEQ ID NO:1, the amino acid sequence of Mut25 is shown in SEQ ID NO:47, the amino acid sequence of Mut26 is shown in SEQ ID NO:48, the amino acid sequence of Mut27 is shown in SEQ ID NO:49, the amino acid sequence of Mut28 is shown in SEQ ID NO:50, and the amino acid sequence of Mut29 is shown in SEQ ID NO:51.

[0436] Example 8. Screening of TIGIT mutant peptides

[0437] Select 6-8 week old female NCG mice and use 3E5 (3*10 5 ) were injected into the peritoneal cavity to form tumors for 3 days to establish a tumor-bearing model. On the 3rd day after tumor formation, 5E5 (5*10 5 ) CAR-T cells were injected into the mice, and in vivo imaging of the mice was performed every 7 days after administration to detect the retention of tumor cells in the mice. The imaging fluorescence values ​​were statistically analyzed using T Test for significance; the Control T group was re-infused with the same total number of T lymphocytes on the 3rd day. The results are shown in Figures 16A and 16B. Figure 16A is a visual graph of mice using CAR-T cells expressing CARs with different TIGIT mutant peptides as extracellular recognition regions to kill tumors in an immunodeficient mouse model of acute lymphoblastic leukemia; Figure 16B is a statistical curve of fluorescence values. The results show that CAR53 corresponding to the Mut29 mutant peptide is the optimal CAR-T, and CAR-T containing Mut26 also has an in vivo killing ability that is superior to the wild type (CAR54), indicating that the mutation contained in Mut29 can significantly improve the effectiveness of wild-type TIGIT as an extracellular recognition domain.

[0438] In addition to the amino acid sequences described in SEQ ID NO: 48 and SEQ ID NO: 51, the above-mentioned Mut26 and Mut29 can be further truncated to retain the amino acid sequences shown in SEQ ID NO: 52 and SEQ ID NO: 53. Through the same experiments described above in this example, it can be seen that the above-mentioned truncated Mut26 and truncated Mut29 have comparable capabilities to Mut26 and Mut29, and can be used as extracellular recognition domains to construct CAR structures. The CAR structure can be an extracellular recognition domain, a hinge structure, a transmembrane structure, an intracellular signaling region, or an extracellular recognition domain, a transmembrane structure, and an intracellular signaling region. CAR-T, CAR-NK and other engineered immune cells expressing CAR constructed with truncated Mut26 and truncated Mut29 have effective in vivo killing of CD155-positive solid tumors.

[0439] Example 9. In vitro functional verification of CAR-T cells using TIGIT mutant peptides as antigen binding domains

[0440] Different CAR structures were designed based on the Mut29 mutant peptide, including different hinges including 7h, G4h and 8h, different transmembrane domains including CD28TM and CD8TM, different co-stimulatory signal domains including CD28, 4-1BB and CD28-4-1BB, different CD3s including Z and z, and the structural designs of second-generation CAR and third-generation CAR were selected to form different CAR structures as shown in Table 14. The sequences of the various CAR structural elements are shown in the sequence table at the end of the article; wherein, the amino acid sequence of Mut29 is shown in SEQ ID NO: 51, the amino acid sequence of 28TM (i.e., CD28TM, derived from the human CD28 transmembrane sequence) is shown in SEQ ID NO: 36, the amino acid sequence of 28 (i.e., the costimulatory receptor molecule CD28, derived from the human CD28 intracellular signal sequence) is shown in SEQ ID NO: 37, the amino acid sequence of Z or z is shown in SEQ ID NO: 41, the amino acid sequence of WT is shown in SEQ ID NO: 1, the amino acid sequence of 8h (i.e., the hinge region derived from the human CD8 hinge sequence) is shown in SEQ ID NO: 32, the amino acid sequence of 8TM (i.e., CD8TM, the transmembrane domain derived from the human CD8 transmembrane sequence) is shown in SEQ ID NO: 35, the amino acid sequence of BB (i.e., 4-1BB, a costimulatory receptor molecule derived from the 4-1BB or CD137 intracellular signal sequence) is shown in SEQ ID NO: 38, and the amino acid sequence of 7h (i.e., the hinge region derived from the human CD7 molecule) is shown in SEQ ID NO: The amino acid sequence of G4h (i.e., the G4h hinge region) is shown in SEQ ID NO: 33, the amino acid sequence of 28-4-1BB (i.e., the CD28-4-1BB intracellular signal) is shown in SEQ ID NO: 40.

[0441] Table 14: CAR structure and name correspondence

[0442] HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP cells expressing CD155 for different indications were used as positive target cells. HPAC-Luc-GFP is indicated for human pancreatic cancer, PC3-PSCA-Luc-GFP is indicated for human prostate cancer, and HT-1376-Luc-GFP is indicated for human bladder cancer. CAR-T cells were plated on target cells at a 1:1 ratio, and the cytotoxicity of different CAR-T cells against target cells was assessed using the ACEA xCELLigence RTCA MP instrument. The ACEA xCELLigence RTCA MP instrument measures the electrical resistance index of tumor cells attached to the bottom of the wells every 15 minutes. The electrical resistance index is used to determine the proliferation or death of adherent target cells. The electrical resistance index analysis formula 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%.

[0443] The results are shown in Figure 17. CAR-T cells expressing CAR53 with a TIGIT mutant peptide as the recognition domain kill three target cells in vitro. The horizontal axis represents the killing of different target cells by CAR53, and the vertical axis represents the percentage of in vitro killing. The results show that CAR53 has a significant killing effect on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP, proving that CAR53 has a killing function in different target cells.

[0444] Furthermore, the CAR-T cells were activated by target cells and assayed for cytokine secretion. IFN-γ was detected using an Elisa assay using a BD kit. Kit catalog number: 555142, batch number: 6266958. Specific steps are described in the kit instructions. Cytokine secretion results are shown in Table 15 below.

[0445] Table 15: IFN-γ secretion by CAR53 during the killing of three target cells

[0446] It can be seen that the secretion of IFN-γ factor by CAR53 during the in vitro killing of three target cells was significantly higher than that of the control group, proving that CAR53 exerts effector function in different target cells.

[0447] In summary, CAR with TIGIT mutant peptide as the recognition domain has excellent function, and the constructed CAR-T cells have a killing effect on solid tumors such as pancreatic cancer, prostate cancer and bladder cancer that express CD155.

[0448] The expression of CD155 on target cells was detected by flow cytometry. The results are shown in Table 16.

[0449] Table 16: Expression of CD155 in HT1376-Luc-GFP and HPAC-Luc-GFP cells

[0450] Female NCG mice aged 6-8 weeks were selected, and HPAC-Luc-GFP and HT1376-Luc-GFP (HPAC and HT1376 cell lines were purchased from Beijing Beina Chuanglian Biotechnology Research Institute) 3E5 tumor cells were used to form tumors in the abdominal cavity for 3 days to construct a tumor-bearing model. On the 3rd day of tumor formation, 5E5 CAR-T cells were intraperitoneally administered and infused. Every 7 days after administration, mice were imaged in vivo to detect the retention of tumor cells in the mice. The imaging fluorescence values ​​were statistically analyzed using T Test for significance. The Control T group was infused with the same total number of T lymphocytes on the 7th day. The results are shown in Figures 18 and 19, which verify the effectiveness of CAR-T with mutant TIGIT peptide as the extracellular recognition domain against pancreatic cancer and bladder cancer, respectively. It can be seen that HPAC-Luc-GFP and HT1376-Luc-GFP were used for peritoneal tumor formation, and the results showed that the in vivo efficacy of CAR53 was significantly better than that of CAR54, proving that the CAR-T constructed with the mutant TIGIT peptide as the extracellular recognition domain of the present invention has better effectiveness against pancreatic cancer and bladder cancer expressing CD155 than the wild-type TIGIT peptide. The TIGIT peptide of the present invention can be used as the extracellular recognition domain of the CAR structure for CD155-expressing immune cell therapy.

[0451] In addition to being applied to the 28TM-28z structure described in CAR53, the mutant TIGIT peptide described in the present invention is also applicable to a variety of CAR structure combinations. Here, 8h-8TM-BBZ, 7h-28TM-28z, and G4h-28TM-28-4-1BBZ are used as examples to verify the applicability of the TIGIT mutant peptide to the CAR structure combination:

[0452] The CAR-T cells of CAR55, CAR56, and CAR57 were prepared using the aforementioned method, with HT1376-Luc-GFP, PC3-Luc-GFP, and HPAC-Luc-GFP as target cells, respectively. The effector-target ratio was 1:1, and the killing of target cells by CAR-T after 24 hours of co-incubation with target cells was detected. The effectiveness of the mutated TIGIT described in the present invention against bladder cancer, prostate cancer, and pancreatic cancer expressing CD155 was verified under different CAR structures. The results are shown in Figure 20, which shows the killing of malignant tumors of different indications by CAR-T cells constructed with TIGIT mutant peptides as extracellular recognition domains in different CAR structures. Further, the above-mentioned CAR-T (CAR55, CAR56, and CAR57) was tested for cytokines after being activated by target cells. The cytokine IFN-γ detection was performed using the Elisa method using a BD company kit. Detection kit item number: 555142, production batch number 6266958, specific steps are shown in the kit instructions. The cytokine secretion results are shown in Table 17 below.

[0453] Table 17: IFN-γ secretion of three CARs with different structures during the killing of three target cells

[0454] Figure 20 and Table 17 demonstrate that the optimal mutant peptide of mut29, when combined with different hinges 7h, G4h, and 8h, different transmembrane CD28TM and CD8TM, different co-stimulatory signals CD28, 4-1BB, and CD28-4-1BB, different CD3 Z and z, and the structural designs of second-generation CAR and third-generation CAR, can exert in vitro killing and effector functions in different target cells, namely PC3-Luc-GFP, HT1376-Luc-GFP, and HPAC-Luc-GFP.

[0455] Based on this, those skilled in the art can reasonably infer that the mutated TIGIT peptide described in the present invention can be used as the extracellular recognition domain of the CAR structure, and the constructed therapeutic immune cells or products or drugs containing therapeutic immune cells can play an effective killing effect on CD155-expressing malignant tumor cells. In some embodiments, the therapeutic immune cells refer to CAR-T cells, in some embodiments, the therapeutic immune cells refer to CAR-NK cells, and in some embodiments, the therapeutic immune cells refer to CAR-macrophages, etc. In some embodiments, the CD155-expressing malignant tumor cells include breast cancer, bladder cancer, prostate cancer and pancreatic cancer, as well as brain glioma, melanoma, bile duct cancer, non-small cell lung cancer, colorectal cancer, etc.

[0456] The above-mentioned Mut26, Mut29, truncated Mut26, and truncated Mut29 can also be combined with different CAR structures, including but not limited to hinge 7h, G4h, and 8h, different transmembrane CD28TM and CD8TM, different co-stimulatory signals CD28, 4-1BB, and CD28-4-1BB, different CD3 Z and z, as well as the structural designs of second-generation CAR and third-generation CAR, all of which can exert in vitro killing and effector functions in different CD155-positive tumors such as prostate cancer (PC3-Luc-GFP), bladder cancer (HT1376-Luc-GFP), pancreatic cancer (HPAC-Luc-GFP), acute lymphoblastic leukemia, etc.

[0457] Through the same test as above in this example, it can be seen that the mutated TIGIT peptides of the present invention, such as the truncated peptides shown in SEQ ID NO: 52 (Mut26 truncation) and SEQ ID NO: 53 (Mut29 truncation), can be used as the extracellular recognition domain of the CAR structure, and the constructed therapeutic immune cells or products or drugs containing therapeutic immune cells can play an effective killing effect on CD155-expressing malignant tumor cells. In some embodiments, the therapeutic immune cells refer to CAR-T cells, in some embodiments, the therapeutic immune cells refer to CAR-NK cells, and in some embodiments, the therapeutic immune cells refer to CAR-macrophages, etc. In some embodiments, the CD155-expressing malignant tumor cells include breast cancer, bladder cancer, prostate cancer and pancreatic cancer, as well as: brain glioma, melanoma, bile duct cancer, non-small cell lung cancer, colorectal cancer, etc.

[0458] Example 10. Verification of the application of mutant peptides as antigen binding domains in CAR or fusion proteins

[0459] 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 an engineered receptor or fusion protein containing a TIGIT mutant peptide in the antigen binding domain (in the embodiments of this application, the fusion protein 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.

[0460] 1) The inventors designed a mut29-28TM-28 fusion protein and, based on this, designed structures combining CARs that bind to different target proteins with the fusion protein to verify the adaptability of the TIGIT mutant peptide to different transmembrane and intracellular signals. The TIGIT mutation has adaptability to multiple peptide transmembrane and intracellular signals.

[0461] 2) The inventors also made different designs for the CAR structure and considered the compatibility of CARs with different targets such as CD123 and CEA with engineered receptors containing TIGIT mutant peptides in the antigen binding domain, wherein the engineered receptor is a fusion protein that does not contain a primary signal transduction domain, and its specific structural design is shown in Table 18 below. The sequences of the elements in the CAR structure involved are shown in the sequence listing at the end of the article; wherein, 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 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 or 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 mut29 is shown in SEQ ID NO: 51, 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 CD123 (i.e., the antigen binding domain that binds to CD123) is shown in SEQ ID NO: 30, and the amino acid sequence of 2B4 (i.e., the 2B4 intracellular signal) is shown in SEQ ID NO: 39.

[0462] Table 18: CAR structure design

[0463] CAR-T cells were prepared using the protocol of Example 7, and the effectiveness of a single fusion protein with the mutant TIGIT extracellular domain as the antigen binding domain and a CAR with the mutant TIGIT extracellular domain as the antigen binding domain were compared. The results are shown in Figure 21, which shows the in vitro killing of target cells of three different indications by CAR53 and CAR61. Table 19 shows the secretion of IFN-γ factors by CAR53 and CAR61 during the in vitro killing experiment.

[0464] Table 19

[0465] As shown in Figure 21 and Table 19 above, the fusion protein without the primary signal transduction domain formed by the mutant peptide is expressed in CAR61 prepared by T cells. Its structure includes an extracellular recognition domain with a TIGIT mutant peptide, a transmembrane structure and an intracellular co-stimulatory signal domain, but does not contain a primary signal domain. CAR61 has no killing effect on the three malignant tumor cells expressing CD155, but the CAR-T cell CAR53 constructed with the TIGIT mutant peptide has a killing effect on the three malignant tumor cells expressing CD155. This proves that the mutant TIGIT peptide constructs a fusion protein form as shown in CAR61 that cannot exert a killing function alone.

[0466] Select CAR (CAR58) targeting the CEA target, design CAR59 with a fusion protein of the TIGIT mutant peptide, use DLD-1-CEA-Luc-GFP and DLD1-Luc-GFP as target cells, and select 1:1 for the effect-target ratio, where DLD-1-CEA-Luc-GFP highly expresses the CEA target molecule, and DLD1-Luc-GFP has weak CEA expression. The method of Example 9 was used to verify the in vitro killing ability of CAR58 (T cells expressing CAR targeting CEA alone without expressing the fusion peptide carrying TIGIT) and CAR59 (T cells simultaneously expressing CAR targeting CEA and the fusion peptide shown in CAR61) to evaluate the effectiveness of an engineered receptor expressing an antigen binding domain containing a TIGIT mutant peptide without a primary signal transduction domain in CAR-T cells. The results are shown in Figure 22, showing the in vitro effectiveness data of CAR-T cells expressing an engineered receptor expressing an antigen binding domain containing a TIGIT mutant peptide without a primary signal transduction domain. The horizontal axis represents CAR58 and CAR59, and the vertical axis represents the killing percentage of the two cells against DLD-1-CEA-Luc-GFP and DLD-1-Luc-GFP. The results showed that CAR58 and CAR59 had obvious killing effects on DLD-1-CEA-Luc-GFP, and the killing effect of CAR59 on DLD-1-Luc-GFP was significantly higher than that of the CAR58 group. It can be seen that CAR-T that simultaneously expresses CAR targeting CEA and the fusion peptide shown by CAR61 is superior to CAR-T that does not express fusion protein in terms of effective killing ability.

[0467] Female NCG mice aged 6-8 weeks were selected, and subcutaneous tumor formation was performed using DLD-1-CEA-Luc-GFP. CAR-T cells were reinfused into the tail vein 12 days after tumor formation. In vivo imaging of the mice was performed every 7 days after administration to detect the retention of tumor cells in the mice. The imaging fluorescence values ​​were statistically analyzed and significance analysis was performed using T Test. The results are shown in Figures 23 and 24. Figure 23 is a graph showing the in vivo effectiveness of CAR-T cells expressing a fusion protein containing a TIGIT mutant peptide in the antigen binding domain against colorectal cancer tumors in colorectal cancer-indication tumor-bearing mice, and Figure 24 is a graph showing the in vivo tumor fluorescence curve of CAR-T cells expressing a fusion protein containing a TIGIT mutant peptide in the antigen binding domain against colorectal cancer tumors in colorectal cancer-indication tumor-bearing mice. The results showed that CAR59 CAR-T cells expressing mut29-28TM-28 fusion protein had better in vivo efficacy than CAR-T cells CAR58 which did not express fusion protein containing TIGIT mutant peptide in the antigen binding domain, indicating that mut29-28TM-28 fusion protein, or TIGIT mutant peptide, as an engineered receptor of the extracellular recognition domain combined with CAR can promote the in vivo efficacy of CAR-T cells.

[0468] The experimental mice were bled at the endpoint, and the CAR-T copy number in the blood was detected by fluorescent quantitative PCR. The results are shown in Figure 25. The CAR-T cells expressing the fusion protein containing the TIGIT mutant peptide in the antigen binding domain killed the tumor in colorectal cancer-bearing mice. The results showed that the CAR-T copy number in the blood of CAR59 was significantly higher than that of CAR58 on the 7th, 20th and 27th days after CAR-T administration, proving that the simultaneous expression of CAR and the fusion protein containing the TIGIT mutant peptide in the antigen binding domain on the T cell membrane, namely CAR13, can significantly improve the persistence of CAR in the in vivo model.

[0469] The above results demonstrate that CAR59, a fusion protein containing a TIGIT mutant peptide in the antigen-binding domain, can significantly enhance the in vivo efficacy and improve the effectiveness of CAR-T cells in vivo. Furthermore, the effectiveness and sustainability of this effect are derived from the co-expression of the fusion protein containing a TIGIT mutant peptide in the antigen-binding domain of the present invention with CAR.

[0470] Furthermore, the inventors have also verified the effectiveness of fusion proteins containing TIGIT mutant peptides in multiple indications, such as acute myeloid leukemia, pancreatic cancer, bladder cancer, and other hematological tumors and solid tumors, in CAR-T cells expressing multiple indication targets against CD155-expressing malignant tumors. The results are shown in Figure 26. The effective killing of CD123 CAR-T cells expressing fusion proteins containing TIGIT mutant peptides in the antigen binding domain against acute myeloid leukemia was verified using the acute myeloid leukemia tumor cell line Molm-13.

[0471] Further, through the same experiments in this example, it can be seen that Mut26, Mut29, truncated Mut26, and truncated Mut29 can also construct fusion proteins or engineered receptors of various structures to match the CAR structure to form an engineered receptor combination, which is expressed on the cell surface of immune cells such as T cells, NK cells, DC cells, macrophages, etc., for the treatment of solid tumors and acute lymphoblastic leukemia that express CD155.

[0472] It can be seen that the fusion protein whose antigen binding domain contains TIGIT mutant peptide itself does not play an effector function in the killing process, which ensures the safety of its application. However, when the fusion protein is co-expressed in immune cells with CEA or CD123 CAR, it can significantly improve the in vivo efficacy and at the same time improve the persistence of immune cells (such as CAR-T) in the blood. At the same time, the in vitro killing and factor secretion data show that the fusion protein whose antigen binding domain contains TIGIT mutant peptide and CAR molecules targeting different target proteins can play obvious killing and effector functions when co-expressed on the surface of immune cells, indicating that this fusion protein can be applied in combination with different targets.

[0473] The corresponding sequences of the components used in the examples of this application are shown in the following sequence table.

[0474] Sequence Listing

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 at position 48 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. The TIGIT extracellular domain polypeptide according to any one of claims 1 to 6, further comprising a mutation at the following site relative to the reference sequence: 1) No. 57 and No. 86; or 2) No. 71 and No. 88; The reference sequence is an amino acid sequence as shown in SEQ ID NO: 1, and the numbering of amino acid positions is defined by the reference sequence.

8. The TIGIT extracellular domain polypeptide according to claim 7, comprising the following mutation combinations relative to the reference sequence: 1) C48W, S57P and F86S; or 2)C48W, G71D and I88V.

9. The TIGIT extracellular region polypeptide according to claim 8, comprising a combination of amino acid sites of any one of 1) to 4): 1)34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S and 88I; 2) 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F and 88V; 3) 9T, 20I, 21I, 34T, 39E, 48W, 57P, 61K, 70L, 71G, 80N, 86S, 88I, 101I and 110V; or 4)9T, 20I, 21I, 34T, 39E, 48W, 57S, 61K, 70L, 71D, 80N, 86F, 88V, 101I and 110V. 10 . The TIGIT extracellular region polypeptide according to claim 7 , comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47-53 or a conservatively substituted variant thereof.

11. A fusion protein comprising the TIGIT extracellular domain polypeptide according to any one of claims 1-10.

12. The fusion protein according to claim 11, 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.

13. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the TIGIT extracellular region polypeptide of any one of claims 1-10, or the fusion protein of claim 11 or 12.

14. An engineered receptor according to claim 13, further comprising a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

15. The engineered receptor according to claim 13 or 14, which is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell antigen coupling agent (TAC) or a fusion protein.

16. The engineered receptor of claim 14 or 15, wherein the co-stimulatory domain comprises a signal transduction domain selected from 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), 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.

17. An engineered receptor according to any one of claims 14 to 16, wherein the primary signaling domain comprises a signaling 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.

18. The engineered receptor according to claim 17, further comprising a transmembrane domain between the antigen binding domain and the signal transduction domain, wherein the transmembrane domain comprises a transmembrane domain selected from any one or more of the following molecules: ICOS, CD4, CD8α, CD28, CD3ζ and TIGIT.

19. An engineered receptor according to claim 18, 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.

20. The engineered receptor according to any one of claims 13 to 19, comprising or being: 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, The co-stimulatory domain comprises or is a signal transduction domain selected from 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), 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; 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 The hinge region comprises or is the hinge region of TIGIT, CD7, IgG, IgD, CD8α or CD28, or a combination thereof.

21. The engineered receptor according to claim 20, which comprises or is, from N-terminus to C-terminus, TIGIT extracellular domain polypeptide, CD28 transmembrane domain and CD28 signal transduction domain.

22. The engineered receptor according to any one of claims 13 to 19, which comprises or is any one of 1) to 10) from N-terminus to C-terminus: 1) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signal transduction domain and CD3ζ signal transduction domain; 2) TIGIT extracellular domain polypeptide, CD8 hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain; 3) TIGIT extracellular domain polypeptide, CD8 hinge region, CD28 transmembrane domain, 4-1BB signaling domain, and CD3ζ signaling domain; 4) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain; 5) TIGIT extracellular domain polypeptide, ICOS transmembrane domain, ICOS signal transduction domain and CD3ζ signal transduction domain; 6) TIGIT extracellular domain polypeptide, CD8 transmembrane domain, CD134 signaling domain, and CD3ζ signaling domain; 7) TIGIT extracellular domain polypeptide, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain; 8) TIGIT extracellular domain polypeptide, CD7 hinge region, C28 transmembrane domain, CD28 signaling domain, and CD3ζ signaling domain; 9) TIGIT extracellular domain polypeptide, G4h hinge region, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, and CD3ζ signaling domain; and 10) TIGIT extracellular domain polypeptide, CD8 hinge region, CD8 transmembrane domain, 4-1BB signaling domain and CD3ζ signaling domain.

23. An engineered nucleic acid molecule comprising a protein encoding the TIGIT extracellular domain polypeptide of any one of claims 1-10, the fusion protein of claim 11 or 12, or the engineered receptor of any one of claims 13-22.

24. An engineered cell comprising the TIGIT extracellular domain polypeptide of any one of claims 1-10, the fusion protein of claim 11 or 12, the engineered receptor of any one of claims 13-22 and / or the engineered nucleic acid molecule of claim 23.

25. An engineered cell according to claim 24, comprising an engineered receptor that binds the target molecule CD155.

26. An engineered cell according to claim 24 or 25, comprising two or more engineered receptors that bind to the same target molecule or different target molecules.

27. The engineered cell according to claim 26, 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, The engineered receptor that binds to the target molecule CD155 is an engineered receptor according to any one of claims 13 to 22, 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.

28. An engineered cell according to claim 27, wherein The antigen binding domain of the engineered receptor that binds to the target molecule CD155 comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-26, 47-53 or a conservatively substituted variant thereof; 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; 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, 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.

29. The engineered cell according to any one of claims 24-28, which is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof.

30. Use of the TIGIT extracellular domain polypeptide of any one of claims 1-10, the fusion protein of claim 11 or 12, or the engineered receptor of any one of claims 13-22, the nucleic acid molecule of claim 23, or the engineered cell of any one of claims 24-29 for preparing a drug for treating cancer.

31. The use according to claim 30, wherein the cancer is selected from one or more of the following: 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.

32. A method for extending the in vivo persistence of CAR-T cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of claims 1-10, the fusion protein described in claim 11 or 12, or the engineered receptor described in any one of claims 13-22 on the CAR-T cell membrane.

33. A method for enhancing the in vivo expansion ability of CAR-T cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of claims 1-10, the fusion protein described in claim 11 or 12, or the engineered receptor described in any one of claims 13-22 on the CAR-T cell membrane.

34. A method for enhancing the in vivo killing ability of CAR-T on target cells, comprising expressing the TIGIT extracellular domain polypeptide described in any one of claims 1-10, the fusion protein described in claim 11 or 12, or the engineered receptor described in any one of claims 13-22 on the CAR-T cell membrane.

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