Antibodies targeting PSCA and uses thereof

Abstract

The present disclosure provides an isolated antibody that binds to prostate stem cell antigen (PSCA), preferably the antibody is a single chain antibody (scFv). Further provided are engineered receptors and engineered cells using the antibodies as antigen binding domains.

Description

Technical Field

[0001] The present application relates to antibodies and engineered receptors comprising antigen-binding fragments of antibodies, as well as engineered cells comprising the engineered receptors. Background Art

[0002] Chimeric antigen receptor (CAR) is an artificial receptor that simulates TCR function. CAR molecules generally contain an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain is usually the CD3ζ chain or FcRγ, or is connected to one or more co-stimulatory molecules, such as 4-1BB, CD28, and ICOS (CD278).

[0003] PSCA (prostate stem cell antigen) is a glycosylphosphatidylinositol (GPI)-linked cell membrane antigen that is only expressed in the prostate and bladder in normal tissues. It is overexpressed in most prostate tumors, bladder cancer, pancreatic cancer, non-small cell lung cancer, and gastric cancer. It is a good therapeutic target for solid tumors. Faced with the current problem of poor efficacy and poor prognosis of solid tumors, immunotherapy targeting PSCA may be a breakthrough in the future treatment of prostate cancer and a variety of solid tumors expressing PSCA.

[0004] The extracellular antigen recognition domain can also be called the extracellular antigen binding domain, which can specifically recognize tumor surface antigens. The CAR molecule transmits immune cell activation signals through the hinge region and transmembrane region by using the characteristics of specific recognition of tumor antigens by the antigen binding domain. The most common extracellular antigen recognition domain is ScFv (single-chain antibody) that recognizes tumor antigens. The selection of ScFv is crucial for the function of CAR. Therefore, for immunotherapy targeting PSCA, the screening of anti-PSCA antibodies or anti-PSCA ScFv is very necessary. Summary of the invention

[0005] The present application obtains a fully human single-chain antibody suitable for targeting PSCA through screening of a fully human phage library, and the fully human single-chain antibody can target PSCA protein and PSCA-positive cells with high specificity; the fully human single-chain antibody can be used as an antigen binding domain of a chimeric antigen receptor (CAR) and an engineered receptor, and has broad application prospects in the treatment of solid tumors. In one aspect, the present disclosure provides a separated antibody (or its antigen binding fragment), which, when used as an antigen binding domain of a CAR, can enable a CAR-T cell containing the CAR to have a higher positive rate of CAR targeting PSCA, and a higher target cell killing ability.

[0006] Specifically, this application relates to:

[0007] 1. An antibody that binds to prostate stem cell antigen (PSCA), comprising an immunoglobulin heavy chain variable region (VH) sequence and an immunoglobulin light chain variable region (VL) sequence, wherein the VH comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), and a heavy chain variable region complementarity determining region 3 (HCDR3), and the VL comprises a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3);

[0008] Where VL and VH contain:

[0009] 1) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1, 2, 3 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 7;

[0010] 2) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 9, 10, 11 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14, 15;

[0011] 3) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, 18, 19 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 21; or

[0012] 4) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 23, 2, 24 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 6, 26,

[0013] Among them, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by the IMGT system.

[0014] 2. The antibody according to item 1, which is a chimeric antibody or a human antibody. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure further comprises a human acceptor framework. In certain embodiments, the human acceptor framework is derived from a human immunoglobulin framework or a human common framework. In certain embodiments, the human acceptor framework comprises a subtype κI framework sequence of VL, and a subtype III framework sequence of VH. In general, the subtype of the sequence is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication 91-3242, Bethesda MD (1991), 1-3 volumes. In certain embodiments, for VL, the subtype is as described in subtype κI of Kabat et al. (supra). In certain embodiments, for VH, the subtype is as described in subtype III of Kabat et al. (supra). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human common framework. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human consensus framework with changes in the amino acid sequence, e.g., changes of 1-15, 1-10, 2-9, 3-8, 4-7, or 5-6 amino acids.

[0015] 3. An antibody according to item 1 or 2, wherein the amino acid sequence of VH comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27.

[0016] 4. An antibody according to any one of items 1 to 3, wherein the amino acid sequence of VL comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25.

[0017] 5. The antibody according to any one of items 1 to 4, comprising the VL and VH of any of the following:

[0018] 1) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO: 25, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO: 27, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 27;

[0019] 2) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8;

[0020] 3) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO:12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16; or

[0021] 4) the amino acid sequence of the VL comprises or is the amino acid sequence as shown in SEQ ID NO:20, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the amino acid sequence of the VH comprises or is the amino acid sequence as shown in SEQ ID NO:22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22.

[0022] 6. The antibody according to any one of items 1 to 5, which is a full-length antibody or an antigen-binding fragment.

[0023] 7. The antibody according to item 6, wherein the antigen-binding fragment is a single-chain antibody (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb, diabody or a complex formed thereof;

[0024] The full-length antibody is IgG, IgE, IgA, or IgM.

[0025] In some embodiments, the antibody is of IgG1, IgG2, or IgG4 isotype.

[0026] 8. The antibody according to item 7, wherein VH and VL in the scFv are connected via a chemical bond or a linker.

[0027] 9. The antibody according to item 8, wherein the chemical bond is a peptide bond, and the linker is an amino acid linker, preferably the amino acid linker is a peptide chain comprising 4 to 25 amino acids.

[0028] 10. An antibody according to claim 9, wherein the peptide chain comprises or is an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0029] 11. An antibody according to any one of items 1 to 10, which comprises or is the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 28.

[0030] 12. The antibody according to any one of items 1 to 11 is a bispecific or multispecific antibody.

[0031] 13. An engineered receptor comprising an antigen binding domain, wherein the antigen binding domain comprises the antibody according to any one of items 1 to 12.

[0032] 14. An engineered receptor according to item 13, further comprising a signal transduction domain, wherein the signal transduction domain comprises a primary signal transduction domain and / or a co-stimulatory domain.

[0033] 15. An engineered receptor according to item 13 or 14, which is a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a T cell antigen coupling agent (TAC).

[0034] 16. An engineered receptor according to any one of items 13 to 15, wherein the antibody is a single chain antibody (scFv).

[0035] 17. An engineered receptor according to any one of items 13 to 16, wherein the primary signal transduction domain comprises a signal transduction domain of one or more molecules selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, FcRγ, FcRβ, FcεRIγ, FcεRIβ, FcγRIIa, CD79α, CD79β, CD66d, DAP10 and DAP12.

[0036] 18. An engineered receptor according to any one of items 13 to 17, wherein the co-stimulatory domain comprises a signal transduction domain of one or more molecules selected from 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, ITG AD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83.

[0037] 19. An engineered receptor according to any one of items 14-18, which further comprises a transmembrane domain located between the signal transduction domain and the antigen binding domain, preferably the transmembrane domain is the transmembrane domain of ICOS, CD4, CD8α, CD28 or CD3ζ or a combination thereof.

[0038] 20. An engineered receptor according to claim 19, wherein the one or more antigen binding domains and the transmembrane domain are connected by a hinge region, preferably the hinge region is the hinge region of IgG, IgD, CD7, CD8α or CD28 or a combination thereof.

[0039] In some embodiments, the engineered receptor comprises or is, from N-terminus to C-terminus:

[0040] 1) The antibody, transmembrane structure, and primary signal transduction domain according to any one of the preceding items 1 to 12;

[0041] 2) the antibody, transmembrane structure, co-stimulatory domain, or primary signal transduction domain according to any one of items 1 to 12 above;

[0042] 3) the antibody, hinge region, transmembrane structure, or primary signal transduction domain according to any one of 1 to 12 above; or

[0043] 4) The antibody, hinge region, transmembrane structure, co-stimulatory domain, and primary signal transduction domain according to any one of Items 1 to 12 above.

[0044] In some embodiments, the engineered receptor comprises or is, from N-terminus to C-terminus:

[0045] 1) the antibody, CD8 hinge region, CD28 transmembrane domain, CD28 signaling domain, 4-1BB signaling domain, CD3ζ signaling domain according to any one of the preceding items 1 to 12; or

[0046] 2) The antibody, CD8 hinge region, CD8 transmembrane domain, 4-1BB signaling domain, and CD3ζ signaling domain according to any one of the preceding items 1 to 12.

[0047] In some embodiments, the CD8 hinge region comprises or is an amino acid sequence as shown in SEQ ID NO: 36 or 37. In some embodiments, the CD28 transmembrane domain comprises or is an amino acid sequence as shown in SEQ ID NO: 35. In some embodiments, the CD8 transmembrane domain comprises or is an amino acid sequence as shown in SEQ ID NO: 34. In some embodiments, the CD28 signaling domain comprises or is an amino acid sequence as shown in SEQ ID NO: 39. In some embodiments, the 4-1BB signaling domain comprises or is an amino acid sequence as shown in SEQ ID NO: 38. In some embodiments, the CD3 zeta signaling domain comprises or is an amino acid sequence as shown in SEQ ID NO: 41.

[0048] 21. A nucleic acid molecule comprising a nucleic acid molecule encoding an antibody according to any one of items 1 to 12, or an engineered receptor according to any one of items 13 to 20. In some embodiments, the engineered nucleic acid molecule is DNA, RNA (eg, mRNA), or a hybrid molecule of RNA and DNA.

[0049] In some embodiments, the engineered nucleic acid molecules are chemically modified. For example, in some embodiments, one or more thymidines of the engineered nucleic acid molecules are replaced by uridines. In some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by thymidines. In some embodiments, one or more guanosines of the engineered nucleic acid molecules are replaced by creatinine. In some embodiments, the chemical modification is to replace one or more nucleotides of the nucleic acid molecules with their corresponding nucleotide derivatives. For example, in some embodiments, one or more uridines of the engineered nucleic acid molecules are replaced by one or more selected from the following: 5-(carboxyhydroxymethyl)uridine (CHM5U), 5-carboxymethylaminomethyluridine (CMNM5U), 5-carboxymethylaminomethyl-2-thiouridine (CMNM5S2U), dihydrouridine (DHU), 2'-O-methylpseudouridine (FM), 1-methylpseudouridine (M1F), 3-(3-amino-3-carboxy-propyl)uridine ((ACP3)U), uridine-5-oxyacetic acid (O5U), uridine-5-oxyacetic acid methyl ester (MV), 5-methoxycarbonyl uridine (M5U), 5-methoxycarbonylmethyl-2-thiouracil (MCM5S2U), 5-methoxyuridine (MO5U), 5-methyl-2-thiouridine (S2T), 2-thiouridine (S2U), 4-thiouridine (S4U), 5-methyluridine (M5U), 2'-O-methyl-5-methyluridine (TM), 2'-O-methyluridine (UM), 5-methylaminomethyluridine (MAM5U), 5-methylaminomethyl-2-thiouridine (MAM5S2U), pseudouridine (P), and 5-methoxycarbonylmethyl-2-thioguanosine (MCM5S2U). In some embodiments, one or more guanosines of the engineered nucleic acid molecule are replaced by one or more selected from the following: wybutoxosine (osyw), ybutoxosine (yw), 1-methylinosine (m1i), 2'-O-methylguanosine (gm), 1-methylguanosine (m1g), 2,2-dimethylguanosine (m22g), 2-methylguanosine (m2g), 7-methylguanosine (m7g) and β, D-galactose Q nucleoside (gal q), Q nucleoside (q) and β, D-mannose Q nucleoside (man q).In some embodiments, one or more adenosines of the engineered nucleic acid molecule are replaced by one or more selected from the following: N6-isopentenyl adenosine (i6a), 1-methyladenosine (m1a), 2-methyladenosine (m2a), N6-methyladenosine (m6a), 2-methylthio-N6-isopentenyl adenosine (ms2i6a), N-((9-β-D-ribofuranosyl-2-thiomethylpurin-6-yl)carbamoyl)threonine (ms2t6a), N-((9-β-D-ribofuranosylpurin-6-yl)N-methylcarbamoyl)threonine (mt6a), N-((9-β-D-ribofuranosylpurin-6-yl)-carbamoyl)threonine (t6a) and β, Q nucleoside (q) and D-mannose Q nucleoside (man q). In some embodiments, one or more cytidines of the engineered nucleic acid molecule are replaced by one or more selected from the following: 4-acetylcytidine (ac4c), 2'-O-methylcytidine (cm), 3-methylcytosine (m3c), N4-methylcytidine (m4c), 5-methylcytidine (m5c), β, and 2-thiocytidine (s2c). In some embodiments, the chemical modification includes 2'-O-methylation modification on the ribose of the nucleotide or 3'thiophosphate bond modification between nucleotides or both. In some embodiments, the modification is 2'-O-methylation modification on the first three nucleotide riboses at the 5' end, 2'-O-methylation modification on the last three nucleotide riboses at the 3' end, 3'thiophosphate modification between the first three nucleotides at the 5' end, and 3'thiophosphate modification between the last three nucleotides at the 3' end.

[0050] 22. An engineered immune cell comprising the engineered receptor according to any one of items 13 to 20, or the nucleic acid molecule according to item 21.

[0051] 23. The engineered immune cell according to item 22 comprises two or more engineered receptors that bind to the same target molecule or different target molecules.

[0052] In some embodiments, the engineered receptor comprises or does not comprise a primary signaling domain. In some embodiments, the engineered receptor comprises or is, from N-terminus to C-terminus:

[0053] Antigen binding domain, transmembrane region and signal transduction domain; or

[0054] antigen binding domain, hinge region, transmembrane region and signal transduction domain;

[0055] In some embodiments, at least one of the two or more engineered receptors that bind to the same target molecule or different target molecules does not comprise a primary signal transduction domain, and from the N-terminus to the C-terminus comprises or is:

[0056] antigen binding domain, transmembrane region and co-stimulatory domain, or

[0057] antigen binding domain, hinge region, transmembrane region and co-stimulatory domain;

[0058] And at least one other engineered receptor comprises a primary signal transduction domain, and from the N-terminus to the C-terminus, comprises or is:

[0059] antigen binding domain, transmembrane region, co-stimulatory domain and primary signal transduction domain, or

[0060] Antigen binding domain, hinge region, transmembrane region, co-stimulatory domain and primary signal transduction domain.

[0061] In some embodiments, at least one of the two or more engineered receptors that bind to the same target molecule or different target molecules is an engineered receptor according to any one of 13 to 23.

[0062] In some embodiments, the at least another engineered receptor is an engineered receptor according to any one of 13 to 23.

[0063] 24. The engineered immune cell according to item 23, wherein one of the target molecules is PSCA, and one or more other target molecules are selected from tumor antigens and / or immune checkpoint proteins, wherein,

[0064] The tumor antigen is selected from the group consisting of: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA) CAM5, CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit α (IL-13Rα); interleukin 11 receptor α (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 β (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, EG FRvIII); 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)bDGalp(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); uroplakin2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding Tie2, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, p53 mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, V-myc avian myelocytic disease viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), cytochrome 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 receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155;

[0065] The immune checkpoint protein is selected from: 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, CD 48, 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.

[0066] 25. The engineered immune cell according to item 24, wherein the target molecules are PSCA and CD155. In some embodiments, the target molecule of at least one of the two or more engineered receptors that bind to the same target molecule or different target molecules is CD155, which does not contain a primary signal transduction domain, and which from N-terminus to C-terminus comprises or is:

[0067] antigen binding domain, transmembrane region and co-stimulatory domain, or

[0068] antigen binding domain, hinge region, transmembrane region and co-stimulatory domain;

[0069] And wherein at least another engineered receptor is an engineered receptor according to any one of items 13 to 20.

[0070] 26. An engineered immune cell according to item 24 or 25, wherein the engineered receptor is a CAR against the target molecule PSCA and an engineered receptor against the target molecule CD155.

[0071] In some embodiments, the antigen binding domain of the CAR is a scFv against the target molecule PSCA, and the scFv comprises:

[0072] 1) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1, 2, 3 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 7;

[0073] 2) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 9, 10, 11 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14, 15;

[0074] 3) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, 18, 19 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 21; or

[0075] 4) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 23, 2, 24 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 6, 26,

[0076] Furthermore, the antigen binding domain contained in the engineered receptor for the target molecule CD155 includes the extracellular region of TIGIT or the domain of TIGIT specifically binding to CD155, or the extracellular region of CD226 or the domain of CD226 specifically binding to CD155.

[0077] In some embodiments, the engineered cell comprises:

[0078] An engineered receptor 1, wherein the engineered receptor 1 is an engineered receptor according to any one of items 13 to 23; and an engineered receptor 2, wherein the engineered receptor 2 comprises or is, from the N-terminus to the C-terminus:

[0079] 1) CD226 or its domain that specifically binds to CD155, CD28 transmembrane domain, CD28 signal transduction domain; or

[0080] 2)TIGIT or TIGIT extracellular region, CD28 transmembrane domain, CD28 signaling domain.

[0081] 27. The engineered immune cell according to item 26, wherein the TIGIT extracellular region comprises an amino acid sequence as shown in SEQ ID NO: 46, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0082] The CD226 comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0083] 28. The engineered cell according to item 24, wherein the target molecules are PSCA and CD47. In some embodiments, the target molecule of at least one of the two or more engineered receptors that bind to the same target molecule or different target molecules is CD47, which does not comprise a primary signal transduction domain, and which comprises or is from N-terminus to C-terminus:

[0084] antigen binding domain, transmembrane region and co-stimulatory domain, or

[0085] antigen binding domain, hinge region, transmembrane region and co-stimulatory domain;

[0086] And wherein at least another engineered receptor is an engineered receptor according to any one of items 13 to 20.

[0087] 29. The engineered immune cell according to item 24 or 28, wherein the engineered receptor is a CAR for the target molecule PSCA and an engineered receptor for the target molecule CD47,

[0088] Furthermore, the antigen binding domain of the CAR is a scFv for the target molecule PSCA, and the scFv comprises:

[0089] 1) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1, 2, 3 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 7;

[0090] 2) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 9, 10, 11 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14, 15;

[0091] 3) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, 18, 19 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 21; or

[0092] 4) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 23, 2, 24 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 6, 26,

[0093] Furthermore, the antigen binding domain contained in the engineered receptor for the target molecule CD47 includes the SIRPγ extracellular region.

[0094] In some embodiments, the engineered cell comprises:

[0095] An engineered receptor 1, wherein the engineered receptor 1 is an engineered receptor according to any one of items 13 to 23; and an engineered receptor 2, wherein the engineered receptor 2 comprises or is, from the N-terminus to the C-terminus:

[0096] SIRPγ or its domain that specifically binds to CD47, CD28 transmembrane domain, or CD28 signal transduction domain.

[0097] 30. The engineered immune cell of claim 29, wherein the SIRPγ extracellular region comprises an amino acid sequence as shown in SEQ ID NO: 31 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0098] 31. The engineered immune cell according to any one of items 22 to 30, which is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof.

[0099] 32. Use of an antibody according to any one of items 1-12, an engineered receptor according to any one of items 13 to 20, a nucleic acid molecule according to item 21, or an engineered immune cell according to any one of items 22 to 31 for the preparation of a medicament for treating cancer. In some embodiments, the cancer is a human cancer.

[0100] 33. The method according to claim 32, wherein the cancer is selected from one or more of the following:

[0101] 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 bladder cancer, prostate cancer, or pancreatic acinar epithelial cancer.

[0102] In addition, the present application also provides a method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of items 1-12, an engineered receptor according to any one of items 13 to 20, a nucleic acid molecule according to item 21, or an engineered immune cell according to any one of items 22 to 31. In some embodiments of the method for treating cancer, the cancer is selected from one or more of the following:

[0103] 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 bladder cancer, prostate cancer, or pancreatic acinar epithelial cancer. In some embodiments, the subject or patient is a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 .Detection of PSCA expression in target cells.

[0105] Figure 2 :In vitro killing of CAR1 on target cells of three different indications: prostate cancer, pancreatic cancer and bladder cancer;

[0106] Figure 3A :In vivo efficacy of CAR1 in pancreatic cancer tumorigenesis model;

[0107] Figure 3B :In vivo efficacy of CAR1 in prostate cancer tumorigenesis model;

[0108] Figure 3C :In vivo efficacy of CAR1 in bladder cancer tumorigenesis model.

[0109] Figure 4 : Changes in tumor volume of CAR1 in HPAC-Luc-GFP subcutaneous tumor model.

[0110] Figure 5 :In vitro effectiveness verification of PSCA CAR combined with TIGIT fusion protein in pancreatic cancer, prostate cancer, and bladder cancer.

[0111] Fig. 6A :In vivo efficacy of CAR2~CAR4 in HPAC-Luc-GFP (pancreatic cancer) peritoneal tumor model;

[0112] Figure 6B :In vivo efficacy of CAR2-CAR4 in the HT1376-Luc-GFP (bladder cancer) peritoneal tumor model;

[0113] Figure 6C :In vivo efficacy of CAR2~CAR4 in the PC3-PSCA-Luc-GFP (prostate cancer) peritoneal tumor model.

[0114] Figure 7 : Changes in tumor volume of CAR2~CAR4 in HPAC-Luc-GFP subcutaneous tumor model.

[0115] Fig. 8A : AK-15-H02 recognition and detection of PSCA;

[0116] Figure 8B : AK-24-A08 and AK25-H08 recognize and detect PSCA;

[0117] Figure 8C : Recognition and detection of PSCA by AK-27-B10. DETAILED DESCRIPTION

[0118] The present disclosure provides antibodies (including antigen-binding fragments thereof), especially single-chain antibodies (scFv), that bind to prostate stem cell antigen (PSCA) proteins, especially human PSCA proteins or polypeptides. The present disclosure also relates to engineered receptors comprising the antibodies in the antigen-binding domain, such as chimeric antigen receptors CAR, and engineered cells comprising the antibodies or engineered receptors. Furthermore, the present application further provides uses of the antibodies, engineered receptors, and engineered cells.

[0119] definition

[0120] It should be understood that the present disclosure is not limited to the aspects described herein, which may of course vary in nature. It should also be understood that the terminology used herein is only used to describe particular aspects and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. All technologies and patents cited herein are incorporated herein by reference in their entirety. Unless otherwise indicated, those skilled in the art will adopt the conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA within the technical scope of the art. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Harlow and Lane (1999) Antibodies, A Laboratory Manual. MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH (Shepherd, P. et al., 2000) Oxford University Press, USA, New York NY.

[0122] The term "PSCA" or prostate stem cell antigen, when naturally present, is a glycosylphosphatidylinositol-anchored cell membrane glycoprotein, which was first discovered in prostate cancer. In addition to having high prostate tissue specificity, it is also expressed in organs such as bladder, placenta, colon, kidney and stomach. Later studies have found that it is widely overexpressed on the surface of various solid tumors, such as gastric cancer, pancreatic cancer, bladder cancer, etc. Therefore, the antibodies or engineered receptors of the present application can be used to target the aforementioned organs and solid tumors. In the present application, unless otherwise specified, PSCA is human PSCA, and the exemplary PSCA protein is encoded by the gene with ID 8000 in the NCBI database.

[0123] The term "TIGIT" is an abbreviation for T cell immunoreceptor with Ig and ITIM domains, also known as WUCAM, Vstm3, VSIG9. It consists of an extracellular immunoglobulin variable group (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with typical immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoglobulin tyrosine-based tail (ITT) motifs. 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 immunosurveillance. TIGIT competes with the immune activator receptor CD226 (DNAM-1) for the same set of ligands: CD155 (PVR or poliovirus receptor) and CD112 (nectin-2 or PVRL2). However, the binding affinity of TIGIT to PVRL2 and PVRL3 is much weaker than that of TIGIT to PVR.

[0124] As used herein, a "variant" has at least one amino acid difference relative to a reference amino acid sequence, for example, at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution may be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" may be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Gln); u); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met). In some other embodiments, the variant may also include non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared to the amino acid sequence, the "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range consisting of any two of the foregoing values. As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one or more amino acid residues in which the overall conformation and function of the protein or enzyme are changed by amino acid substitution, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes with more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90% is optimal, and have the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0125] As used herein, "amino acid" refers to any monomer unit that can be incorporated into a peptide, polypeptide or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). In some embodiments, the application also includes non-natural amino acids or amino acid analogs derived or modified on the basis of any of the above natural amino acids. As used herein, "amino acid" also includes non-natural amino acids, modified amino acids (e.g., with modified side chains and / or backbones) and amino acid analogs. To further illustrate, amino acids are generally organic acids comprising substituted or unsubstituted amino, substituted or unsubstituted carboxyl and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photosensitive crosslinker, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing metals, amino acids containing novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photolabile (photocaged) and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.The amino acids described in the present application include, but are not limited to, 20 natural amino acids and 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminoalanine (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or pipecolic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptaneic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisomethacrylic acid (bAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), deoxyephedrine (Des), 2,2'-diaminopimelic acid (Dp m), 2,3-diaminopropanesulfonic acid (Dpr), ethylglycine (EtGly), N-ethylaspartic acid (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine or sarcosine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orm). Therefore, in some embodiments, after the mutation, the amino acid mutation at the site comprises a substitution mutation converted to any one of the above 20 natural amino acids and the above non-natural amino acids. In some embodiments, the amino acid mutation comprises a substitution mutation to any one of the amino acids selected from the group consisting of G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H, Aad, bAad, bAla, Abu, 4Abu, Acp, Ahe, Aib, bAib, Apm, Dbu, Des, Dpm, Dpr, EtGly, EtAsn, Hyl, aHyl, 3Hyp, 4Hyp, Ide, aIle, MeGly, MeIle, MeLys, MeVal, Nva, Nle, and Orm.

[0126] A "conjugate" is an antibody conjugated to one or more heterologous molecules including, but not limited to, a cytotoxic agent.

[0127] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0128] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials. For example, a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or that is substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally exist as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to polypeptides separated from other cellular proteins, and is intended to cover purified and recombinant polypeptides.

[0129] As used herein, the percentage of "identity", such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to the degree of similarity between amino acid sequences or between nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, the proportion of the number of positions with the same base or amino acid residue to the total number of positions determined after two sequences have the same residue at as many positions as possible by introducing spaces, etc. The percentage of "identity" can be determined using software programs known in the art. Preferably, the comparison is performed using default parameters. A preferred comparison program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. In the context of the present invention, the terms "DNA" and "RNA" refer to single-stranded or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms "DNA" and "DNA molecule" refer to double-stranded DNA molecules consisting of A, C, G and / or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules consisting of A, C, G and / or U nucleotides. In this article, the A, C, G, T and U nucleotides refer to nucleotides containing adenine, guanine, cytosine, thymine and uracil as their respective nitrogenous bases.

[0130] RNA molecules include coding RNA or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long noncoding RNA (lncRNA).

[0131] 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:

[0132] Replace one or more deoxyribonucleotides in DNA with ribonucleotides;

[0133] Substituting one or more ribonucleotides in the RNA with deoxyribonucleotides; or

[0134] De novo synthesis using deoxyribonucleotides and ribonucleotides as raw materials by biological or chemical synthesis. It should be noted that the method of obtaining a hybrid molecule of DNA and RNA is not limited to the above method, and the hybrid molecule of DNA and RNA obtained by any method belongs to the category of "hybrid molecule of DNA and RNA" defined in this application.

[0135] As used herein, if two nucleic acid molecules are described as having "the same genetic information", it means that the two nucleic acid molecules are complementary, or contain exactly the same base sequence, or one or more thymines in the base sequence of one of the nucleic acid molecules are converted to uracil to obtain a nucleic acid molecule that is exactly the same as the base sequence of another nucleic acid molecule. Therefore, any two of DNA, RNA, and hybrid molecules of DNA and RNA can have the same genetic information. Among them, the term "base sequence" refers to the order of arrangement of bases in a polynucleotide molecule. Those skilled in the art should know that, unless otherwise specified, the base sequence or polynucleotide sequence described in this application can be used to describe DNA sequences. "T" refers to thymine, but when the base sequence or polynucleotide sequence is used to describe RNA (such as mRNA), "T" will be replaced by "U" (uracil). Therefore, any DNA disclosed by a specific sequence number (SEQ ID NO) herein also discloses an RNA (such as mRNA or Poly (A) tail) sequence that is complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is replaced by "U".

[0136] In this article, "coding" refers to i) genetic information contained in a DNA sequence that can be transcribed into an RNA molecule, and / or ii) genetic information contained in an RNA molecule that can be translated into an amino acid sequence. Therefore, as used herein, "coding sequence" can be used to refer to a ribonucleotide (RNA) sequence or a fragment thereof that can be translated into a protein in an mRNA precursor or mature mRNA, and can also refer to a complementary sequence or a fragment thereof of a deoxyribonucleotide (DNA) sequence that is used as a template to transcribe the mRNA precursor or mature mRNA. In addition, the "coding sequence" of the present application may further include polynucleotide sequences encoding proteins, functional nucleic acids, or fragments thereof, such as miRNA, shRNA, dsRNA, guide RNA, Poly (A) tail, 5'UTR, 3'UTR, etc. Among them, a DNA molecule containing genetic information that can be transcribed into an RNA molecule is called the "coding nucleic acid" of the RNA molecule; an RNA molecule containing genetic information that can be translated into an amino acid sequence is called the "coding nucleic acid" of the amino acid sequence.

[0137] Unless otherwise specified, "peptide", "polypeptide" and "protein" are used interchangeably in this application and can be used to refer to any natural active substance formed by covalently linking two or more amino acids through peptide bonds, which may or may not contain the secondary or tertiary structure of the protein molecule.

[0138] As used herein, the term "contacting" is used in its simple common sense and refers to a process of allowing at least two different substances to become close enough to react, interact or physically contact. It should be understood that the resulting reaction product can be 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 polypeptide (or engineered nucleic acid molecule) and a cell as provided herein. In an embodiment, contacting includes, for example, allowing the engineered nucleic acid molecule or engineered peptide described in the present application to enter a cell.

[0139] The terms "cancer" and "tumor" are used interchangeably in this disclosure and refer to a neoplasm or tumor caused by abnormal, uncontrolled growth of cells that makes them pathogenic to the host organism. In some embodiments, cancer refers to a benign tumor that has become localized. In other embodiments, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In some embodiments, cancer is associated with a specific cancer antigen.

[0140] As used herein, a method of "treating" a disease in a subject, or "therapy" refers to a method for obtaining beneficial or desired results, including, but not limited to, one or more of the following: alleviation or improvement of one or more symptoms, reduction in the scope of the condition (including the disease), a stable (i.e., non-worsening) state of the condition (including the disease), a delay or slowing of the condition (including the disease), progression, improvement or palliation of the condition (including the disease), state and remission (whether partial or complete), whether detectable or undetectable.

[0141] A "pharmaceutically acceptable carrier" is a carrier that constitutes a pharmaceutical preparation with an active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0142] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0143] Antibody

[0144] As used in the present disclosure, the term "antibody", also commonly referred to as "immunoglobulin", covers antibodies with natural antibody structural features and antibody-like molecules with structural features different from natural antibodies but showing binding specificity to antigen molecules. In the present application, the term "antibody" has its broadest meaning, and covers immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules containing antigen binding sites or domains, and can be used to refer to antigen structural fragments (e.g., antigen binding fragments) or complexes (e.g., scFv) of one or more antigen binding fragments with antigen binding activity. Immunoglobulin molecules can be any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0145] The antibody provided in the present application can bind to PSCA, and comprises at least an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL), wherein the VH comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), and a heavy chain variable region complementarity determining region 3 (HCDR3), and the VL comprises a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3);

[0146] Wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 respectively comprise or are:

[0147] 1) the amino acid sequences shown in SEQ ID NOs: 1 to 3, and 5 to 7;

[0148] 2) the amino acid sequences shown in SEQ ID NOs: 9 to 11, and 13 to 15;

[0149] 3) the amino acid sequences shown in SEQ ID NOs: 17 to 19, and 5, 6 and 21; or

[0150] 4) The amino acid sequences shown in SEQ ID NOs: 23, 2, 24, 5, 6 and 26.

[0151] The term "sequentially, respectively", "comprising" or "being" means that the six CDRs LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence corresponding to the order of the six amino acid sequences listed in any one of 1) to 4). For example, when LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 respectively comprise or are: 1) the amino acid sequences shown in SEQ ID NOs: 1 to 3, and 5 to 7, it means that LCDR1 comprises or is the amino acid sequence shown in SEQ ID NO: 1, LCDR2 comprises or is the amino acid sequence shown in SEQ ID NO: 2, LCDR3 comprises or is the amino acid sequence shown in SEQ ID NO: 3, HCDR1 comprises or is the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprises or is the amino acid sequence shown in SEQ ID NO: 6, and HCDR3 comprises or is the amino acid sequence shown in SEQ ID NO: 7.

[0152] In some embodiments, the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by the IMGT system. LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are collectively referred to as complementarity determining regions (CDRs). In this application, unless otherwise specified, CDRs are defined by the IMGT system.

[0153] Those skilled in the art will appreciate that there are many ways to define CDRs. Exemplary definitions are shown in the table below, and different CDR definition methods may also be combined (CDR1 and CDR2 are defined by one CDR method, and CDR3 is defined by another CDR definition method).

[0154]

[0155] in:

[0156] 1The amino acid numbering in the Kabat system definition is based on the Kabat numbering system (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991));

[0157] 2 The amino acid numbering in the Chothia system definition is based on the Chothia numbering system (see, e.g., Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987));

[0158] 3 The amino acid numbering in the MacCallum system definition is based on the MacCallum numbering system (see MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008));

[0159] 4 The amino acid numbering in the IMGT system definition is based on the IMGT numbering system (see, for example, Lefranc MP. (2013) IMGT Unique Numbering. In: Dubitzky W., Wolkenhauer O., Cho KH., Yokota H. (eds) Encyclopedia of Systems Biology. Springer, New York, NY; https: / / doi.org / 10.1007 / 978-1-4419-9863-7_127);

[0160] 5 The amino acid numbering in the AHo system definition (see, for example, Honegger and Plückthun, J. Mol. Biol., 309: 657-670 (2001)) is based on the AHo numbering system.

[0161] Since CDRs are defined in different ways, those skilled in the art can define CDRs in a different way from that used in the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the present application, thereby obtaining CDRs that are somewhat different in sequence from the combination of the same heavy chain variable region and light chain variable region as the present application. Those skilled in the art should be aware that the CDR combination obtained from the antibody of the present application by different definition methods, even if the sequence is different from that of the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the present application, the antibody comprising the CDR combination is still undoubtedly covered within the scope of the present application.

[0162] Therefore, HCDR1, HCDR2 and HCDR3 determined by a non-IMGT method (for example, by Kabat, Chothia, MacCallum, IMGT4, AHo5, Contact or a combination thereof) based on the VH sequences provided in the present application: SEQ ID NOs: 8, 16, 22 and 27, and LCDR1, LCDR2 and LCDR3 determined by a non-IMGT method (for example, by Kabat, Chothia, MacCallum, IMGT4, AHo5, Contact or a combination thereof) based on the VL sequences provided in the present application: SEQ ID NOs: 4, 12, 20 and 25, and VH, VL, scFv or other antibodies or antigen-binding fragments thereof comprising the HCDR1, HCDR2 and HCDR3 and / or LCDR1, LCDR2 and LCDR3 are all within the scope of the present application.

[0163] The terms "heavy chain" ("HC"), "light chain" ("LC"), "light chain variable region" ("VL"), "heavy chain variable region" ("VH"), "framework region" ("FR") refer to domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g., humanized antibodies). The basic structural unit of a naturally occurring immunoglobulin (e.g., IgG) is a tetramer having two light chains and two heavy chains. The amino-terminal ("N") portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminus ("C" portion) of each chain is defined as a constant region, with light chains having a single constant domain and heavy chains typically having three constant domains and a hinge region. Thus, the structure of the light chain of a naturally occurring IgG molecule is VL-CL from N (nitrogen) terminus to C (carbon), and the structure of the IgG heavy chain is VH-CH1-H-CH2-CH3 (where H is the hinge region) from N-terminus to C-terminus. The variable region of an IgG molecule is composed of a complementarity determining region (CDR) (comprising residues that contact the antigen) and a non-CDR segment (called a framework segment or framework region, which maintains the structure and determines the position of the CDR loop). Thus, the VL and VH domains have a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure from N-terminus to C-terminus.

[0164] In natural antibodies, variability is unevenly distributed in the variable region of the antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The CDR on the heavy chain can be called HCDRn, "n" is an integer and does not represent the order of CDRs on the heavy chain. Similarly, the CDR on the light chain can be called LCDRn, "n" is an integer marking CDRs and does not represent the order of CDRs on the light chain. The more highly conserved part in the variable region is called framework (FR). The variable regions of natural heavy and light chains each contain four FR regions connected by three CDRs. The CDRs in each chain are closely connected by the FR region and contribute to the formation of the antigen binding site of the antibody together with the CDRs from another chain [see Kabat, EA et al., Sequences of Proteins of Immunological Interest National Institute of Health, Bethesda, MD (1987)]. The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

[0165] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a PSCA molecule, such as human PSCA). The antibody fragment comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all, functions normally associated with the portion when it is present in an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody (scFv) molecules; and multispecific antibodies formed from antibody fragments.

[0166] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual Fc fragment, whose name reflects its ability to crystallize easily. The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between the "Fab'" fragment and the Fab fragment is that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. "Fab'-SH" refers to Fab' in which the cysteine ​​residues of the constant domains have free thiol groups. The "F(ab')" fragment is produced by the cleavage of the hinge cysteine ​​disulfide bonds of the pepsin digestion product "F(ab')2".

[0167] The "Fd" fragment consists of the VH and CH1 domains. The "dAb" fragment (Ward et al., (1989) Nature 341: 544-546) consists of the VH domain. Isolated complementarity determining regions (CDRs) and combinations of two or more isolated CDRs can optionally be connected by synthetic linkers.

[0168] The "Fv" fragment consists of the VL and VH domains of a single arm of an antibody. Single-chain Fv (scFv) consists of one heavy chain variable region and one light chain variable region, which are covalently linked into a single polypeptide chain by a flexible peptide linker.

[0169] In some embodiments, the VH of the antibody of the present application comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27. In some embodiments, the VL of the antibody of the present application comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25. In some embodiments, the VH of the antibody of the present application comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27, and the VL of the antibody of the present application comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25. In some embodiments, the amino acid sequence having the identity has one or more mutations only in the FR segment compared to the VH and / or VL of the present application, such as deletion mutations, insertion mutations, substitution mutations. In some embodiments, the one or more substitution mutations are conservative substitutions. In some embodiments, the amino acid sequence having the identity has one or more conservative substitutions only in the FR segment compared to the VH and / or VL of the present application.

[0170] In some embodiments, the antibodies provided herein are chimeric antibodies, humanized antibodies, or human antibodies.

[0171] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present technology may include or not include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific in vitro mutagenesis or by somatic mutations in vivo). In some embodiments, the term "human antibody" refers to an antibody, wherein substantially each part of the protein (e.g., CDR, framework, CL, CH domain (e.g., CH1, CH2, CH3), hinge, VL, VH) is substantially non-immunogenic in humans, with only minor sequence changes or variations. Therefore, human antibodies are different from chimeric or humanized antibodies. It should be noted that human antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes.

[0172] As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region from one monoclonal antibody is replaced with the Fc constant region from another antibody (e.g., human Fc constant region) using recombinant DNA technology. See, for example, PCT / US86 / 02269; EP173,494, etc. Methods for preparing chimeric antibodies are known in the art. Other antibodies of the present application can be obtained from the specific sequence of the antibody of the present application using gene shuffling (see, for example, WO93 / 06213) or CDR grafting, wherein the other antibodies of the present application have similar affinity and specificity to the antibody whose specific amino acid sequence is provided in the present application. In some embodiments, the CDRs provided in the present application can be introduced into other human immunoglobulin variable regions with extremely high identity to the variable regions (e.g., VH or VL) of the present application, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 99% or more sequence identity, that is, the CDRs in the human immunoglobulin variable regions with extremely high similarity are replaced with the CDRs of the present application (or the FRs are replaced for the antibodies of the present application), thereby obtaining other antibodies with similar functions to the antibodies of the present application, such as other antibodies with the ability to bind to PSCA, and the other antibodies still fall within the scope of protection of the present application. In some embodiments, backmutation can be performed after replacing the FRs for the antibodies and retaining high affinity for the antigen and other favorable biological properties. In some embodiments, the human immunoglobulin variable regions with extremely high similarity can be prepared or selected by analyzing the parental sequences and a variety of conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the acceptor (e.g., the variable region of a human immunoglobulin having a high degree of similarity) and the input sequence to obtain the desired antibody characteristic, such as increased affinity for PSCA.

[0173] In CDR transplantation, the immunoglobulin or antibody providing the CDR is called a "donor", and the human immunoglobulin or immunoglobulin variable region providing the framework is called an "acceptor". Therefore, all CDRs come from the donor. "Acceptor" means an amino acid sequence comprising any light chain variable region (VL) framework region or heavy chain variable region (VH) framework region derived from a human immunoglobulin framework region or a human consensus framework region. The acceptor "derived from" a human immunoglobulin framework region or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is 1-10, 2-9, 3-8, 4-7 or 5-6. "Human consensus framework" is a framework representing the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from the subtype of the variable region sequence. In general, the subtype of the sequence is as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In certain embodiments, for VL, the subtype is subtype κI as described in Kabat et al. (supra). In certain embodiments, for VH, the subtype is subtype III as described in Kabat et al. (supra).

[0174] In some embodiments, the VH of the antibody of the present application comprises an amino acid sequence as shown in SEQ ID NO: 27, and the VL comprises an amino acid sequence as shown in SEQ ID NO: 25. In some embodiments, the VH is connected to the VL by a chemical bond or a linker. In some embodiments, the chemical bond is a peptide bond, and the linker is an amino acid linker, preferably the amino acid linker is a peptide chain comprising 4 to 25 amino acids. In some embodiments, the VL and VH of the antibody are connected by a flexible linker, such as any GS linker. In some embodiments, the VL and VH of the antibody are connected by an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the antibody is a monospecific antibody or a multispecific antibody. In some embodiments, the VL and VH of the antibody are not connected in series. In some embodiments, the antibody is a single-chain antibody (scFv), Fab, F(ab')2, Fab', Fv, Fd, dAb, IgG, IgE, IgA, IgM, a diabody, or a complex formed by the foregoing antibodies, wherein the VL and VH have the function of binding to PSCA in the antibody. ScFv, Fab, F(ab')2, Fab', Fv, Fd, dAb, etc. can be obtained using conventional techniques known to those skilled in the art, such as by recombinant DNA technology, or by enzymatic or chemical cleavage of intact immunoglobulins. Various techniques have been developed for producing antibodies (or antibody fragments) such as scFv, Fab, F(ab')2, Fab', Fv, Fd, dAb, etc. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies. However, these fragments can now be directly produced by recombinant host cells. Fab, Fv and ScFv antibody fragments can be expressed in E. coli and secreted from E. coli, thus allowing these fragments to be conveniently produced in large quantities. Antibody fragments can be isolated from the above-mentioned antibody phage library. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments. According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Patent US5,869,046 describes Fab and F(ab')2 fragments with increased half-life in vivo. Other techniques for producing antibody fragments are also obvious to those skilled in the art. In some embodiments, the selected antibody is a single-chain antibody (scFv). See WO 93 / 16185; U.S. Patents 5,571,894; and 5,587,458. Fv and scFv are species with complete binding sites without constant regions; therefore, they are suitable for reduced nonspecific binding during in vivo use.scFv fusion proteins can be constructed to produce fusion of effector proteins at the amino or carboxyl terminus of the scFv. The antibody fragment can also be a "linear antibody", for example, as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0175] The antibodies of the present application may be monospecific antibodies, bispecific antibodies or multispecific antibodies. Unless otherwise specified, the multispecific antibodies of the present application include the meaning of bispecific antibodies. As used herein, the term "bispecific antibody" is an artificial hybrid antibody having two different heavy chain / light chain pairs and two different binding sites. For the purposes of the present disclosure, a "bispecific antibody" specifically binds to PSCA and another antigen (or other antigenic epitopes in PSCA that are different from the antigenic epitopes bound by the VL and VH), for example, tumor antigens or immune checkpoint proteins expressed on tumor cells. Exemplary bispecific antibodies can bind to two different epitopes of the PSCA protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing PSCA, in which case the antibody has a PSCA binding arm and a cytotoxic agent binding arm.

[0176] In some embodiments, the bispecific antibody or multispecific antibody has a PSCA binding arm, which comprises an anti-PSCA antibody or fragment thereof of the present disclosure, and an arm that binds to a tumor antigen or immune checkpoint protein. In some embodiments, the tumor antigen is selected from any one or more of the following: A33; ADAM-9; ALCAM; BAGE; β-catenin; CA125; carboxypeptidase M; CD103; CD19; CD20. CD22; CD23; CD25; CD27; CD28; CD36; CD40 / CD154; CD45; CD46; CD5; CD56; CD79a / CD79b; CDK4; CEA; CTLA4; cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2 / GD3 / GM2; HER-2 / neu; human Papillomavirus-E6; human papillomavirus-E7; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosamine transferase; oncostatin M; pl5; PIPA; PSA; PSMA; ROR1; TNF-β receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor and VEGF receptor. In some embodiments, the immune checkpoint protein is selected from any 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; VSIG8.

[0177] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies, di-scFv bispecific antibodies, etc.). Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies are prepared by Knobs-into-holes (KiH) technology, leucine zipper technology, coiled coil technology, technology for forming electrostatic interactions by polar amino acid residues, or a combination thereof, see, for example, US2007 / 0178552, WO96 / 027011, WO98 / 050431, WO2007 / 147901, WO2009 / 089004, WO2010 / 034605, etc. Bispecific antibodies can also be prepared in the form of diabodies, wherein the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable region (VH) connected to a light chain variable region (VL) in the same polypeptide chain (VH-VL). By using a linker (which is too short to allow pairing between the two domains on the same chain), the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097 and WO 93 / 11161.

[0178] In some embodiments, the antibodies of the present application are monoclonal antibodies or polyclonal antibodies.

[0179] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variant antibodies (e.g., containing naturally occurring mutations or mutations that arise during preparation of the monoclonal antibody, such variants generally exist in minor amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope.

[0180] As used in this application, "polyclonal antibody" refers to a mixture or composition of multiple monoclonal antibodies. The polyclonal antibody can be a combination or mixture of multiple antibodies of this application, or a combination or mixture of one or more antibodies of this application and one or more other antibodies.

[0181] Fusion Protein

[0182] On the one hand, the present application also provides a fusion protein comprising the above-mentioned antibody, which can be a protein complex formed by the above-mentioned antibody in any way combined with another one or more proteins, polypeptides or protein functional domains. In some embodiments, the combination is that the antibody is connected to the other one or more proteins, polypeptides or protein functional domains by direct and / or indirect means. In some embodiments, the carbon (C) end of the antibody in the fusion protein is connected to the nitrogen (N) end of the other protein, polypeptide or protein functional domain. In some embodiments, the nitrogen (N) end of the antibody in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the nitrogen (N) end of the antibody in the fusion protein is connected to the nitrogen (N) end of the other protein, polypeptide or protein functional domain. In some embodiments, the carbon (C) end of the antibody in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the antibody in the fusion protein is connected to the carbon (C) end of the other protein, polypeptide or protein functional domain. In some embodiments, the antibody in the fusion protein is connected to the other one or more proteins, polypeptides or protein functional domains in series. In some embodiments, the C-terminus and / or N-terminus of the antibody in the fusion protein are connected to at least two or more other proteins, polypeptides or protein functional domains, and the two or more other proteins, polypeptides or protein functional domains are not connected in series. In some embodiments, the other one or more proteins, polypeptides or protein functional domains bind to the same or different antigens or bind to different antigenic epitopes as the antibody.

[0183] The term "direct" connection or "direct connection" refers to a connection achieved only by chemical bonds, that is, two proteins or polypeptides are not connected by other molecules, and the chemical bond can be a non-covalent bond (such as an ionic bond, a hydrogen bond, a hydrophobic bond or a van der Waals bond), or a covalent bond (such as a peptide bond). The term "indirect" connection or "indirect connection" refers to a connection through a linker, and the two proteins or polypeptides connected to each other using a linker are respectively connected to one end of the linker by a covalent or non-covalent bond. The "linker" can be a peptide linker (i.e., a peptide chain, such as a peptide chain composed of 1 to 50 amino acids or their derivatives) or a non-peptide linker, and the linker can be cleavable (i.e., it can be hydrolyzed by an enzyme in the body, such as a mammalian body) or non-cleavable. Exemplary non-peptide linkers include, but are not limited to, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethers, biodegradable polymers, polymerized lipids, chitin and hyaluronic acid, or derivatives thereof, or combinations thereof.

[0184] In some embodiments, the one or more proteins, polypeptides or protein domains comprise a recognition portion that specifically binds to the one or more proteins, and the one or more proteins may be tumor antigens, such as one or more selected from the following: prostate stem cell antigen (PSCA) carcinoembryonic antigen (CEA) CAM5, CD123, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD30; CD 70; CD 123; CD 138; CD33; 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 beta (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)bDGalp(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); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of tumor necrosis area;G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; 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 virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, breakpoint X 2 (SSX2); CD79a; CD79b; CD72; leukocyte phase Related immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5);Immunoglobulin lambda-like polypeptide 1 (IGLL1) and CD155. ;

[0185] In some embodiments, the one or more other proteins, polypeptides or protein domains comprise a recognition portion that specifically binds to the 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:

[0186] 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, CD 48, 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.

[0187] In some embodiments, the other one or more proteins, polypeptides or protein domains contain a recognition portion that specifically binds to another one or more proteins, and the other one or more proteins include any one or more immune checkpoint proteins selected from the above-mentioned, and any one or more tumor antigens mentioned above.

[0188] In some embodiments, the recognition portion 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.

[0189] Engineered receptors

[0190] The application also provides an engineered receptor comprising the aforementioned antibody, the engineered receptor comprising an antigen binding domain, and the antigen binding domain comprises the aforementioned antibody or the aforementioned fusion protein. In some embodiments, the engineered receptor further comprises a signal transduction domain operably connected to the engineered receptor. Wherein, the term "operably connected" means that when the engineered receptor is combined with its ligand or its target molecule, the antigen binding domain can be allowed to conduct activation or inhibition signals to the cell through the signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain, but does not include a costimulatory domain. In some embodiments, the signal transduction domain comprises a costimulatory domain or a secondary signal transduction domain, but does not include a primary signal transduction domain. In some embodiments, the signal transduction domain comprises a primary signal transduction domain and a costimulatory domain.

[0191] In some embodiments, the engineered receptor comprises or is from N-terminus to C-terminus: the aforementioned antibody-transmembrane domain-costimulatory domain (or secondary signal transduction domain). In some embodiments, the engineered receptor comprises or does not comprise a hinge region, which connects the antigen binding domain and the transmembrane domain or the signal transduction domain. In some embodiments, the hinge is derived from 7h (CD7 hinge region) / G4h / 8h (CD8 hinge region). In some embodiments, the transmembrane domain is from the transmembrane domain of CD4, CD8α, CD28 or CD3ζ. In some embodiments, the co-stimulatory domain or secondary signaling domain comprises one or more of the following, or consists of one or more of the following: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a signal transduction domain of a ligand that specifically binds to CD83.

[0192] In some embodiments, the engineered receptor comprises or is from N-terminus to C-terminus: the aforementioned antibody-transmembrane domain-costimulatory domain-primary signal transduction domain. In some embodiments, the engineered receptor comprises or is from N-terminus to C-terminus: the aforementioned antibody-transmembrane domain-primary signal transduction domain. In some embodiments, the engineered receptor is: a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a T cell antigen coupling agent (TAC).

[0193] 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) a signal transduction domain. Among them, the "signal transduction domain" comprises a primary signal transduction domain and / or a co-stimulatory domain. In some embodiments, the antigen binding domain is selected from one or more of the following groups: an extracellular domain of a 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 signal transduction domain comprises a primary signal transduction domain, which is from a molecule selected from any one of the following groups: CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10 and DAP12. In some embodiments, the primary signal transduction domain is from CD3ζ. As used herein, the "primary signal transduction domain" generally comprises an immune receptor tyrosine activation motif (ITAM), the basic composition of which is: YXXL / V. Wherein Y is tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to the corresponding ligand, the tyrosine in the ITMA connected thereto can be phosphorylated under the action of a class of protein tyrosine kinase PTK connected to the cell membrane, thereby recruiting other free protein kinases or adapter proteins in the cell to transmit activation signals into the cell.

[0194] In some embodiments, the signal transduction 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 antibody. In some embodiments, the antigen binding domain is the aforementioned antibody. As used herein, "costimulatory domain" is generally derived from the costimulatory receptors of immune cells, providing a second signal or secondary intracellular signal of activated immune cells (eg, T cells).

[0195] As used herein, an engineered "TCR" is an engineered T cell receptor comprising: (a) an antigen binding domain or a ligand binding domain comprising 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 an extracellular domain or portion thereof of a first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε); (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) a signal transduction domain comprising a third TCR subunit (e.g., TCRα, TCRβ); wherein the first, second and third TCR subunits are independently selected from any one of the following groups: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ and CD3ζ. In some embodiments, the first, second and third TCR subunits are the same (e.g., all CD3ε, all TCRα or all TCRβ). In some embodiments, the first, second and third TCR subunits are different. In some embodiments, the engineered TCR further comprises a hinge domain between the C-terminus of the antigen binding domain or the ligand 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 or the ligand binding domain comprises the aforementioned antibody. In some embodiments, the antigen binding domain or the ligand binding domain is the aforementioned antibody.

[0196] 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 antibody. In some embodiments, the antigen binding domain is the aforementioned antibody. In some embodiments, the TAC comprises: (a) an antigen binding domain or a ligand binding domain comprising 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 the extracellular domain of a TCR subunit (e.g., CD3ε); (d) optionally a second linker; (e) optionally a first TCR co-receptor ( For example, CD4, CD8) extracellular domain or part thereof; (f) a transmembrane domain comprising a second TCR co-receptor (e.g., CD4, CD8) transmembrane domain; and (g) optionally a signal transduction domain comprising a third TCR co-receptor (e.g., CD4, CD8) signal transduction domain; 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 or the ligand binding domain and the N-terminus of the transmembrane domain. In some embodiments, the antigen binding domain or the ligand binding domain comprises the aforementioned antibody. In some embodiments, the antigen binding domain or ligand binding domain is the aforementioned antibody.

[0197] As used herein, the terms "antigen binding domain" and "ligand binding domain" are used interchangeably, which are usually located in the extracellular segment of a cell (especially an immune cell) receptor and can specifically bind to a certain protein, and the scope of the protein is not subject to any restrictions. Therefore, in some embodiments, the certain protein is a certain receptor, and the "antigen binding domain" or "ligand binding domain" is a ligand of the certain receptor or a part of the ligand specifically recognized by the receptor; in some embodiments, the protein is a certain ligand, and the "antigen binding domain" or "ligand binding domain" is a receptor for the certain ligand, or a part of the receptor that specifically recognizes the ligand; in some embodiments, the protein is an antibody or an antigen binding domain of the antibody.

[0198] The present application also provides a chimeric antigen receptor (CAR) that binds to PSCA, which comprises one or more antigen binding domains, a transmembrane domain, and a signal transduction domain, wherein the one or more antigen binding domains comprise the aforementioned antibodies. In some embodiments, the one or more antigens, in addition to PSCA that binds to the aforementioned antibodies, further comprise one or more tumor antigens and / or immune checkpoint proteins. Among them, in some embodiments, the one or more tumor antigens are selected from: PSCA, CEACAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSE A-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, Claudin 6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, kappa light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, carcinoembryonic variant of tumor necrosis area, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K , OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155.

[0199] Wherein, in some embodiments, the one or more immune checkpoint proteins are selected from:

[0200] 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, CD 48, 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.

[0201] In some embodiments, the CAR comprises multiple antigen binding domains, and the multiple antigen binding domains are connected in series with each other. In some embodiments, the CAR comprises multiple antigen binding domains, and at least two of the multiple antigen binding domains are connected to the transmembrane domain of the CAR in parallel with each other. In some embodiments, the transmembrane domain comprises a transmembrane domain of CD4, CD8α, CD28 or CD3ζ.

[0202] 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 antibody that binds to CD155.

[0203] 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 or CD8α / CD28. In some embodiments, the signal transduction domain comprises a signal transduction domain, and the signal transduction domain is a signal transduction domain of the intracellular region 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 derived from the intracellular region 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, ITGA D, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and ligands that specifically bind to CD83. In some embodiments, the signal transduction domain is the signal transduction domain of the intracellular region of the CD3ζ molecule, and the co-stimulatory domain is from the intracellular region of 4-1BB, CD134, ICOS or CD28.In some embodiments, the signal transduction domain is the signal transduction domain of the intracellular region of the CD3ζ molecule, and the co-stimulatory domain is from the intracellular region of 4-1BB and CD28.

[0204] In addition, the present application also provides a composition comprising any one or more of the above-mentioned fusion proteins or engineered receptors. In some embodiments, the composition is a combination of the engineered receptor or fusion protein and one other engineered receptor, and the other engineered receptor targets an antigen or antigenic epitope different from the aforementioned antibody. In some embodiments, the composition is a composition of a CAR, TCR or TAC and the engineered receptor, the engineered receptor comprising an antigen binding domain and a costimulatory domain, and the CAR, TCR or TAC binds to a different target molecule than the aforementioned engineered receptor, or binds to different antigenic epitopes of the same target molecule; in some cases, a hinge region is also included between the antigen binding domain and the costimulatory domain.

[0205] Engineered Nucleic Acid Molecules

[0206] The present application also provides an engineered nucleic acid molecule encoding the aforementioned antibody, fusion protein or engineered receptor, the nucleic acid molecule comprising a target protein coding sequence, the target protein coding sequence can be expressed as the antibody, fusion protein or engineered receptor. The term "engineered nucleic acid molecule" is used to distinguish it from a "natural nucleic acid molecule". "Natural nucleic acid molecule" refers to a nucleic acid molecule that exists in a natural form in nature. "Engineered nucleic acid molecule" is a restriction on the source or preparation method of the nucleic acid molecule, and does not constitute any restriction on its function or structure. Therefore, an engineered nucleic acid molecule can be used to refer to any nucleic acid molecule that can be obtained by any or multiple bioengineering means, which can have a polynucleotide sequence that is exactly the same as a natural nucleic acid molecule, have a modification that is exactly the same as a natural nucleic acid molecule, and even form a structure that is exactly the same as a natural nucleic acid molecule, but the difference between an engineered nucleic acid molecule and its corresponding natural nucleic acid molecule, or a natural nucleic acid molecule with the same polynucleotide sequence as it at least includes: the engineered nucleic acid molecule is not directly purified or extracted from an animal or plant that exists naturally in nature in its natural form.

[0207] In some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the DNA molecule can be replicated and / or expressed in a cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a eukaryotic cell. In some embodiments, the DNA molecule can be replicated and / or expressed in a prokaryotic cell. In some embodiments, the DNA molecule can be expressed in a eukaryotic cell and can be replicated in a prokaryotic cell. Therefore, the DNA molecule, in addition to comprising the target protein coding sequence, also comprises a gene manipulation or regulatory element for replication and / or expression in a prokaryotic and / or eukaryotic cell.

[0208] The necessary structural elements that make the engineered DNA molecule replicate or efficiently replicate in cells are known in the art, including, for example, an origin of replication (ORI). In some embodiments, the engineered DNA molecule further comprises a marker gene or its fragment and / or a reporter gene or its fragment and a unique restriction endonuclease site that allows insertion of DNA elements, preferably a restriction endonuclease site in the form of a multiple cloning site (MCS). The marker gene is conducive to identifying cells containing a plasmid comprising the marker gene, and can be selected from, for example, an antibiotic resistance gene. Each restriction endonuclease site in the MCS can be specifically recognized by different restriction endonucleases.

[0209] In some embodiments, the DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid composed of double-stranded DNA molecules. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" can also encompass linear DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, cutting a circular plasmid with a restriction endonuclease, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid, and linear molecules that can be replicated in prokaryotes. Plasmids can replicate, i.e., amplify in cells independently of the genomic genetic information stored in the nucleoid or nucleoid of prokaryotes, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the present invention is a medium copy or high copy plasmid, more preferably a high copy plasmid. Examples of such high copy plasmids are such vectors: they are based on pUC, pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) etc. that contain an ORI that supports high copies of plasmids.

[0210] In some embodiments, the engineered DNA molecule is a DNA molecule or a fragment thereof constituting a prokaryotic nucleoid or nucleoid, or a DNA molecule or a fragment thereof constituting a eukaryotic genome, that is, the target protein coding sequence or its complementary sequence can be replicated along with the prokaryotic genome.

[0211] In some embodiments, the engineered DNA molecule can be transcribed into mRNA. In some embodiments, the engineered DNA molecule also includes a coding sequence of an element that can be used to start or regulate the expression of the protein, polypeptide or its fragment after transcription, and the element includes but is not limited to 5'UTR, 3'UTR, poly (A) tail (or tailing signal), etc. In some embodiments, the engineered DNA molecule includes a coding sequence of at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR and the target protein coding sequence. In some embodiments, the engineered DNA molecule includes at least the coding sequence of 5'UTR, the target protein coding sequence, the coding sequence of 3'UTR, the tailing signal (or the DNA sequence corresponding to the Ploy (A) tail sequence) from 5' to 3', and the start codon (5' end) and the stop codon (3' end) at both ends of the target protein coding sequence may be included, respectively, which are the first three nucleotides and the last three nucleotides that can be translated of the mRNA molecule, respectively. The 5'UTR usually contains at least one ribosome binding site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. The RBS promotes the efficient and accurate translation of mRNA molecules by recruiting ribosomes at the start of translation. Its activity can be optimized by changing the length and sequence of a given RBS or translation revelation site and the distance from the start codon. Alternatively or optionally, the 5'UTR includes an internal ribosome entry site or IRES. The 3'UTR may contain one or more regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for regulatory RNA molecules (such as miRNA molecules), and / or signal sequences that participate in the intracellular transport of mRNA molecules.

[0212] On the basis of the aforementioned embodiments, in some embodiments, the target gene fragment further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance the stability of mRNA molecules, binding sites for amino acid sequences that enhance the translation of mRNA molecules, regulatory elements (such as riboswitches), and / or nucleotide sequences that have a positive impact on translation initiation. In addition, within the 5'UTR, preferably there is no functional upstream open reading frame, an out-of-frame upstream translation initiation site, an out-of-frame upstream start codon, and / or a nucleotide sequence that produces a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5'UTR can have a negative impact on translation.

[0213] The target protein coding sequence comprises codons that can be translated into an amino acid sequence. All the codons contained in the target protein coding sequence may be naturally occurring codons encoding amino acids, or may be partially or entirely composed of artificially synthesized codons. In some embodiments, some or all of the codons have been codon-optimized. In some embodiments, some or all of the codons encode non-natural amino acids.

[0214] In some embodiments, the engineered DNA molecule further comprises a structural element necessary for initiating or regulating transcription of the RNA on the 5' end side of the target gene fragment, and the structural element is known in the art. In some embodiments, the structural element at least comprises a promoter. Promoters and their sequences are known in the art, including weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, etc. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in prokaryotes. In some embodiments, the promoter can initiate transcription of the target protein coding sequence in eukaryotic cells. The "promoter" comprises at least one transcription recognition site and a subsequent transcription factor binding site. The recognition and binding site can interact with an amino acid sequence that mediates or regulates transcription. Compared with the recognition site, the binding site is closer to the aforementioned target gene fragment. The binding site can be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when using the Pribnow box, the transcription recognition site can be located at about 35bp upstream of the transcription start site, and the transcription factor binding site can be located at about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises at least one other regulatory element, such as an upstream element rich in AT at about 40 and / or 60 nucleotides before the transcription start site, and / or an additional regulatory element of the enhancing promoter activity between the recognition site and the binding site. In some embodiments, the promoter is a strong promoter, that is, the promoter comprises a sequence that promotes the transcription of the aforementioned target protein coding sequence. Strong promoters are known to those skilled in the art, such as OXB18, OXB19 and OXB20 promoters derived from the RecA promoter of Escherichia coli, or can be identified or synthesized by conventional laboratory procedures. In some embodiments, the promoter is a T7 promoter. In some embodiments, the promoter also comprises other regulatory elements before it, such as an enhancer that can promote the transcription of the aforementioned target protein coding sequence in a DNA plasmid.

[0215] In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the DNA molecule is a yeast display vector.

[0216] In addition, the present application also provides an engineered RNA molecule comprising the aforementioned antibody, fusion protein or engineered receptor. In some embodiments, the engineered RNA molecule is obtained by transcription of the aforementioned engineered DNA molecule. In some embodiments, the engineered RNA molecule has the same sequence as the RNA molecule obtained by transcription of the aforementioned engineered DNA molecule. In some embodiments, the engineered RNA is mRNA. As used herein, "mRNA" (messenger RNA) is any RNA that encodes at least one protein, polypeptide or fragment thereof, naturally occurring, non-naturally occurring or modified, and the mRNA has the ability to be translated to produce the encoded protein, polypeptide or fragment thereof in vitro, in vivo, in situ or in vitro. Therefore, the mRNA can be a mature mRNA or a pre-mature mRNA, and the elements or structures that must be included or selectively included are known in the art. In some embodiments, the mRNA includes the coding sequence of multiple necessary functional components to express, regulate, or enhance the expression level of the protein, polypeptide or fragment thereof. The functional components include but are not limited to 5' cap, 5' UTR, 3' UTR, etc. Both the 5'UTR and the 3'UTR are usually transcribed from genomic DNA and are elements present in the pre-mature mRNA.

[0217] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate connection with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II respectively. Type O refers to the ribose of the terminal nucleotide is not methylated, type I refers to the ribose of one terminal nucleotide is methylated, and type II refers to the ribose of both terminal nucleotides is methylated. In some embodiments, the 5' cap can be accomplished simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCapAG) according to the manufacturer's protocol. For example, in some embodiments, 5' capping of the modified RNA can be accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated from Type I cap structures followed by 2'-O-methylation of the 5'-third to last nucleotide using 2'-O methyl-transferase. Type III cap structures can be generated from Type II cap structures followed by 2'-O-methylation of the 5'-fourth to last nucleotide using 2'-O methyl-transferase.

[0218] In some embodiments, the mRNA further comprises a stabilizing element. Stabilizing elements may include, for example, a histone stem-loop. In some embodiments, the mRNA comprises a coding region, at least one histone stem-loop and optionally a poly(A) sequence or a polyadenylation signal. The poly(A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the mRNA comprises a combination of a poly(A) sequence or a polyadenylation signal and at least one histone stem-loop, although both have alternative mechanisms in nature, their synergistic effect can increase protein expression to a level that exceeds that observed for any single element. The synergistic effect of the combination of poly(A) and at least one histone stem-loop is independent of the order of the elements or the length of the poly(A) sequence. In some embodiments, the histone stem-loop is generally derived from a histone gene and comprises two adjacent partial or complete reverse complementary sequences separated by a spacer (composed of a short sequence) and the intramolecular base pairing forms a loop. Unpaired loop regions are generally unable to base pair with any of the stem-loop elements. The stability of the stem-loop structure generally depends on the length, the number of mismatches or bulges, and the base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may occur. In some embodiments, the at least one histone stem-loop sequence comprises 15 to 45 nucleotides in length.

[0219] 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.

[0220] In some embodiments, the mRNA is configured in a lipid nanoparticle (LNP). In some embodiments, lipids are mixed with the mRNA to form lipid nanoparticles. In some embodiments, RNA is formulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles are first formed into empty lipid nanoparticles and are combined or wrapped with the mRNA of the vaccine before being administered (e.g., within a few minutes to an hour).

[0221] The lipid nanoparticles generally include ionizable lipids, non-cationic lipids, sterols and PEG lipid components and target nucleic acids, such as the above-mentioned mRNA. Lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 037551, PCT / US2017 / 037552, PCT / US2017 / 037551 ... S2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575 and PCT / US2016 / 069491, all of which are incorporated herein by reference in their entirety.

[0222] In some embodiments, the engineered nucleic acid molecule may also be a hybrid molecule of DNA and RNA, wherein the hybrid molecule of DNA and RNA has the same genetic information as the engineered DNA molecule or the engineered RNA molecule.

[0223] Engineered immune cells

[0224] The present application also provides engineered immune cells, which express or contain the aforementioned antibodies, fusion proteins, engineered receptors, or engineered nucleic acid molecules on their cell membranes.

[0225] In some embodiments, the engineered immune cell is a T cell, a NK cell, a macrophage, a DC cell, a B cell, or a precursor cell thereof. In some embodiments, the engineered immune cell is a CAR-T or CAR-NK cell targeting one or more antigenic epitopes of PSCA. In some embodiments, the engineered immune cell is a TCR-T cell targeting one or more antigenic epitopes of PSCA. In some embodiments, the engineered immune cell is a TAC-T cell targeting one or more antigenic epitopes of PSCA.

[0226] In some embodiments, the engineered immune cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises an engineered receptor as described above, and one or more other engineered receptors (e.g., CAR, TAC, and TCR) targeting other tumor antigens. In some embodiments, the engineered immune cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises an engineered receptor as described above, and one or more other engineered receptors (e.g., CAR, TAC, and TCR) targeting immune checkpoint proteins. In some embodiments, the engineered immune cell is a CAR-T or CAR-NK cell targeting multiple different antigens, which comprises an engineered receptor as described above, one or more other engineered receptors targeting other tumor antigens, and one or more other engineered receptors targeting immune checkpoint proteins.

[0227] In some embodiments, the engineered immune cell is a TCR-T cell targeting multiple different antigens, which comprises a TCR in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TCR comprises the aforementioned antibody, and the TCR-T further comprises one or more TCRs targeting other tumor antigens. In some embodiments, the engineered immune cell is a TCR-T cell targeting multiple different antigens, which comprises a TCR in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TCR comprises the aforementioned antibody, and the TCR-T further comprises a TCR targeting one or more immune checkpoint proteins. In some embodiments, the engineered immune cell is a TCR-T cell targeting multiple different antigens, which comprises a TCR in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TCR comprises the aforementioned antibody, and the TCR-T further comprises one or more TCRs targeting other tumor antigens, and one or more TCRs targeting immune checkpoint proteins.

[0228] In some embodiments, the engineered immune cell is a TAC-T cell targeting multiple different antigens, which comprises a TAC in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TAC comprises the aforementioned antibody, and the TAC-T further comprises one or more TACs targeting other tumor antigens. In some embodiments, the engineered immune cell is a TAC-T cell targeting multiple different antigens, which comprises a TAC in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TAC comprises the aforementioned antibody, and the TAC-T further comprises a TAC targeting one or more immune checkpoint proteins. In some embodiments, the engineered immune cell is a TAC-T cell targeting multiple different antigens, which comprises a TAC in the aforementioned engineered receptor, the antigen binding domain or ligand binding domain of the TAC comprises the aforementioned antibody, and the TAC-T further comprises one or more TACs targeting other tumor antigens, and one or more TACs targeting immune checkpoint proteins.

[0229] In some embodiments, the engineered immune 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 and TAC, and at least one of the engineered receptors comprises the aforementioned antibody, which activates or inhibits the downstream signaling pathway of the engineered receptor after binding to PSCA. In some embodiments, the engineered immune 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 and TAC, and at least one of the engineered receptors comprises the aforementioned engineered receptor containing the aforementioned antibody, which activates or inhibits the downstream signaling pathway of the engineered receptor after binding to PSCA. In some embodiments, the engineered immune cell is selected from: T cells, NK cells, macrophages, DC cells, B cells, or their precursor cells.

[0230] In some embodiments, the engineered immune cell comprises the aforementioned engineered receptor and one or more other recognition polypeptides on its cell membrane, and the other recognition polypeptides comprise an antigen binding domain and a signal transduction domain. In some embodiments, a transmembrane domain is further included between the antigen binding domain and the signal transduction domain. In some embodiments, the transmembrane domain is connected to the antigen binding domain by a hinge or by a peptide bond. In some embodiments, the signal transduction domain comprises a primary transduction domain. In some embodiments, the signal transduction domain comprises a secondary transduction domain. In some embodiments, the signal transduction domain comprises a primary transduction domain and a stimulation transduction domain. In some embodiments, the other recognition polypeptides comprise, from N-terminus to C-terminus, an antigen binding domain-a transmembrane domain-a co-stimulatory domain, or an immune checkpoint-related ligand or receptor binding domain peptide-a transmembrane domain-a co-stimulatory domain, wherein the antigen is selected from: PSCA, CEACAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSE A-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, Claudin 6, 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 area, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor , cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1 and CD155, the immune checkpoint-related ligand or receptor is selected from: 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, CD 48, 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, wherein the transmembrane domain is selected from the transmembrane domain of CD4, CD8α, CD28 or CD3ζ,The co-stimulatory domain is selected from the signal transduction domains 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, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and a ligand that specifically binds to CD83. In some embodiments, the other recognition polypeptides include, from N-terminus to C-terminus, SIRPγ extracellular region peptide segment-transmembrane domain-co-stimulatory domain. In some embodiments, the other recognition polypeptides include, from N-terminus to C-terminus: SIRPγ-28TM-28, wherein SIRPγ represents the SIRPγ extracellular region peptide, 28TM represents the transmembrane domain of CD28, and 28 represents the signal transduction domain of CD28. In some embodiments, the SIRPγ extracellular region peptide comprises or is the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the other recognition polypeptides include, from N-terminus to C-terminus: CD226 extracellular region peptide-transmembrane domain-co-stimulatory domain. In some embodiments, the other recognition polypeptides include, from N-terminus to C-terminus: CD226-28TM-28, wherein CD226 represents the extracellular region peptide of CD226, 28TM represents the transmembrane domain of CD28, and 28 represents the signal transduction domain of CD28. In some embodiments, the CD226 extracellular region peptide comprises or is the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the other recognition polypeptides include, from N-terminus to C-terminus, TIGIT extracellular peptide segment-transmembrane domain-co-stimulatory domain. In some embodiments,The other recognition polypeptides include, from N-terminus to C-terminus, TIGIT-28TM-28, wherein TIGIT represents the extracellular peptide segment of TIGIT, 28TM represents the transmembrane domain of CD28, and 28 represents the signal transduction domain of CD28. In some embodiments, the TIGIT extracellular peptide segment includes or is the amino acid sequence shown in SEQ ID NO: 29.

[0231] In some embodiments, the tumor antigens described herein are selected from one or more of the following:

[0232] PSCA, CEACAM5, CD123, TSHR, CD171, CS-1, C-type lectin-like molecule-1, ganglioside GD3, Tn antigen, CD19, CD20, CD 22, CD 30, CD 70, CD 123, CD 138, CD33, CD44, CD44v7 / 8, CD38, CD44v6, B7H3 (CD276), B7H6, CD117, IL-13Rα, IL-11Rα, PSMA, NY-ESO-1, HIV-1Gag, MART-1, gp100, tyrosinase, mesothelin, EpCAM, PRSS21, vascular endothelial growth factor receptor, Lewis (Y) antigen, CD24, PDGFR-β, SSE A-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, Claudin 6, Claudin18.2, Claudin18.1, ASGPR1, CDH16, 5T4, 8H9, αvβ6 integrin, BCMA), CA9, kappa light chain, CSPG4, EGP2, EGP40, FAP, FAR, FBP, embryonic AchR, HLA-A1, HLA-A2, MAGEA1, MAGE3, KDR, MCSP, NKG2D ligand, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, fibronectin, tenascin, carcinoembryonic variant of tumor necrosis area, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, (PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K , OR51E2, TARP, WT1, ETV6-AML, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53 mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, CYP1B1, BORIS, SART3, (PAX5, OYTES1, (LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, and CD155.

[0233] 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, CD 48, 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.

[0234] use

[0235] The present application also provides the use of the aforementioned antibodies, fusion proteins, engineered 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:

[0236] 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.

[0237] In some embodiments, the cancer involves tumor cells that highly express PSCA.

[0238] In some embodiments, the antibodies, fusion proteins, engineered receptors, or engineered cells may be administered in combination with other anti-cancer agents.

[0239] Example

[0240] Example 1. Construction and screening of a fully human scFv library

[0241] Ficoll separation solution is used for PBMC separation. Ficoll separation solution is slowly added to the collected normal human blood so that the Ficoll separation solution and the normal human blood maintain a clear separation interface. The 50mL centrifuge tube containing the blood and separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid. There is a narrow band of white cloud layer dominated by PBMC at the junction of the upper and middle layers, i.e., the PBMC cell layer. PBMC is sucked out with a new sterile Pasteur pipette to obtain separated PBMC.

[0242] Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Based on the similarity of the heavy chain and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region, and PCR was performed to obtain the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody. The ScFv nucleic acid fragment was amplified by conventional overlapping PCR, and the ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss. The product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

[0243] (2) Preparation of phage display fully human single-chain antibody library

[0244] The library bacterial solution was added to fresh LB liquid medium for recovery, and VSCM13 helper phage was added at a multiplicity of infection of 50:1 for VCSM13: bacteria, and the mixture was thoroughly mixed and continued to be cultured in a shaker. The culture was centrifuged and the supernatant was discarded. The precipitate was resuspended in SOB medium with ampicillin and kanamycin resistance and cultured overnight. The bacterial solution was centrifuged at 4°C and 8000rpm for 20min, the supernatant was collected and 1 / 5 volume of 20% PEG 8000 2.5mmol / L NaCl solution was added, incubated on ice for 1 hour, and then centrifuged at 4°C and 12000rpm for 30min. The precipitated phage was resuspended in PBS and filtered with a 0.22μm filter membrane.

[0245] (3) Panning using PSCA antigen

[0246] PSCA protein with Fc tag (Human PSCA Protein, hFc Tag, dimabio, PME100084)) was co-incubated with proteinG magnetic beads to prepare PSCA-proteinG coupled magnetic beads, which were then drawn into the prepared fully human single-chain antibody library phage panning. After 4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen were enriched.

[0247] (4) Screening of positive clones

[0248] After the panning, the monoclonal plaques finally released from the library were selected for ELISA screening. The test results were as follows: Figure 1 As shown, a phage clone binding to PSCA antigen was obtained, and a ScFv targeting PSCA was obtained by panning, which had the specific binding ability to human PSCA antigen.

[0249] In the embodiment of the present invention, the specific implementation process of antigen panning is as follows:

[0250] (1) Blocking: Dissolve 5% skim milk powder in PBS, filter and use as blocking solution. Resuspend phage and CD70-protein G-coupled magnetic beads in appropriate amount of blocking solution and mix by rolling.

[0251] (2) Co-incubation: Place PSCA-protein G-coupled magnetic beads on a magnetic rack, discard the supernatant, resuspend the magnetic beads with phage, and roll over for co-incubation;

[0252] (3) Washing: Place the magnetic bead-phage mixture on a magnetic rack, discard the supernatant and add a washing solution to wash the magnetic beads. The number of washing times is determined according to different rounds of panning;

[0253] (4) Elution: Place the washed beads on a magnetic rack, aspirate the supernatant, add Gly-HCl 4, mix well, incubate at room temperature for 7 min, add Tris-HCl to adjust the pH to near neutral, finally place the mixture on a magnetic rack, transfer the phage supernatant to a new 1.5 mL EP tube to complete one round of panning;

[0254] (5) Enrichment: The phages were inoculated into TGI bacterial solution for infection, centrifuged after resting, the precipitate was resuspended, spread on 2YTAG plates, and inverted for overnight culture;

[0255] (6) Plate washing: The bacterial plaques on the plates cultured overnight are washed off with the culture medium and used as the seed bacterial solution for the next round of library packaging.

[0256] In the embodiment of the present invention, the specific implementation process of ELISA detection is as follows:

[0257] (1) Coating: Dilute PSCA antigen to 1 μg / mL with carbonate coating buffer, add 100 μL / well to a 96-well plate, and cover and incubate overnight at 4°C;

[0258] (2) Obtaining phage samples: Centrifuge the overnight cultured phage monoclonal recombinant bacterial solution for 10 min and take the supernatant as the test sample;

[0259] (3) Blocking: Wash the antigen-coated plate three times with PBS on a plate washer, add skim milk powder blocking solution to each well, and block at 37°C;

[0260] (4) Antibody incubation: After washing the plate three times with a plate washer, add the monoclonal recombinant bacterial solution of the phage to be tested to each well and incubate at 37°C;

[0261] (5) Adding secondary antibody: Wash the plate three times with a plate washer, add 100 μL of 1:5000 Anti-M13-HRP secondary antibody to each well, and incubate at 37°C;

[0262] (6) Color development: Wash the plate six times with a plate washer, add color developing solution to each well, and place at room temperature away from light for 25 min to develop color;

[0263] (7) Termination: Add H2SO4 to each well to terminate the reaction;

[0264] (8) Detection: Place the test plate in a microplate reader to detect the OD450 absorbance. Phage clones that are 2.5 times higher than the negative control are considered positive clones.

[0265] In the embodiments of the present invention, the sequences of the carrier structural elements are shown in the sequence table at the end of the text; 8h / 7h / 8hdc / G4H are different hinge structures; 8TM / 28TM are different transmembrane structures; 28 / BB / CD134 are different intracellular signal structures.

[0266] The ScFv sequence that specifically recognizes PSCA and is screened using the above-mentioned method for preparing a fully human antibody is also shown in the sequence table at the end of the article.

[0267] In the examples of the present invention, PSCA (11) is the ScFv AK-27-B10 targeting PSCA, and TIGIT (mut5) is a mutated TIGIT peptide.

[0268] The above ScFv was obtained by prokaryotic expression and purification method. The specific method is as follows:

[0269] The pComb3xss plasmids constructed with the above ScFv gene sequences were transformed into Rosetta gami B competent cells, spread on LB plates containing ampicillin resistance, and cultured at 37°C overnight; the next day, single clones were picked and placed in 4ml LB medium containing ampicillin resistance for 8h, and then the bacterial solution was transferred to 200mL LB medium for further culture. When the bacterial solution OD reached 0.5-1.0, IPTG with a final concentration of 1mM was added, and expression was induced at 37°C for 12h. The bacteria were collected by centrifugation and PBS was added to resuspend the precipitate, ultrasonically broken, centrifuged for 10min, the precipitate was discarded, and the supernatant was collected for protein purification, which was purified using a GE Ni Sepharose excel purification column.

[0270] The ScFv protein that recognizes the PSCA antigen was further examined, and the specific method is as follows:

[0271] 293T and 293T-PSCA cells (293T cells expressing PSCA antigen) were each dispensed into 1.5 mL Eppendorf tubes, with 1×10^6 cells in each tube as target cells. After centrifugation at 400 g for 5 min, the supernatant was discarded and the cells were resuspended with 100 μL of 50 μg / mL, 25 μg / mL and 12.5 μg / mL of the above ScFv solutions, respectively. The group without ScFv and secondary antibody was used as the Blank group, and the group without ScFv and only with secondary antibody was used as the Control group. The cells were incubated at 4°C, and after 30 min, 1 mL of PBS was added to resuspend the cells. The cells were centrifuged at 400 g for 5 min, the supernatant was discarded, 30 μL of Anti-His-647 fluorescent secondary antibody for detection was added to resuspend the cells, and the cells were incubated at 4°C in the dark for 30 min; the cells were resuspended with 1 mL of PBS and washed twice at 400 g for 5 min, the supernatant was discarded and the cells were resuspended with 100 μL of PBS, and the flow cytometer was used to detect the positive rate of flow staining. The results are shown in Figure 8. Fig. 8A The recognition of PSCA antigen by ScFv clone AK-15-H02; Figure 8B The recognition of PSCA antigen by ScFv clones AK-24-A08 and AK25-H08; Figure 8CThe figure shows the recognition of PSCA antigen by ScFv of clone number AK-27-B10; the above ScFv (specific sequence is shown in the sequence table at the end of the article) can significantly recognize PSCA antigen, can be used to detect the expression of PSCA, and can be used as a component of a kit for detecting the expression of PSCA.

[0272] Example 2: Screening for ScFv targeting PSCA suitable as an extracellular recognition domain of CAR-T

[0273] In order to verify the function of the screened fully human antibodies, we constructed CAR vectors to prepare CAR-T cells to verify the ability of the screened fully human antibodies to recognize PSCA expressed by cells and kill tumor cells expressing PSCA.

[0274] 1) Screening for scFv with excellent killing ability against PSCA-expressing tumor cells

[0275] The scFv sequence of the PSCA antibody was obtained by PCR amplification, and then the sequence was connected to the lentiviral vector of the CAR structure by restriction endonuclease digestion. Here, the scFv was mainly connected to the N-terminus of the two structures of 8hdc-28TM-28BBz and 8h-8TM-BBz for screening. The use of these two structures can fully reflect the function of ScFv. The constructed lentiviral vector was verified by sequencing comparison, confirming that the construction was successful.

[0276] The present embodiment packages the lentivirus using the calcium phosphate method, specifically: 293T cells are cultured to an optimal state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5 centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and the medium is changed to 10mL of DMEM medium containing 10% FBS again after 3-5h, and the cell supernatant is collected after 48h or 72h, and the virus is purified. 15 virus particles in Example 1 are obtained.

[0277] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with activated PBMCs at a certain multiplicity of infection (MOI). The positive rate of CAR-T was detected on the 8th day of viral infection. The detection method was flow cytometry, and the antibody was: Protein-L-PE. Protein-L can recognize the light chain of the antibody. The light chain of the ScFv sequence of the CAR antigen binding domain can be recognized by Protein-L. Therefore, Protein-L can be used to detect the positive rate and expression intensity of CAR. anti-CD3 FITC (Bio legend, Lots: B378781, Cat: 300440), Protein-L-PE (Sino Biological, Lot: HR16SE0901, Cat: 11044-H07E-P).

[0278] The CAR-T cells prepared above were plated with PSCA-positive target cells RT4-Luc-GFP at an effector-target ratio of 1:1, and the ACEA xCELLigence RTCA MP instrument was used to detect the killing ability of different CAR-Ts on target cells. The experimental steps were carried out according to the instrument manual. The principle of ACEA xCELLigence RTCA MP is to record the resistance index of tumor cells attached to the bottom of the well every 15 minutes, and judge the proliferation or death of the attached target cells by the resistance index. The formula for analyzing the results using the resistance index is: CAR-T cell killing rate = baseline resistance index - real-time resistance index.

[0279] The abbreviation and structure of CAR and the results of CAR expression and killing detection are shown in Table 1 below. PSCA(n) represents the corresponding scFv in the table, for example, PSCA(11) represents the scFv of AK-27-B10.

[0280] Table 1: CAR structure, abbreviation, virus titer, CAR expression and CAR-T killing rate

[0281]

[0282] It can be found from Table 1 that among the four ScFvs that can normally construct CAR: AK-25-H08, AK-27-B10, AK-15-H02, and AK-24-A08, the constructed CARs can all be expressed normally, and the CAR-T constructed by AK-27-B10 (also known as PSCA (11)) has a significantly higher killing effect on tumor cells expressing PSCA, and the killing result of 76.22% is significantly higher than that of other ScFvs. Finally, the inventors screened and obtained the ScFv AK-27-B10 (also known as PSCA (11)) that can recognize PSCA and has a significant killing effect on tumor cells expressing PSCA.

[0283] Furthermore, the inventors verified the ability of AK-27-B10 (also known as PSCA (11)) to recognize PSCA antigen: human bladder cancer cell line HT1376 and human pancreatic cancer cell line HPAC, which theoretically express PSCA, were selected, and human prostate cancer cell line PC3-PSCA, which exogenously expressed high expression, was constructed to test the ability of AK-27-B10 (also known as PSCA (11)) to recognize PSCA antigen.

[0284] After the above cell culture, the cells were harvested and centrifuged at 300 g / min for 5 min. The supernatant was discarded to collect the cells. The cells were resuspended in PBS solution containing 1% fetal bovine serum and the cell density was adjusted to 1×10 6 / ml; the collected cells were divided into the final labeled antibody, incubated at 4°C for 30 minutes, washed twice with PBS solution, and the supernatant was discarded to collect the cells; the cells were resuspended with PBS solution containing 1% fetal bovine serum, added with secondary antibody, incubated at 4°C for 30 minutes, washed twice with PBS solution, and detected by flow cytometry. Labeled antibody: (Manufacturer: Santa Cruz, item number: Sc-80654, Lot: J 1821, PSCA intermediate label secondary antibody is Alexa Fluor 647, goat anti-mouse IgG (H+L) RFF: A21235, Lot: 2482945) to detect the expression of PSCA antigen, the results are as follows Figure 1 And as shown in Table 2.

[0285] Table 2: Expression of PSCA in HPAC-Luc-GFP and PC3-PSCA-Luc-GFP cells

[0286] cell PSCA expression positive rate PC3-PSCA-Luc-GFP 100％ HPAC-Luc-GFP 60％ RT4-Luc-GFP 100％

[0287] like Figure 1As shown in Table 2, the positive expression rate of PSCA in HT1376-Luc-GFP was about 80%, PSCA was 100% expressed in PC3-PSCA-Luc-GFP, PSCA was 60% expressed in HPAC-Luc-GFP, and 100% expressed in RT4-Luc-GFP.

[0288] In combination with Table 1 and Table 2, AK-27-B10 (also known as PSCA (11)) screened by the present invention can specifically identify PSCA-expressing tumors such as human bladder cancer, human pancreatic cancer, and human prostate cancer, and can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high PSCA expression, effectively killing malignant tumors with high PSCA expression and controlling tumor proliferation, such as application in CAR-T cell therapy.

[0289] Example 3. Functional verification of fully human single-chain antibodies

[0290] Different CAR structures were designed for the optimal PSCA (11) anti-PSCA fully human ScFv screened above to verify the adaptability of PSCA (11) anti-PSCA fully human ScFv to different CAR structures. 8hdc / G4h / 8h was selected, transmembrane CD28TM was selected, different co-stimulatory signals including CD28, CD27, 4-1BB were selected, and Z (3) was selected for structural design. Specifically, the structure of PSCA (11)-8h-8TM-BBZ was used in this embodiment to verify the recognition and functionality of PSCA (11) against PSCA antigens, as well as the application as a chimeric antigen receptor (CAR). The virus containing the CAR vector was prepared using the method of Example 2, CAR-T cells were prepared, and CAR expression detection was completed, as shown in Table 3 below. Among them, the CAR with PSCA (11) as the extracellular recognition domain is represented by CAR1.

[0291] Table 3: CAR titer and expression data

[0292]

[0293] The CAR-T cells prepared above were plated with PSCA-positive target cells HT1376-Luc-GFP, PC3-PSCA-Luc-GFP, and HPAC-Luc-GFP PSCA at a ratio of 1:1. The killing ability of different CAR-T cells on target cells was detected using the ACEA xCELLigence RTCA MP instrument. The experimental steps were carried out according to the instrument manual. The killing results are statistically shown as follows Figure 2As shown: the horizontal axis represents CAR, and the vertical axis represents the in vitro killing percentage in different cells. The results show that CAR1 has significant killing effects on HPAC-Luc-GFP, HT1376-Luc-GFP and PC3-PSCA-Luc-GFP, and the killing percentages are all higher than 50%, proving that CAR1 can exert significant in vitro pharmacodynamic effects in various solid tumor cell models. It is proved that PSCA (11) can recognize PSCA antigens expressed by HPAC-Luc-GFP, HT1376-Luc-GFP and PC3-PSCA-Luc-GFP, and can transmit intracellular signals of CAR structure. The constructed CAR-T targeting PSCA has killing effects on pancreatic cancer, bladder cancer and prostate cancer, and can be used to prepare drug combinations for the treatment of pancreatic cancer, bladder cancer and prostate cancer.

[0294] 6-8 week old NCG female mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were used to establish an intraperitoneal tumor model using HPAC-Luc-GFP or HT1376-Luc-GFP or PC3-PSCA-Luc-GFP. Three days after the intraperitoneal injection of 3E5 tumor cells, CAR1:5E5 CAR-T / CAR-T cells were intraperitoneally administered. Control-T was activated control T cells that were cultured in the same manner as CAR-T but were not transduced with CAR structures. Live imaging of mice was performed every 7 days after administration, and the imaging fluorescence values ​​were statistically analyzed using T Test for significance. The results are shown in Figure 3 below. Figure 3A As shown, HPAC-Luc-GFP was used for peritoneal tumor formation. The results showed that CAR1 had significant efficacy in the HPAC-Luc-GFP tumor formation model. PSCA (11) can function as an extracellular recognition domain in the CAR structure. The prepared CAR-T cells can be used as a drug or drug combination against pancreatic cancer. Figure 3B and Figure 3C As shown, CAR1 has significant efficacy in both PC3-PSCA-Luc-GFP tumor model and HT1376-Luc-GFP tumor model.

[0295] At the same time, HPAC-Luc-GFP was used for subcutaneous tumor formation. 3E6 CAR-T cells were intraperitoneally administered 7 days after tumor formation. The mice were imaged in vivo every 7 days after administration. The fluorescence values ​​of the imaging were statistically analyzed using T Test for significance. The results are shown in the figure. Figure 4 As shown, Figure 4 Tumor proliferation curves drawn for tumor volume measurements after CAR-T and Control-T infusion, showing that CAR1 has significant efficacy in the HPAC-Luc-GFP subcutaneous tumor model

[0296] According to Figure 3 and Figure 4 CAR1 has significant in vivo efficacy results in pancreatic cancer peritoneal tumor model, bladder cancer peritoneal tumor model, prostate cancer peritoneal tumor model and pancreatic cancer subcutaneous tumor model, proving that CAR1 has significant therapeutic prospects in different solid tumors. PSCA (11) can be used as the extracellular recognition domain of the CAR structure, and the cell therapy product prepared by the CAR can be used for the treatment of solid tumors expressing PSCA antigens such as pancreatic cancer, bladder cancer, and prostate cancer.

[0297] PSCA (11) can be used with different promoters, i.e., normal promoter, hypoxia promoter, different hinges, i.e., 8h, 8hdc, and G4h, different transmembrane 8TM and 28TM, and different intracellular signals CD28, 4-1BB, and CD27 to recognize PSCA-positive tumor cells and kill them in in vitro functional validation, and can also play a significant role in killing tumor cells in different solid tumor models. Through the validation of the above-mentioned multiple CAR structures and tumor indications, PSCA (11) can be used as the antigen binding domain of chimeric antigen receptors, and can be used for the treatment of PSCA-expressing tumors such as pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma, and gallbladder cancer, including the treatment of pancreatic cancer, prostate cancer, bladder cancer, gastric cancer, lung cancer, breast cancer, oral squamous cell carcinoma, and gallbladder cancer, etc., with the constructed CAR-T cells of multiple structures.

[0298] In some embodiments, the structure of "chimeric antigen receptor" or "CAR" comprises: an antigen binding domain (such as ScFv, which in the present invention refers to ScFv that recognizes PSCA), a hinge structure (such as 8hinge derived from human CD8), a transmembrane structure (such as CD8TM derived from human CD8 transmembrane) and an intracellular signaling domain. There may also be multiple structures, such as comprising secretable or membrane-expressed cytokines and antibody gene sequences; such as comprising a structure that can be regulated for activation or inactivation, wherein the structure that regulates activation or inactivation comprises: a suicide switch such as inducible caspase-9 (iCasp9), thymidine kinase in herpes simplex virus (HSV-TK) and suicide epitope, truncated EGFR (EGFRt), Fas-FasL apoptosis structure; an inducible CAR (InducibleCAR) structure: Peptide neo-epitope (PNE), fluorescein (FITC), 10amino acids (5B9 tag), FITC-HM-3bifunctional molecule (FHBM) and scFv, Leucine ZipFv linked toantibody, Streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR induction structure, biotin-targeted immune receptor (BBIR) system; "logic gate" regulatory system combined with SynNotch receptor, etc. In some embodiments, the structure described in "chimeric antigen receptor" or "CAR" may also include an antigen binding domain, a transmembrane domain and an intracellular signal transduction domain, and the antigen binding domain may be a complete and continuous extracellular segment of the cell membrane of the expressed molecule / polypeptide, or a fusion form of the expressed molecule and other polypeptides such as polypeptides derived from human CD8 and CD4.The intracellular signaling domain of the CAR structure can be a primary intracellular signaling domain containing only an immunoreceptor tyrosine-based activation motif or ITAM: Examples of primary intracellular signaling sequences containing ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10 and DAP12, etc., and can also contain one or two or more costimulatory signaling domains in addition to the primary intracellular signaling domain, and the costimulatory signaling domain can be selected from any one or more of the following molecules and functional variants derived therefrom: CD28, 41BB, OX40, CD27, DAP10 , 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc. The derived functional molecule is an amino acid or nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with the above-mentioned molecule.

[0299] In addition to the above-mentioned single CAR design, the PSCA (11) described in the present invention can also be used for multi-target CAR structure design, for different PSCA-positive indications, including but not limited to human pancreatic cancer, prostate cancer and bladder cancer, any dual CAR structure containing PSCA (11) has significant killing function. The targets of these dual CAR structures can be PSCA and solid tumor targets including but not limited to CD155, MOv-γ, PSMA, IL13Rα2, EGFRvIII, EGFR, EPCAM, GD2, MUC1, HER2, GPC3, CEA, Meso, CD133, NKG2D, CD138, LeY, k-Light, ROR1, CD70, CD47, CLDN18.2, CDH17, Trop2, B7H3, etc.

[0300] Example 4 Verification of the application of fully human single-chain antibodies as antigen binding domains in CAR

[0301] In terms of CAR structural design, in addition to the traditional single CAR structure and dual CAR structure, there are also CAR structures and engineered receptors without primary signal transduction domains (used in the embodiments of the present application to refer to engineered receptors composed of the following structures from N-terminus to C-terminus: antigen binding domain-hinge region-transmembrane region-co-stimulatory domain or antigen binding domain-transmembrane region-co-stimulatory domain, where "-" indicates direct connection through peptide bonds), and functional polypeptide combination modes. These combinations can be CAR and engineered receptors without primary signal transduction domains, and functional polypeptides constructed on the same vector for expression, or CAR targeting PSCA and engineered receptors without primary signal transduction domains, and functional polypeptides are divided into two vectors and transferred to target immune cells. The engineered receptor or functional polypeptide without a primary signal transduction domain may be an engineered receptor without a primary signal transduction domain comprising an extracellular segment or extracellular recognition functional domain of an immune checkpoint such as PD1 / PDL1, TIGIT, SIRPγ, SIRPα, etc., or an engineered receptor without a primary signal transduction domain comprising the full length, functional domain and / or signal domain of a cytokine such as IL15, IL2, IL21, IL12, IL7, etc., or an engineered receptor without a primary signal transduction domain comprising the full length of a chemokine or chemokine receptor such as CXCR3, CXCR2, CCL19, CCL21, etc.

[0302] Here, we take the engineered receptor without primary signal transduction domain and TIGIT as an example to verify the feasibility of the combination model of CAR containing single-chain antibody PSCA (11) and engineered receptor without primary signal transduction domain and functional polypeptide.

[0303] Here, an engineered receptor without a primary signal transduction domain with TIGIT as an antigen binding domain is used as an example to verify the feasibility of the combined mode of a CAR containing a single-chain antibody PSCA (11) and an engineered receptor without a primary signal transduction domain and a functional polypeptide. The CAR structure design is shown in Table 4 below. Different CARs are designed based on PSCA (11)-8h-8tm-BBZ. In this embodiment, the inventors selected an engineered receptor design without a primary signal transduction domain for immune checkpoints such as CD47 and CD155. The specific design is shown in Table 1, where SIRPγ recognizes CD47, CD226 and TIGIT recognizes CD155. According to the schemes of Examples 1 and 2, CAR virus preparation, CAR-T preparation and in vitro killing verification are completed, and the expression results of CAR titer are shown in Table 4.

[0304] Table 4: CAR structure design combined with engineered receptors without primary signaling domain

[0305] CAR Name structure CAR2 PSCA(11)-8h-8TM-BBZ-P2A-SIRPγ-28TM-28 CAR3 PSCA(11)-8h-8TM-BBZ-P2A-CD226-28TM-28 CAR4 PSCA(11)-8h-8TM-BBZ-P2A-TIGIT(mut5)-28TM-28

[0306] In vitro killing results Figure 5 As shown, the horizontal axis represents different CARs, and the vertical axis represents the killing of three cell types PC3-PSCA-Luc-GFP, HPAC-Luc-GFP, and HT1376-Luc-GFP for three indications of prostate cancer, pancreatic cancer, and bladder cancer by CARs with different structures combined with engineered receptors without primary signal transduction domains and CAR-T expressing CAR alone. The horizontal axis represents different CARs, and the vertical axis represents the in vitro killing percentage in different cells. The results showed that CAR2, CAR3, and CAR4 all had significant killing effects on HPAC-Luc-GFP, PC3-PSCA-Luc-GFP, and HT1376-Luc-GFP: It proves that engineered receptors without primary signal transduction domains combined with PSCA (11) single CAR that bind to different targets (such as different immune checkpoint proteins) can exert significant in vitro pharmacodynamic effects in various solid tumor cell models.

[0307] 6-8 week old NCG female mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used to establish an intraperitoneal tumor model using HPAC-Luc-GFP or HT1376-Luc-GFP or PC3-PSCA-Luc-GFP. 3 days after intraperitoneal injection of 3E5 tumor cells, 5E5 CAR-T / CAR-T cells were intraperitoneally administered. Control-T was activated control T cells that were cultured in the same manner as CAR-T but were not transduced with CAR structures. Live imaging of mice was performed every 7 days after administration, and the imaging fluorescence values ​​were statistically analyzed using T Test for significance. The results are shown in Figure 6. Fig. 6A The tumor fluorescence curve of CAR2 to CAR4 in the HPAC-Luc-GFP (pancreatic cancer) peritoneal tumor model reflects the in vivo efficacy of CAR-T; Figure 6B The tumor fluorescence curve of CAR2 to CAR4 in the HT1376-Luc-GFP (bladder cancer) abdominal tumor model reflects the in vivo efficacy of CAR-T; Figure 6C The in vivo efficacy of CAR2 to CAR4 in the PC3-PSCA-Luc-GFP (prostate cancer) peritoneal tumor model. The results showed that CAR2, CAR3 and CAR4 had significant efficacy in the PC3-PSCA-Luc-GFP, HPAC-Luc-GFP and HT1376-Luc-GFP tumor models.

[0308] At the same time, HPAC-Luc-GFP was used for subcutaneous tumor formation. 3E6 CAR-T cells were intraperitoneally administered 7 days after tumor formation. The mice were imaged in vivo every 7 days after administration. The fluorescence values ​​of the imaging were statistically analyzed using T Test for significance. The results are shown in the figure. Figure 7 As shown, the tumor proliferation curve was drawn based on the tumor volume measurement results after CAR-T and Control-T were reinfused. Control-T was activated control T cells that were cultured in the same manner as CAR-T but were not transduced with the CAR structure. The results showed that CAR2, CAR3, and CAR4 had significant efficacy in the HPAC-Luc-GFP subcutaneous tumor model.

[0309] As shown in Figure 6 above Figure 7 As described above, CAR2, CAR3, and CAR4 all showed significant in vivo efficacy results in the pancreatic cancer peritoneal tumor model, bladder cancer peritoneal tumor model, prostate cancer peritoneal tumor model, and pancreatic cancer subcutaneous tumor model, respectively, demonstrating that engineered receptors without primary signal transduction domains that bind to different targets (e.g., different immune checkpoint proteins) combined with PSCA (11) single CAR have significant therapeutic prospects in different solid tumors.

[0310] The sequences used in the above examples of the present application are shown in the following sequence table. It should be understood that the following sequences are only exemplary sequences of the present application embodiment, rather than any limitation to the present application scheme. The nucleic acid sequences in the following sequence table may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "T" therein represents uridine. Moreover, in the present application, a single T also refers to uracil or uridine in the context of RNA.

[0311] Sequence Listing:

[0312]

[0313]

[0314]

[0315] Note: CDRs in the sequence listing are defined using the IMGT numbering system.

Claims

1. An isolated antibody that binds to prostate stem cell antigen (PSCA), comprising an immunoglobulin heavy chain variable region (VH) sequence and an immunoglobulin light chain variable region (VL) sequence, wherein: The VH comprises a heavy chain variable region complementarity determining region 1 (HCDR1), a heavy chain variable region complementarity determining region 2 (HCDR2), and a heavy chain variable region complementarity determining region 3 (HCDR3), and the VL comprises a light chain variable region complementarity determining region 1 (LCDR1), a light chain variable region complementarity determining region 2 (LCDR2), and a light chain variable region complementarity determining region 3 (LCDR3); Where VL and VH contain: 1) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1, 2, 3 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 7; 2) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 9, 10, 11 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13, 14, 15; 3) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, 18, 19 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, 6, 21; or 4) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 23, 2, 24 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 5, 6, 26, Among them, LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are defined by the IMGT system. The antibody according to claim 1 , which is a chimeric antibody or a human antibody.

3. The antibody according to claim 1 or 2, wherein the amino acid sequence of VH comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence selected from any one of SEQ ID NOs: 8, 16, 22 and 27.

4. The antibody according to any one of claims 1-3, wherein the amino acid sequence of VL comprises or is an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25, or an amino acid sequence selected from any one of SEQ ID NOs: 4, 12, 20 and 25 that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

5. The antibody according to any one of claims 1 to 4, comprising VL and VH, wherein: 1) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO:25, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO:27, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:27; 2) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8; 3) the amino acid sequence of the VL comprises or is the amino acid sequence as set forth in SEQ ID NO:12, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:12, and the amino acid sequence of the VH comprises or is the amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16; or 4) the amino acid sequence of the VL comprises or is the amino acid sequence as shown in SEQ ID NO:20, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:20, and the amino acid sequence of the VH comprises or is the amino acid sequence as shown in SEQ ID NO:22, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

22.

6. The antibody according to any one of claims 1 to 5, which is a full-length antibody or an antigen-binding fragment.

7. An engineered receptor comprising an antigen binding domain, wherein: The antigen binding domain comprises the antibody according to any one of claims 1 to 6.

8. A nucleic acid molecule comprising a nucleic acid encoding the antibody according to any one of claims 1 to 6, or the engineered receptor according to claim 7.

9. An engineered immune cell comprising the engineered receptor according to claim 7 or the nucleic acid molecule according to claim 8.

10. Use of the antibody according to any one of claims 1 to 6, the engineered receptor according to claim 7, the nucleic acid molecule according to claim 8 or the engineered immune cell according to claim 9 for preparing a drug for treating cancer.

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