Viral vectors and use thereof in adoptive cellular therapy

Abstract

A vector comprising: a first nucleotide sequence S1 encoding protein Z1; a second nucleotide sequence S2 encoding protein Z2; a third nucleotide sequence S3 encoding protein Y1; and a fourth nucleotide sequence S4 encoding protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, wherein the first dimer Z1Z2 is different from the second dimer Y1Y2.

Description

Technical Field

[0001] This invention relates to T cell manufacturing. In one instance, the invention relates to the manufacturing of T cells expressing multiple proteins in a single vector using a polycistronic cassette. More specifically, the invention relates to the manufacturing of T cells co-expressing TCRαβ and CD8αβ and the application of such T cells in receptive cell therapy. Prior Technology

[0002] Genetic engineering of human lymphocytes as potential therapies for hereditary, acquired, or infectious diseases requires the efficient transfer and expression of transgenes. In the case of receptive immunotherapy for cancer, naturally occurring and / or recombinant antitumor T-cell receptors (TCRs) have been used to confer antitumor responsiveness to normal T cells or tumor-infiltrating lymphocytes.

[0003] Morgan et al. (J Immunol. 2003 Sep 15; 171(6): 3287–32%) revealed an anti-gp100 TCR expressed by bicistronic RNA, in which the expression of the first gene encoding the TCRβ chain is controlled by a long terminal repeat (LTR) and the expression of the second gene encoding the TCRα chain is manipulated by the internal ribosome entry site (IRES). Antigen reactivity of CD4+ T cell lines engineered with this anti-gp100 TCR gene was also demonstrated.

[0004] Cohen et al. (J Immunol. 1 Nov 2005; 175(9): 5799–5808) revealed a bicistronic retroviral vector for co-expressing both the TCRα and TCRβ chains of the p53 epitope. Expression of the first gene encoding the TCRα chain was controlled by the LTR, and expression of the second gene encoding the TCRβ chain was controlled by the IRES. Lymphocytes transduced by p53 TCRs were able to specifically recognize the peptide pulse APC and HLA-A2.1+ cells transfected with wild-type or mutant p53 protein using high avidity.

[0005] Hughes et al. (Hum Gene Ther. April 2005; 16(4): 457–472) revealed various bicistronic retroviral vectors for co-expressing anti-MART-1 TCR. Expression of the first gene encoding the TCRα chain was controlled by the LTR, and the second gene encoding the TCRβ chain was manipulated by the IRES, or vice versa. Alternatively, expression of the first gene encoding the TCRα chain was controlled by the LTR, and the second gene encoding the TCRβ chain was manipulated by the PGK promoter, or vice versa. T cells transduced with these vectors exhibited highly active T cell effector functions.

[0006] Zhao et al. (J Immunol. April 1, 2005; 174(7): 4415–4423) revealed a bicistronic retroviral vector for co-expressing NY-ESO-1 TCR. Expression of the first gene encoding the TCRα chain was controlled by the LTR, and the second gene encoding the TCRβ chain was manipulated by the IRES, or expression of the first gene encoding the TCRα chain was controlled by the LTR, and the second gene encoding the TCRβ chain was manipulated by the PGK promoter. Transduced lymphocytes effectively identified and killed HLA-A2- and NY-ESO-1-positive melanoma cell lines.

[0007] Morgan et al. (Gene Therapy (2008) 15, 1411–1423) revealed bicistronic lentiviral vectors that combine a furin cleavage site with an amino acid spacer (GSG or SGSG (SEQ ID NO: 8)) followed by a 2A ribosomal jumping peptide to express anti-gp100 TCR or anti-MART-1 TCR. When the spacer sequence is enlarged by adding a synthetic V5 peptide, tag sequence protein processing is accelerated, resulting in a lentiviral vector capable of modulating high levels of TCR expression in transduced lymphocytes.

[0008] Gene delivery systems are still needed to achieve safe and efficient transgenic expression in receptive cell therapy. Summary of the Invention

[0009] In one embodiment, the present invention provides a gene delivery system comprising a vector, the vector comprising: a first nucleotide sequence S1 encoding protein Z1, a second nucleotide sequence S2 encoding protein Z2, a third nucleotide sequence S3 encoding protein Y1, and a fourth nucleotide sequence S4 encoding protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, wherein the first dimer Z1Z2 is different from the second dimer Y1Y2, and wherein the gene delivery system is used in adaptive cell therapy.

[0010] In another configuration, S1, S2, S3, and S4 can be selected from the following 5' to 3' oriented series arrangements: S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S3-S4-S1, S2-S 4-S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S2, S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3 -S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2 or S4-S3-S2-S1.

[0011] In another embodiment, the vector may further include a fifth nucleotide sequence S5 encoding a 2A peptide and a sixth nucleotide sequence S6 encoding a linker peptide, wherein S5 and S6 are located between S1 and S2, between S1 and S3, between S1 and S4, between S2 and S3, between S2 and S4, and / or between S3 and S4.

[0012] In another state, the 2A peptide may be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5) or F2A (SEQ ID NO: 6).

[0013] In another state, the linker peptide may be GSG or SGSG (SEQ ID NO: 8).

[0014] In another embodiment, the vector may include the seventh nucleotide sequence S7 encoding furin peptide (SEQ ID NO: 2), wherein S7 is located between S1 and S2, between S1 and S3, between S1 and S4, between S2 and S3, between S2 and S4, and / or between S3 and S4.

[0015] In another variant, the vector may further include a post-transcriptional regulatory element (PRE) sequence selected from woodchuck PRE (WPRE) or hepatitis B virus (HBV) PRE (HPRE).

[0016] In another embodiment, the vector may further include promoter sequences controlling transcription of S1, S2, S3, S4, S5, S6 and / or S7, wherein the promoter sequences are selected from the following: cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR (MNDU3) containing myeloproliferative sarcoma virus enhancer, ubiquitin C promoter, EF-1 α promoter, or murine stem cell virus (MSCV) promoter.

[0017] In another state, the first dimer Z1Z2 can be selected from SEQ ID NO: 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88 or 89 and 90.

[0018] In another state, the second dimers Y1 and Y2 are described in SEQ ID NO: 11 and 12.

[0019] In another state, the orientation is S2-S1-S4-S3.

[0020] In another state, the vector has a sequence selected from the following: PTE WPRE (SEQ ID NO: 91), TPE WPRE (SEQ ID NO: 92), or PTE fn WPRE (SEQ ID NO: 93).

[0021] In another state, the orientation is S4-S3-S2-S1.

[0022] In another sample, the vector has the sequence PTE CD8 TCR WPRE (SEQ ID NO: 94).

[0023] In another variant, the viral vector is selected from adenovirus, poxvirus, alpha virus, arenavirus, flavivirus, rod-shaped virus, retrovirus, lentivirus, herpesvirus, paramyxovirus, or microRNA virus.

[0024] In another variant, the vector is pseudotyped with an envelope protein selected from the following viruses: native feline endogenous virus (RD114), a chimeric form of RD114 (RD114TR), gibberish ape leukemia virus (GALV), a chimeric form of GALV (GALV-TR), murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), baboon retroviral envelope glycoprotein (BaEV), or lymphocytic choriomeningitis virus (LCMV).

[0025] In another variant, the vector was pseudotyped by the envelope protein of vesicular stomatitis virus (VSV-G).

[0026] In one embodiment, the present invention relates to a method for preparing T cells for immunotherapy, the method comprising the steps of: isolating T cells from a blood sample of a human subject; activating the isolated T cells in the presence of an aminobisphosphonate; transducing the activated T cells using a vector described herein; and expanding the transduced T cells.

[0027] In another case, these T cells can be isolated from human samples using leukocyte ablation.

[0028] In another embodiment, the amino bisphosphonate may be selected from pamidronic acid, alendronic acid, zoledronic acid, risedronic acid, ibandronic acid, incadronic acid, salts of the aforementioned phosphonates, and / or hydrates of the aforementioned phosphonates.

[0029] In another state, this activation can be further carried out in the presence of recombinant human interleukin 2 (IL-2) and recombinant human interleukin 15 (IL-15).

[0030] In another state, the amplification can be performed in the presence of IL-2 and IL-15.

[0031] In another phenotype, these T cells may be γδ T cells.

[0032] In another state, the first dimer Z1Z2 and the second dimer Y1Y2 are co-present on the surface of the expanded T cells.

[0033] In another embodiment, the present invention relates to an expanded T cell population prepared by the method described above.

[0034] In one embodiment, the present invention relates to a method for treating a patient with cancer, the method comprising the steps of: administering to the patient a composition comprising the population of expanded T cells as described herein, wherein the T cells kill cancer cells that exhibit a peptide complexed with an MHC molecule on their surface, wherein the peptide is selected from any one of SEQ ID NO: 98 to 255, wherein the cancer is selected from the group consisting of: non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0035] In another embodiment, the composition further comprises an adjuvant.

[0036] In another variant, the adjuvant is selected from one or more of the following: anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon α, interferon β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil, a particle formulation containing poly(lactide co-glycolide); PLG, virion, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0037] In one embodiment, the present invention relates to a method for eliciting an immune response in a patient with cancer, the method comprising the steps of: administering to the patient a composition comprising the population of expanded T cells as described herein, wherein the T cells kill cancer cells that exhibit a peptide complexed with an MHC molecule on their surface, wherein the peptide is selected from any one of SEQ ID NO: 98 to 255, and wherein the cancer is selected from the group consisting of: non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

[0038] In another variant, the immune response includes a cytotoxic T-cell response.

[0039] In one embodiment, the present invention provides a method for preparing T cells using statin in accordance with the methods described herein. In another embodiment, the present invention provides a method for preparing T cells by activating T cells in the presence of statin.

[0040] In another embodiment, the present invention relates to a method for preparing T cells for immunotherapy, the method comprising the steps of: activating the T cells in the presence of statin; transducing the activated T cells with a vector of the present invention, wherein the vector is pseudotyped by a vesicular stomatitis virus (VSV-G) envelope protein; and amplifying the transduced T cells.

[0041] In another variant, these T cells may include αβ T cells, γδ T cells, and / or natural killer T cells.

[0042] In another variant, statin may be selected from atorvastatin, cerivastatin, dalvastatin, fluindostatin, fluvastatin, mevastatin, pravastatin, simvastatin, velostatin, and rosuvastatin.

[0043] In one embodiment, the present invention relates to a method for preparing T cells for immunotherapy, the method comprising the steps of: activating the T cells; transducing the activated T cells using the vector of the present invention; and expanding the transduced T cells.

[0044] In another state, activation can occur in the presence of both anti-CD3 and anti-CD28 antibodies.

[0045] In another state, the amplification can be performed in the presence of IL-7 and IL-15. Simple Explanation of the Diagram

[0046] Figure 1 illustrates a γδ T cell manufacturing procedure according to one embodiment of the present invention. γδ T cell manufacturing may include: collecting or obtaining leukocytes or PBMCs (e.g., leukocyte ablation products); consuming αβ T cells from PBMCs or leukocyte ablation products, followed by activation, transduction, and expansion of γδ T cells.

[0047] Figure 2 illustrates a transduction strategy using open reading frame (ORF) shuffling according to some embodiments of the present invention.

[0048] Figure 3 illustrates a lentivirus construct according to some embodiments of the present invention.

[0049] Figure 4 shows lentiviruses pseudotyped with RD114TR for transducing γδ T cells after activation with zoledronic acid, IL-2, and IL-15 for 3 or 6 days. Transduction efficiency was assessed using antibodies specific to TCR (Vβ8) and CD8 (CD8α) via flow cytometry.

[0050] Figure 5A shows a structure according to one embodiment of the present invention.

[0051] Figure 5B illustrates a structure according to another embodiment of the present invention.

[0052] Figure 5C shows a structure according to another embodiment of the present invention.

[0053] Figure 5D shows a structure according to another embodiment of the present invention.

[0054] Figure 6A illustrates a structure according to another embodiment of the present invention.

[0055] Figure 6B illustrates a structure according to another embodiment of the present invention.

[0056] Figure 7 shows a schematic diagram of a structure according to some embodiments of the present invention.

[0057] Figure 8A shows a structure according to one embodiment of the present invention.

[0058] Figure 8B shows a structure according to another embodiment of the present invention.

[0059] Figure 8C shows a structure according to another embodiment of the present invention.

[0060] Figure 8D shows a structure according to another embodiment of the present invention.

[0061] Figure 9A shows a schematic diagram of a structure according to some embodiments of the present invention.

[0062] Figure 9B shows a schematic diagram of a structure according to some embodiments of the present invention.

[0063] Figure 10 shows %CD8+TCR+γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control.

[0064] Figure 11 shows the median fluorescence intensity (MFI) of CD8 and TCR in γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control.

[0065] Figure 12 illustrates the tumor-killing activity of γδ T cells obtained from donor 3 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high-antigen-expressing tumor cell lines (e.g., UACC257 (top assay combination)) or low-antigen-expressing tumor cell lines (e.g., U2OS (bottom assay combination)), as determined by Incucyte cytotoxicity assays. Target-only and non-transduced cells served as controls.

[0066] Figures 13A through 13C show the amount of interferon (IFN)-γ cryptic in donor γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high antigen-expressing tumor cell lines (e.g., UACC257 (Figure 13A)), low antigen-expressing tumor cell lines (e.g., U2OS (Figure 13B)), or antigen-negative tumor cell lines (e.g., MCF-7 (Figure 13C)). Untransduced cells served as controls.

[0067] Figure 14 illustrates the tumor-killing activity of γδ T cells obtained from donor 4 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE in high-antigen-expressing tumor cell lines (e.g., UACC257 (top assay combination)) or low-antigen-expressing tumor cell lines (e.g., U2OS (bottom assay combination)), as determined by Incucyte cytotoxicity assays. Target and untransduced cells served as controls only.

[0068] Figures 15A through 15C show the amount of IFN-γ cryptic in γδ T cells obtained from donor 4 transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE, in high antigen-expressing tumor cell lines (e.g., UACC257 (Figure 15A)), low antigen-expressing tumor cell lines (e.g., U2OS (Figure 15B)), or antigen-negative tumor cell lines (e.g., MCF-7 (Figure 15C)). Untransduced cells served as controls.

[0069] Figure 16 shows the copy number of viral vectors in γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Untransduced cells serve as controls.

[0070] Figures 17A and 17B show the fold expansion of γδ T cells obtained from donor 3 (Figure 17A) or donor 4 (Figure 17B) transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as controls.

[0071] Figure 18A illustrates the memory phenotype of γδ T cells as determined by flow cytometry according to some embodiments of the present invention.

[0072] Figure 18B shows the memory phenotype of γδ T cells transduced with viral vectors containing PTE.CD8.TCR.WPRE, PTE.WPRE, PTE.Fn.WPRE, or TPE.WPRE. Non-transduced (NT) cells serve as a control.

[0073] Figure 19 shows a comparison of transduction efficiency between γδ T cells transduced with a single lentiviral vector (LV) containing PTE.CD8.TCR.WPRE (detection combination B (120 µl) and detection combination C (240 µl)) and with two separate lentiviral vectors (increased amounts of viral vectors, e.g., 120 µl each of R11KE.WPRE and CD8,WPRE (detection combination D) and 240 µl each of R11KE.WPRE and CD8,WPRE (detection combination E)). Non-transduced (NT) cells served as a control.

[0074] Figure 20 illustrates the enhanced transduction efficiency in γδ T cells transduced with increased amounts of viral vectors containing PTE.CD8.TCR.WPRE (e.g., 30 µl, 120 µl, and 240 µl). Untransduced cells serve as a control.

[0075] Figure 21 illustrates forced CD8 expression in CD4+ T cells obtained from self-donor 5 and donor 6 using various dilutions of a lentiviral vector (LV) that embodies the 4-in-1 construct of the present invention (e.g., LV-PTE.CD8.TCR.WPRE).

[0076] Figure 22 illustrates the detection of TCR performance in CD4+ T cells using various dilutions of the LV of the 4-in-1 construct (e.g., LV-PTE.CD8.TCR.WPRE) of the present invention.

[0077] Figure 23 shows the % target peptide / MHC complex Dextramer203 (Dex203)+ in CD4+ and / or CD8+ T cells obtained from donor 5 (top detection combo) and donor 6 (bottom detection combo) transduced by the 4-in-1 construct of this invention (e.g., LV-PTE.CD8.TCR.WPRE).

[0078] Figure 24 shows Dex203 MFI in CD4+ and / or CD8+ T cells obtained from donor 5 (top detection assembly) and donor 6 (bottom detection assembly) transduced with the 4-in-1 construct of this invention (e.g., LV-PTE.CD8.TCR.WPRE).

[0079] Figure 25 illustrates an experimental design according to one embodiment of the present invention for testing the functionality of T cells transduced using a 4-in-1 construct or a TCR construct only.

[0080] Figure 26 shows increased % IFN-γ positive cells (top detection group) and increased IFN-γ MFI (bottom detection group) in CD4-CD8α+ T cells obtained from grouped donors transduced with either a lentiviral vector containing R11KE.WPRE (LV-TCR) (TCR) or a lentiviral vector containing PTE.CD8.TCR.WPRE (LV-CD8.TCR) (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor N = 4).

[0081] Figure 27 shows increased % granzyme B-positive cells (top detection group) and increased granzyme B MFI (bottom detection group) in CD4-CD8α+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor N = 3).

[0082] Figure 28 shows increased % IFN-γ positive cells (top detection group) and increased IFN-γ MFI (bottom detection group) in CD4+CD8α+ T cells obtained from self-administered LV-CD8.TCR (TCR+CD8) transduced or untransduced (NT) grouped donors. These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. (Effective cell to target cell ratio = 2:1 and donor group N = 4).

[0083] Figure 29 shows increased % granzyme B-positive cells (top detection group) and increased granzyme B MFI (bottom detection group) in CD4+CD8α+ T cells obtained from self-transduced or untransduced (NT) donors. These T cells were then co-cultured with high-target-expression UACC257 cells to compare them with T cells co-cultured with non-target-expression MCF7 cells. (Effective cell to target cell ratio = 2:1 and donor N = 4).

[0084] Figure 30 shows increased % IFN-γ-positive cells (top detection group) and increased IFN-γ MFI (bottom detection group) in CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor N = 4).

[0085] Figure 31 shows increased % granzyme B-positive cells (top detection group) and increased granzyme B MFI (bottom detection group) in CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor N = 3 in each group).

[0086] Figure 32 shows increased IFN-γ cryptic expression in CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells, UACC257 cells, and MCF7 cells served as controls. (Effective cell to target cell ratio = 2:1 and donor N = 4).

[0087] Figure 33 shows increased IFN-γ cryptic expression in CD3+ T cells obtained from individual donors 5, 6, 7, and 8 transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8). These T cells were then co-cultured with high-target-expressing UACC257 cells to compare them with T cells co-cultured with non-target-expressing MCF7 cells. Non-transduced (NT) cells, UACC257 cells only, and MCF7 cells only served as controls. (Effective cell to target cell ratio = 2:1).

[0088] Figure 34 shows the percentages of CD25+ cells (top panel), CD69+ cells (middle panel), and human low-density lipoprotein receptor (hLDLR)+ cells (bottom panel) in CD3+CD4+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Pre-activated cells, cells activated without atorvastatin or DMSO (controls), and DMSO serve as controls.

[0089] Figure 35 shows the percentages of CD25+ cells (top assay), CD69+ cells (middle assay), and hLDLR+ cells (bottom assay) in CD3+CD8+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Pre-activated cells, cells activated without statin or DMSO (controls), and DMSO serve as controls.

[0090] Figure 36 illustrates the titration of a lentiviral vector according to one embodiment of the present invention.

[0091] Figure 37 illustrates a T-cell manufacturing process according to one embodiment of the present invention. Implementation

[0092] As used herein, the term "self-cleaving 2A peptide" refers to a relatively short peptide (approximately 20 amino acids in length, depending on viral origin) that co-translates to prevent the formation of a normal peptide bond between glycine and the last proline, thereby causing the ribosome to jump to the next codon and cleavage the nascent peptide between Gly and Pro. Following cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while the proline is added to the N-terminus of the "downstream" protein. Self-cleaving 2A peptides can be selected from porcine cephalovirus-1 (P2A), equine rhinitis A virus (E2A), tungsten brown-winged moth virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (see, for example, Kim et al., PLOS One 6:e18556, 2011, the contents of which, including the 2A nucleic acid and amino acid sequences, are incorporated herein by reference in their entirety). By adding a linker sequence (GSG or SGSG (SEQ ID NO: 8)) before self-cleaving the 2A sequence, efficient synthesis of bioactive proteins (e.g., TCR) can be achieved.

[0093] As used herein, the term "promoter" refers to a regulatory region of DNA typically located upstream of a gene (towards the 5′ region of the sense strand) that allows for gene transcription. A promoter contains a specific DNA sequence and a responder recognized by proteins called transcription factors. These factors bind to the promoter sequence, thereby selecting an RNA polymerase to synthesize RNA from the coding region of the gene. For example, promoter sequences used herein may be selected from cytomegalovirus (CMV) promoters, phosphoglycerate kinase (PGK) promoters, myelin basic protein (MBP) promoters, glial fibrillary acidic protein (GFAP) promoters, modified MoMuLV LTR (MNDU3) containing myeloproliferative sarcoma virus enhancers, ubiquitin C promoters, EF-1 α promoters, or murine stem cell virus (MSCV) promoters.

[0094] As used herein, the term "constitutive promoter" can include regulatory sequences that guide the transcription of genes in most cells or tissues for most of the time. In some non-limiting embodiments, constitutive promoters may be selected from the group consisting of: MSCV promoter, ubiquitin C (Ubc) promoter, CMV promoter, EF-1 α promoter, PGK promoter, β-actin promoter, and ROSA26 promoter.

[0095] In some embodiments, the promoter may be an inducible promoter. The activity of an inducible promoter may increase or decrease in response to a signal. For example, an inducible promoter may promote transcription in response to the presence of a signal such as T cell activation or isopropyl β-D-1-thiogalactopyranoside (IPTG). An inducible promoter may promote transcription in response to the absence of a signal such as sulfate. In either case, the amount of transcription may or may not be proportional to the amount of signal or the absence of signal. Examples of inducible promoters suitable for prokaryotic host cells may include (but are not limited to) NFAT, CD69, lac, tac, trc, trp, pho, recA, tetA, nar, phage PL, cspA, T7, and PBAD promoters (see Terpe K. 2006 Appl. Microbiol. Biotechnol. 72:211; the contents of the references are incorporated herein by reference in their entirety).

[0096] In some embodiments, an inducible promoter may include a nuclear factor of activated T cell (NFAT) / AP1 transcriptional response element (TRE). Upon recognition of a homologous peptide / MHC1 complex, NFAT may undergo a Ca2+-dependent translocation to the nucleus, whereby NFAT promotes transcription of genes protecting NFAT TREs. Suitable NFAT TREs are well known in this art and include the human IL2 promoter NFAT TRE (Macian et al. (2001) Oncogene, April 30, 2001; 20(19):2476-89). Zhang et al. ("Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the Immunotherapy of Metastatic Melanoma", Clin. Cancer Res. 21:2278-2288, 2015) described the application of genetically engineered human tumor-infiltrating lymphocytes (TILs) with a hidden single-stranded IL12, driven by an inducible NFAT promoter, in clinical trials. The contents of these cited references are incorporated herein by reference in their entirety.

[0097] In some embodiments, inducible promoters may include, for example, the CD69 promoter disclosed in U.S. Patent 5,759,805, the contents of which are incorporated herein by reference in their entirety. CD69 may be present in these newly synthesized cell-surface activating molecules induced early on activated T cells. CD69 expression can be observed within 60 minutes of T cell stimulation, but may not appear in dormant cells. CD69 expression can also be induced on thymocytes, B cells, natural killer (NK) cells, and neutrophils. Four non-coding regions, known as CNS1 to CNS4, located within 50 kb upstream of the mouse CD69 promoter, are useful for the developmental and temporal control of CD69 activation on T cells and B cells. The CNS2 region may act as an intensity enhancer. Kulemzin et al. ("Design and analysis of stably integrated reporters for inducible transgene expression in human T cells and chimeric antigen receptor (CAR) NK cell lines", BMC Medical Genomics 2019, 12(Suppl 2):44, 88-95; references are incorporated herein by reference in their entirety) describe that, in the case of native T cells, activated inducible CD69 promoter variants provide the highest fold induction. This promoter can therefore be used to express proteins in activated but not dormant human T cells or CAR T cells.

[0098] In some embodiments, an inducible promoter may be an IPTG-inducible promoter. An IPTG-inducible promoter may refer to any polynucleotide sequence that promotes transcription in response to IPTG or any other lactose derivative (e.g., allolactose) that promotes transcription of the lactose operon. Many examples of IPTG-inducible promoters are known in the art, including (but not limited to) tac (e.g., tacI, tacII, etc.) promoters, lac promoters and their derivatives (e.g., lacUV5, taclac, etc.).

[0099] In one state, the expression of a 4-in-1 viral vector (e.g., a lentiviral vector) containing sequences encoding CD8 α, CD8 β, TCR α, and TCR β chains can be driven by a constitutive or inducible promoter. For example, Figure 5A shows a 4-in-1 viral vector containing PTE CD8 TCR WPRE (SEQ ID NO: 94), which has codon-optimized sequences encoding CD8 α (SEQ ID NO: 12) and CD8 β (SEQ ID NO: 13) upstream of the sequences encoding TCR (e.g., TCR R11KE α chain (SEQ ID NO: 13) and R11KE β chain (SEQ ID NO: 14)) and is driven by a constitutive MSCV promoter (SEQ ID NO: 1). The same coding sequence described above can also be driven by an inducible promoter (e.g., NFAT, CD69, or IPTG promoter).

[0100] In another variant, the expression of a 3-in-1 viral vector containing sequences encoding a fusion protein, a TCR α chain, and a TCR β chain can be driven by a constitutive or inducible promoter. For example, Figure 5B shows a viral vector containing CD8aCD4Fusion.TCR WPRE (SEQ ID NO: 256), which has: a codon-optimized sequence encoding a fusion protein, wherein the extracellular domain of CD8α is fused with the transmembrane domain and the intracellular domain of CD4; and sequences encoding the TCR R11KE α chain (SEQ ID NO: 13) and the R11KE β chain (SEQ ID NO: 14), driven by the MSCV promoter (SEQ ID NO: 1). Figure 5C shows a viral vector containing CD8bCD4Fusion.TCR WPRE (SEQ ID NO: 257). CD8bCD4Fusion.TCR WPRE has: a codon-optimized sequence encoding a fusion protein, wherein the CD8β extracellular domain is fused with the CD4 transmembrane domain and the CD4 intracellular domain; and sequences encoding the TCR R11KE α chain (SEQ ID NO: 13) and R11KE β chain (SEQ ID NO: 14), driven by the MSCV promoter (SEQ ID NO: 1). Figure 5D shows a viral vector containing CD8bCD8aFusion.TCR WPRE (SEQ ID NO: 258), which has: a sequence encoding a fusion protein in which the CD8β extracellular domain is fused with the CD8α transmembrane domain and the CD8α intracellular domain; and sequences encoding the TCR R11KE α chain (SEQ ID NO: 13) and the R11KE β chain (SEQ ID NO: 14), driven by the MSCV promoter (SEQ ID NO: 1). The same coding sequences described above can also be driven by inducible promoters (e.g., NFAT, CD69, or IPTG promoters).

[0101] In one embodiment, the expression of the 4-in-1 viral vector of the present invention can be achieved by bidirectional constitutive and / or inducible promoters. For example, Figure 6A illustrates a 4-in-1 viral vector containing PGK.CD8.EF1a.TCR (SEQ ID NO: 259), wherein PGK.CD8.EF1a.TCR has: codon-optimized sequences encoding the CD8 α and CD8 β chains located upstream of the sequences encoding the TCR R11KE α and R11KE β chains, wherein the sequences encoding the CD8 α and CD8 β chains and the sequences encoding the TCR R11KE α and R11KE β chains can be separated by bidirectional promoters (e.g., the PGK promoter and the EF-1 α promoter). The PGK promoter can be positioned at the 3' end of the codon-optimized sequences encoding the CD8 α and CD8 β chains to drive the expression of the CD8 α and CD8 β chains. The EF-1 α promoter can be located at the 5' end of the sequence encoding the TCR R11KE α chain and R11KE β chain to drive the behavior of the TCR R11KE α chain and R11KE β chain.

[0102] Figure 6B illustrates another 4-in-1 viral vector containing PGK.TCR.EF1a.CD8 (SEQ ID NO: 260). PGK.TCR.EF1a.CD8 has sequences encoding the TCR R11KE α and R11KE β chains located upstream of the codon-optimized sequences encoding the CD8 α and CD8 β chains. These sequences, along with the sequences encoding the CD8 α and CD8 β chains, can be separated by bidirectional promoters (e.g., the PGK promoter and the EF-1 α promoter). The PGK promoter can be positioned at the 3' end of the sequences encoding the TCR R11KE α and R11KE β chains to drive their expression. The EF-1 α promoter can be positioned at the 5' end of the codon-optimized sequence encoding the CD8 α and CD8 β chains to drive the behavior of the CD8 α and CD8 β chains.

[0103] Some embodiments of the present invention may include viral vectors containing sequences encoding the TCR α and TCR β chains and sequences encoding other proteins, such as intercytokines (including but not limited to IL-1, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21), IL-15 / IL-15 receptor (IL-15 receptor; IL-15R) fusion proteins, dominant-negative TGF-β receptors (DN TGFbRII), and / or the extracellular domains of transforming growth factor-β receptors. In some embodiments, these coding sequences may be driven by a promoter or a bidirectional promoter.

[0104] Figure 7 illustrates a viral vector containing sequences encoding the TCR α and TCR β chains located upstream of the sequence encoding a cytokine, wherein the sequences encoding the TCR α and TCR β chains and the sequence encoding the cytokine can be separated by bidirectional promoters. The bidirectional promoters can be aligned constitutively-constitutively, constitutively-induciblely, inducible-constitutively, or inducible-induciblely in a 5' to 3' orientation. For example, a constitutively defined promoter (e.g., the MSCV, PGK, or EF1 α promoter) can be positioned at the 3' end of the sequences encoding the TCR α and TCR β chains to drive the expression of the TCR α and TCR β chains. An induciblely defined promoter (e.g., the NFAT, CD69, or IPTG promoter) can be positioned at the 5' end of the sequence encoding the cytokine to drive the expression of the cytokine. Figure 8A illustrates an inducible NFAT promoter in which the minimal IL-2 promoter is located at the 5' end of the sequence encoding IL-12 (e.g., the IL-12 α (p35) / IL-12β (p40) fusion protein (SEQ ID NO: 261)) to drive the expression of the 12 α (p35) / IL-12β (p40) fusion protein in the viral vector shown in Figure 7. Figure 8B illustrates an inducible CD69 promoter in which the CNS1 and CNS2 enhancer elements are located at the 5' end of the sequence encoding IL-12 (e.g., the IL-12 α (p35) / IL-12β (p40) fusion protein (SEQ ID NO: 262)) to drive the expression of the 12 α (p35) / IL-12β (p40) fusion protein in the viral vector shown in Figure 7. Figure 8C illustrates an inducible NFAT promoter, wherein the minimal IL-2 promoter is located at the 5' end of a sequence encoding IL-18 (e.g., IL-18 variant 1 (SEQ ID NO: 263)) to drive the expression of IL-18 variant 1 in the viral vector shown in Figure 7. Figure 8D illustrates an inducible CD69 promoter, wherein CNS1 and CNS2 enhancer elements are located at the 5' end of a sequence encoding IL-18 (e.g., IL-18 variant 1 (SEQ ID NO: 264)) to drive the expression of IL-18 variant 1 in the viral vector shown in Figure 7.

[0105] In one embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation of CD8β-CD8α-TCRβ-TCRα. In another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation of CD8β-CD8α-TCRα-TCRβ. In yet another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation of CD8α-CD8β-TCRβ-TCRα. In yet another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation of CD8α-CD8β-TCRα-TCRβ.

[0106] In one embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation excluding TCRβ-TCRα-CD8α-CD8β. In another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation excluding TCRβ-TCRα-CD8β-CD8α. In yet another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation excluding TCRα-TCRβ-CD8α-CD8β. In yet another embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation excluding TCRα-TCRβ-CD8β-CD8α.

[0107] In one embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation of CD8β-CD8α-TCRβ-TCRα. In a non-limiting embodiment, the present invention provides a 4-in-1 structure having a 5' to 3' end orientation excluding TCRβ-TCRα-CD8α-CD8β.

[0108] In some embodiments, the viral vector of the present invention may contain sequences encoding the TCR α chain and the TCR β chain, and sequences encoding TGF-β repressors (e.g., the extracellular domain of the dominant-negative TGF β receptor (DN TGFbRII) and / or the transforming growth factor-β receptor). Figure 9A illustrates a viral vector containing sequences encoding the TCR α chain and the TCR β chain located upstream of the sequence encoding DN TGFbRII, wherein the sequences encoding the TCR α chain and the TCR β chain and the sequence encoding DN TGFbRII may be separated by bidirectional promoters. For example, Figure 9A shows that a constitutive promoter (e.g., MSCV, Ubc, CMV, EF-1 α, and PGK promoters) can be located at the 3' end of the sequence encoding the TCR α and TCR β chains to drive the expression of the TCR α and TCR β chains; and another constitutive promoter can be located at the 5' end of the sequence encoding DN TGFbRII to drive the expression of DN TGFbRII.

[0109] Alternatively, Figure 9B illustrates a viral vector containing a constitutive promoter (e.g., the MSCV, Ubc, CMV, EF-1 α, and PGK promoters) located at the 5' end of the sequence encoding DN TGFbRII, upstream of the sequences encoding the TCR α and TCR β chains, to drive the expression of DN TGFbRII, the TCR α chain, and the TCR β chain. The same coding sequences described above can also be driven by inducible promoters (e.g., the NFAT, CD69, or IPTG promoters).

[0110] As used herein, the term "cistron" refers to a slice of a DNA molecule that defines the formation of one polypeptide chain (i.e., encodes one polypeptide chain). For example, "bicistron" refers to two slices of a DNA molecule that define the formation of two polypeptide chains (i.e., encodes two polypeptide chains); "tricistron" refers to three slices of a DNA molecule that define the formation of three polypeptide chains (i.e., encodes three polypeptide chains); and so on.

[0111] As used herein, the term "polycistronic RNA" or "polycistronic RNA" refers to RNA containing genetic information for translation into several proteins. In contrast, monocistronic RNA contains genetic information for translation into only a single protein. In the context of this invention, the polycistronic RNA transcribed by the lentiviruses in Examples 2 to 4 can be translated into four proteins (4-in-1): the TCRα chain, the TCRβ chain, the CD8α chain, and the CD8β chain; or into two proteins (2-in-1): the TCRα chain and the TCRβ chain or the CD8α chain and the CD8β chain.

[0112] As used herein, the term "tandem arrangement" refers to the arrangement of genes in a nucleic acid sequence, one after another, or consecutively following one another. Genes are linked together consecutively in the nucleic acid sequence, and the coding strands (meaningful strands) of each gene are linked together in the nucleic acid sequence.

[0113] As used herein, the term "meaningful strand" refers to the DNA strand of a gene that is translated or can be translated into a protein. When a gene is oriented in a "meaningful orientation" relative to the promoter in a nucleic acid sequence, the "meaningful strand" is located at the 5' end downstream of the promoter, where the first codon of the nucleic acid encoding the protein is close to the promoter and the last codon is far from the promoter.

[0114] As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the ability to encapsulate into a viral vector particle, and encodes at least one exogenous nucleic acid. Vectors and / or particles can be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Numerous forms of viral vectors are known in this art. The term "virosome" is used to refer to a single infectious viral particle. "Viral vector," "viral vector particle," and "viral particle" also refer to a complete viral particle with a DNA or RNA core and protein coat as if it were present outside the cell. For example, viral vectors may be selected from adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviviruses, rod-shaped viruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, or picornaviruses.

[0115] The terms "T cell" or "T lymphocyte" are technically recognized and intended to include thymocytes, naïve T lymphocytes, immature T lymphocytes, mature T lymphocytes, dormant T lymphocytes, or activated T lymphocytes. The illustrative group of T cells suitable for use in a particular embodiment includes (but is not limited to) helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, natural killer T cells, T cells expressing αβ TCR (αβ T cells), T cells expressing γδ TCR (γδ T cells), or any other subset of T cells. Other illustrative populations of T cells suitable for use in specific embodiments include (but are not limited to) T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, and may be further isolated by positive or negative selection techniques if necessary.

[0116] The terms "statin," "vastatin," or, as used interchangeably herein, "3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor," refer to agents that inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme is involved in the conversion of HMG-CoA to mevalonate, a step in cholesterol biosynthesis. This inhibition is readily determined using standard tests familiar to those skilled in the art.

[0117] The preferred statins that can be used according to the present invention include: atorvastatin, disclosed in U.S. Patent No. 4,681,893; atorvastatin calcium, disclosed in U.S. Patent No. 5,273,995; sivastatin, disclosed in U.S. Patent No. 5,502,199; davastatin, disclosed in U.S. Patent No. 5,316,765; fludostatin, disclosed in U.S. Patent No. 4,915,954; fluvastatin, disclosed in U.S. Patent No. 4,739,073; and lovastatin, disclosed in U.S. Patent No. 4,739,073. The contents of each of these references are incorporated herein by reference in their entirety. The references include: U.S. Patent No. 4,231,938; mevastatin, disclosed in U.S. Patent No. 3,983,140; pravastatin, disclosed in U.S. Patent No. 4,346,227; simvastatin, disclosed in U.S. Patent No. 4,444,784; velotasone, disclosed in U.S. Patent Nos. 4,448,784 and 4,450,171; and rosuvastatin, disclosed in U.S. Patent Nos. 6,858,618 and 7,511,140. Representative 3-hydroxy-3-methylpentadiazine coenzyme A reductase inhibitors may include atorvastatin, atorvastatin calcium (also known as Liptor®), lovastatin (also known as Mevacor®), pravastatin (also known as Pravachol®), simvastatin (also known as Zocor®), and rosuvastatin.

[0118] In one embodiment, the invention relates to the activation, transduction, and / or expansion of T cells (e.g., tumor-infiltrating lymphocytes, CD8+ T cells, CD4+ T cells, and γδ T cells) that can be used for transgenic expression. In another embodiment, the invention relates to the activation, transduction, and expansion of γδ T cells when α-TCR and / or β-TCR-positive cells are consumed.

[0119] In one state, γδ T cells can be isolated from complex samples cultured in vitro. In another state, the entire PBMC population can be activated and expanded, including specific cell populations such as monocytes, αβ T cells, B cells, and NK cells that have not been previously depleted. In another state, an enriched γδ T cell population can be generated prior to its specific activation and expansion. In yet another state, γδ T cell activation and expansion can be performed in the absence of native or engineered APCs. In yet another state, the isolation and expansion of γδ T cells from tumor specimens can be performed using: immobilized γδ T cytokinins, including antibodies specific to γδ TCRs; and other γδ TCR activators, including lectins. In yet another state, the isolation and expansion of γδ T cells from tumor specimens can be performed in the absence of: γδ T cytokinins, including antibodies specific to γδ TCRs; and other γδ TCR activators, including lectins.

[0120] In one state, γδ T cells were isolated from leukocyte isolates of a subject (e.g., a human subject). In another state, γδ T cells were not isolated from peripheral blood mononuclear cells (PBMCs).

[0121] In a single-state sample, isolated γδ T cells can rapidly proliferate in response to contact with one or more antigens. Some γδ T cells (such as Vγ9Vδ2+ T cells) can rapidly proliferate in vitro in response to contact with certain antigens (such as terpene pyrophosphates, alkylamines, and metabolites or microbial extracts from tissue culture). Stimulated γδ T cells can exhibit numerous antigen presentation, co-stimulatory, and adhesion molecules that promote γδ T cell isolation from complex samples. γδ T cells in complex samples can be stimulated in vitro with at least one antigen for 1, 2, 3, 4, 5, 6, 7 days, or another suitable time period. Stimulation of γδ T cells with appropriate antigens can lead to the in vitro expansion of γδ T cell populations.

[0122] Non-limiting examples of antigens that can be used to stimulate the amplification of γδ T cells from complex samples in vitro may include the following: terpene pyrophosphates (such as isopentenyl pyrophosphate (IPP)), alkylamines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), and geranylgeranyl pyrophosphate. pyrophosphate (GGPP), isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-methoxy-1-butyl-pyrophosphate (TUBAg 1), X-pyrophosphate (TUBAg 2), 3-methoxy-1-butyl-uridine triphosphate (TUBAg 3), 3-methoxy-1-butyl-deoxythymidine triphosphate (TUBAg 4), monoethylalkylamine, allyl pyrophosphate, crotonyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, crotonyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, secondary butylamine, iso-pentylamine, and nitrogen-containing diphosphates.

[0123] Activation and expansion of γδ T cells can be performed using the activation and co-stimulatory agents described herein to trigger specific γδ T cell proliferation and sustained populations. In one state, activation and expansion of γδ T cells from different cultures can achieve different subsets of monoclonal or mixed multiclonal populations. In another state, different activators can be used to identify agents that provide specific γδ activation signals. In yet another state, agents that provide specific γδ activation signals can be different monoclonal antibodies (MAbs) targeting γδ TCRs. In yet another state, co-stimulatory agents that help trigger specific γδ T cell proliferation without inducing cellular energy or apoptosis can be used. These co-stimulatory agents may include ligands that bind to receptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In another variant, the co-stimulatory agent may be an antibody specific to unique epitopes on CD2 and CD3 molecules. CD2 and CD3 may have different conformations when expressed on αβ or γδ T cells. In yet another variant, specific antibodies against CD3 and CD2 may induce different activations of γδ T cells.

[0124] The γδ T cell population can be expanded in vitro before the γδ T cells are engineered. Non-limiting examples of reagents that can be used to promote the in vitro expansion of γδ T cell populations may include anti-CD3 or anti-CD2, anti-CD27, anti-CD30, anti-CD70, anti-OX40 antibodies, IL-2, IL-15, IL-12, IL-9, IL-33, IL-18 or IL-21, CD70 (CD27 ligand), phytohaemagglutinin (PHA), concavalin A (ConA), pokeweed (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), lentil agglutinin (LCA), pea agglutinin (PSA), helix pomatia agglutinin (HPA), and vicia graminea agglutinin. Lectin (VGA) or another suitable mitogen that can stimulate T cell proliferation.

[0125] The ability of γδ T cells to recognize broadly effective antigens can be enhanced through genetic engineering. In one phenotype, γδ T cells can be engineered to provide universal allogeneic therapy that recognizes antigens selected in vivo. Genetic engineering of γδ T cells may include the stable integration of constructs, antigen-binding fragments, or lymphocyte activation domains of tumor-recognizing portions of a single receptor (such as αβ TCR, γδ TCR, chimeric antigen receptor; CAR) into the genome of isolated γδ T cells (cytokines (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β)) to enhance T cell proliferation, survival, and function both in vitro and in vivo. Genetic engineering of isolated γδ T cells may also include the deletion or disruption of gene expression of one or more intrinsic genes from the genome of isolated γδ T cells (such as MHC loci (loci)).

[0126] Engineered γδ T cells can be generated using various methods. For example, polynucleotides encoding expression cassettes containing tumor-recognizing or other types of recognition components can be stably introduced into γδ T cells by: transposon / translocase systems or virus-based gene transfer systems, such as lentiviruses or retroviruses; or other suitable methods, such as transfection, electroporation, transduction, lipid transfection, calcium phosphate (CaPO4), nanoengineered materials such as organically modified silicates (Ormosil), viral delivery methods (including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or other suitable methods). Many viral methods have been used in human gene therapy, such as those described in WO 1993020221, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of viral methods that can be used to engineer γδ T cells may include viral methods associated with γ-retroviruses, adenoviruses, lentiviruses, herpes simplex virus, vaccinia virus, vaccinia virus, or adenovirus.

[0127] In this embodiment, the structures and carriers described herein will be used in conjunction with the methods described in U.S. Patent Application No. 16 / 200,308 filed November 26, 2018, the contents of which are incorporated herein by reference in their entirety.

[0128] In this context, "virus" refers to both naturally occurring viruses and artificial viruses. Viruses according to some embodiments of the invention may be enveloped or non-enveloped viruses. Microviruses (such as AAV) are examples of non-enveloped viruses. In a preferred embodiment, the virus may be an enveloped virus. In a preferred embodiment, the virus may be a retrovirus, and particularly a lentivirus. Viral envelope proteins that can promote viral infection of eukaryotic cells may include HIV-1-derived lentiviral vectors (LVs) pseudotyped with envelope glycoproteins (GPs) from vesicular stomatitis virus (VSV-G), modified feline intrinsic retrovirus (RD114TR) (SEQ ID NO: 97), and modified gibberish ape leukemia virus (GALVTR). These envelope proteins can efficiently promote the entry of other viruses, including adeno-associated virus (AAV) such as microviruses, thereby demonstrating the broad-sense efficiency of these viruses. For example, other viral envelope proteins can be used, including Moloney murine leukemia virus (MLV) 4070 env (as described in Merten et al. J. Virol. 79:834-840, 2005; the content of the reference is incorporated herein by reference), RD114 env, chimeric envelope proteins RD114pro or RDpro (which is an RD114-HIV chimera constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / protein shell (MA / CA) cleavage sequence, as described in Bell et al. Experimental Biology and Medicine (2010; 235: 1269–1276); the content of the reference is incorporated herein by reference), baculovirus GP64 env (as described in Wang et al. J. Virol. (81:10869-10878, As described in (2007); the contents of the references are incorporated herein by reference) or GALV env (such as described in Merten et al. J. Virol. (79:834-840, 2005); the contents of the references are incorporated herein by reference) or derivatives thereof.

[0129] The embodiments of the present invention are based on the discovery that a single lentiviral cassette can be used to generate a single lentiviral vector from a single polycistronic mRNA that expresses at least four individual monomeric proteins representing two different dimers, so as to co-express these dimers on the cell surface. For example, integration of a single copy of the lentiviral vector is sufficient to transform γδ T cells to co-express TCRαβ and CD8αβ.

[0130] In one embodiment, the present invention relates to a vector containing a polycistronic cassette, wherein the polycistronic cassette is contained within a single vector capable of expressing more than one, two, three, four, five, or six genes, wherein the polypeptides encoded by these genes can interact with each other or form dimers. The dimers can be homodimers (i.e., two identical proteins forming a dimer) or heterodimers (i.e., two structurally different proteins forming a dimer).

[0131] In one state, the lentiviral vector may contain a first nucleotide sequence S1 encoding protein Z1, a second nucleotide sequence S2 encoding protein Z2, a third nucleotide sequence S3 encoding protein Y1, and a fourth nucleotide sequence S4 encoding protein Y2, wherein Z1 and Z2 form a first dimer and Y1 and Y2 form a second dimer, wherein the first dimer Z1Z2 is different from the second dimer Y1Y2.

[0132] In one lentiviral vector, the first lentiviral vector may contain a bicistronic cassette encoding the dimer Z1Z2 (2-in-1), and the second lentiviral vector may contain a bicistronic cassette encoding the dimer Y1Y2 (2-in-1). In the 2-in-1 vector, S1 and S2 can be tandemly arranged in a 5' to 3' orientation between S1 and S2 or between S2 and S1. Similarly, in the 2-in-1 vector, S3 and S4 can be tandemly arranged in a 5' to 3' orientation between S3 and S4 or between S4 and S3. Z1 and Z2 or Y1 and Y2 can be separated by one or more self-cleaved 2A peptides.

[0133] In another variant, a single lentiviral vector (4-in-1) can encode two different dimers, Z1Z2 and Y1Y2, wherein Z1, Z2, Y1, and Y2 can be separated by one or more self-cleaved 2A peptides. For example, S1, S2, S3, and S4 can be selected from the following 5' to 3' oriented tandem arrangements: S1-S2-S3-S4, S1-S2-S4-S3, S1-S3-S2-S4, S1-S3-S4-S2, S1-S4-S3-S2, S1-S4-S2-S3, S2-S1-S3-S4, S2-S1-S4-S3, S2-S3-S1-S4, S2-S3-S4-S1, S2-S4 -S3-S1, S2-S4-S1-S3, S3-S1-S2-S4, S3-S1-S4-S2, S3-S2-S1-S4, S3-S2-S4-S1, S3-S4-S1-S2, S3 -S4-S2-S1, S4-S1-S2-S3, S4-S1-S3-S2, S4-S2-S1-S3, S4-S2-S3-S1, S4-S3-S1-S2 or S4-S3-S2-S1.

[0134] In the first state, the dimer Z1Z2 can be a TCR with both TCRα and TCRβ chains.

[0135] In one embodiment, TCRs and antigen-binding proteins that can be used with the constructs, methods, and embodiments described herein include, for example, the TCRs and antigen-binding proteins listed in Table 2 (SEQ ID NO: 13 to 90) and the TCRs and antigen-binding proteins described in the following: U.S. Publication No. 20170267738, U.S. Publication No. 20170312350, U.S. Publication No. 20180051080, U.S. Publication No. 20180164315, U.S. Publication No. 20180161396, U.S. Publication No. 20180162922, U.S. Publication No. 20180273602, U.S. Publication No. 20190016801, U.S. Publication No. 20190002556, U.S. Publication No. 201 U.S. Patents 90135914, 10,538,573, 10,626,160, 20190321478, 20190256572, 10,550,182, 10,526,407, 20190284276, 20190016802, and 10,583,573, the contents of each of these publications and the sequence listed therein are incorporated herein by reference in their entirety.

[0136] In another state, the dimer Z1Z2 may be a TCRα chain and a TCRβ chain selected from the following: R11KE (SEQ ID NO: 13 and 14), R20P1H7 (SEQ ID NO: 15 and 16), R7P1D5 (SEQ ID NO: 17 and 18), R10P2G12 (SEQ ID NO: 19 and 20), R10P1A7 (SEQ ID NO: 21 and 22), R4P1D10 (SEQ ID NO: 23 and 24), R4P3F9 (SEQ ID NO: 25 and 26), R4P3H3 (SEQ ID NO: 27 and 28), R36P3F9 (SEQ ID NO: 29 and 30), R52P2G11 (SEQ ID NO: 31 and 32), R53P2A9 (SEQ ID NO: 33 and 34), R26P1A9 (SEQ ID NO: 13 and 24 ... R26P2A6 (SEQ ID NO: 35 and 36), R26P3H1 (SEQ ID NO: 39 and 40), R35P3A4 (SEQ ID NO: 41 and 42), R37P1C9 (SEQ ID NO: 43 and 44), R37P1H1 (SEQ ID NO: 45 and 46), R42P3A9 (SEQ ID NO: 47 and 48), R43P3F2 (SEQ ID NO: 49 and 50), R43P3G5 (SEQ ID NO: 51 and 52), R59P2E7 (SEQ ID NO: 53 and 54), R11P3D3 (SEQ ID NO: 55 and 56), R16P1C10 (SEQ ID NO: 57 and 58), R16P1E8 (SEQ ID NO: 59 and 60), R17P1A9 (SEQ ID NO: 61 and 62), R17P1D7 (SEQ ID NO: 63 and 64), R17P1G3 (SEQ ID NO: 65 and 66), R17P2B6 (SEQ ID NO: 67 and 68), R11P3D3KE (SEQ ID NO: 69 and 70), R39P1C12 (SEQ ID NO: 71 and 72), R39P1F5 (SEQ ID NO: 73 and 74), R40P1C2 (SEQ ID NO: 75 and 76), R41P3E6 (SEQ ID NO: 77 and 78), R43P3G4 (SEQ ID NO: 79 and 80), R44P3B3 (SEQ ID NO: 81 and 82), R44P3E7 ...9 and 60), R17P1D7 (SEQ ID NO: 61 and 62), R17P1G3 (SEQ ID NO: 65 and 66), R17P2B6 (SEQ ID NO:83 and 84), R49P2B7 (SEQ ID NO: 85 and 86), R55P1G7 (SEQ ID NO: 87 and 88), or R59P2A7 (SEQ ID NO: 89 and 90). In one state, the sequences exhibit at least about 90%, at least about 95%, or at least about 98% of any one of SEQ ID NO: 13 to 90.

[0137] Table 1 shows examples of TCR binding to peptides when they are complexed with MHC molecules. Table 1 TCR Name Peptide (Name / Sequence / SEQ ID NO:) R20P1H7, R7P1D5, R10P2G12 MAG-003 (KVLEHVVRV) (SEQ ID NO: 215) R10P1A7 IGF2BP3-001 (KIQEILTQV) (SEQ ID NO: 123) R4P1D10, R4P3F9, R4P3H3 COL6A3-002 (FLLDGSANV) (SEQ ID NO: 238) R36P3F9, R52P2G11, R53P2A9 DCAF4L2-001 (ILQDGQFLV) (SEQ ID NO: 193) R26P1A9, R26P2A6, R26P3H1, R35P3A4, R37P1C9, R37P1H1, R42P3A9, R43P3F2, R43P3G5, R59P2E7 MAGEA1-003 (KVLEYVIKV) (SEQ ID NO: 202) R11KE, R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, R17P2B6, R11P3D3KE PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) R39P1C12, R39P1F5, R40P1C2, R41P3E6, R43P3G4, R44P3B3, R44P3E7, R49P2B7, R55P1G7, R59P2A7 SPINK2-001 (ALSVLRLAL) (SEQ ID NO: 248)

[0138] In one embodiment, tumor-associated antigen (TAA) peptides that can be used with the methods and examples described herein include, for example, the TAA peptides listed in Table 3 and the TAA peptides described in the following: U.S. Publication No. 20160187351, U.S. Publication No. 20170165335, U.S. Publication No. 20170035807, U.S. Publication No. 20160280759, U.S. Publication No. 20160287687, U.S. Publication No. 20160346371, U.S. Publication No. 20160368965, U.S. Publication No. 20170022251, U.S. Publication No. 20170002055, U.S. Publication No. 20170022251, U.S. Publication No. 20170002055, etc. Case No. 20170029486, US Public Case No. 20170037089, US Public Case No. 20170136108, US Public Case No. 20170101473, US Public Case No. 20170096461, US Public Case No. 20170165337, US Public Case No. 20170189505, US Public Case No. 20170173132, US Public Case No. 20170296640, US Public Case No. 20170253633, US Public Case No. 20170260249, US Public Case No. 20180051080 U.S. Publication Nos. 20180164315, 20180291082, 20180291083, 20190255110, U.S. Patents 9,717,774, 9,895,415, 20190247433, 20190292520, 20200085930, 10,336,809, 10,131,703, 10,081,664, 10,093,715, 10,583,573, and 20200085930, the contents of each of these publications, sequences, and the lists of sequences described therein are incorporated herein by reference in their entirety.

[0139] In another morphology, the dimer Z1Z2 can be a T-cell dimer communication module, such as CD3δ / ε, CD3γ / ε and CD247 ζ / ζ or ζ / η, a dimer of the TCRα variable region (Vα) and the TCRβ variable region (Vβ), a dimer of the immunoglobulin heavy chain variable region (VH) and the immunoglobulin light chain variable region (VL), a dimer of Vα and VH, a dimer of Vα and VL, a dimer of Vβ and VH, or a dimer of Vβ and VL.

[0140] In another state, Y1Y2 may be the CD8α chain and CD8β chain or any other suitable dimer membrane receptor, preferably the dimer expressed in CD8+ T cells and / or CD4+ T cells.

[0141] The furin line contains a preproprotein convertase similar to Bacillus subtilis protease, whose natural matrix includes specific serum proteins and growth factor receptors, such as an insulin-like growth factor receptor. The consistent sequence for furin cleavage is RXXR (SEQ ID NO: 7), but the actual cleavage probability depends on the matrix tertiary structure and the amino acids directly surrounding the recognition site. Adding a furin cleavage site along with a linker sequence (GSG or SGSG (SEQ ID NO: 8)) can achieve highly efficient gene expression.

[0142] In one state, the nucleotide sequences of the tandemly arranged furin linker-2A peptide can be located between Z1 and Z2, Z1 and Y1, Z1 and Y2, Z2 and Y1, Z2 and Y2, and / or Y1 and Y2. The furin may have a consistent RXXR sequence (SEQ ID NO: 7), for example, RAKR (SEQ ID NO: 2). The linker sequence may be GSG or SGSG (SEQ ID NO: 8). The 2A peptide may be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), F2A (SEQ ID NO: 6), or any combination thereof.

[0143] In another state, the nucleotide sequence of the tandemly arranged linker-2A peptide can be located between Z1 and Z2, Z1 and Y1, Z1 and Y2, Z2 and Y1, Z2 and Y2, and / or Y1 and Y2. This linker sequence can be GSG or SGSG (SEQ ID NO: 8). The 2A peptide can be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), F2A (SEQ ID NO: 6), or any combination thereof.

[0144] In one state, engineered (or transduced) γδ T cells can be expanded in vitro without stimulation by antigen-presenting cells or aminobisphosphonates. The antigen-responsive engineered T cells of this invention can be expanded both in vitro and in vivo. In another state, an active population of engineered γδ T cells of this invention can be expanded in vitro without stimulation by antigen-presenting cells, antigenic peptides, non-peptide molecules, or small molecule compounds (such as aminobisphosphonates), but using specific antibodies, cytokines, mitogens, or fusion proteins (such as IL-17 Fc fusion protein, MICA Fc fusion protein, and CD70 Fc fusion protein). Examples of antibodies that can be used to expand γδ T cell populations include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies. Examples of interleukins include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7, and / or IL-33. Examples of mitogens include CD70, ligands of human CD27, phytohaemagglutinin (PHA), concavalin A (ConA), pokeweed mitogen (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), lecithin (LCA), pisum sativum agglutinin (PSA), and helix agglutinin. Pomatia agglutinin (HPA), Vicia graminea lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation. In another state, engineered γδ T cell populations can be expanded in less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, or less than 6 days. In another state, engineered γδ T cell populations can be expanded in approximately 7 days to approximately 49 days, approximately 7 days to approximately 42 days, approximately 7 days to approximately 35 days, approximately 7 days to approximately 28 days, approximately 7 days to approximately 21 days, or approximately 7 days to approximately 14 days.

[0145] In another embodiment, the present invention provides a method for in vitro expansion of a population of engineered γδ T cells for receptive transfer therapy. The engineered γδ T cells of the present invention can be expanded in vitro. The engineered γδ T cells of the present invention can be expanded in vitro without APC activation or without co-culture with APC and without aminophosphates.

[0146] Treatment

[0147] Compositions containing engineered γδ T cells as described herein may be administered for preventative and / or therapeutic purposes. In therapeutic applications, the pharmaceutical composition may be administered to a subject suffering from a disease or condition in an amount sufficient to cure or at least partially halt the symptoms of the disease or condition. Engineered γδ T cells may also be administered to reduce the likelihood of the condition developing, shrinking, or worsening. The effective amount of engineered γδ T cell populations used for therapeutic purposes may be based on the severity and course of the disease or condition, prior therapy, the subject's health status, weight, and / or response to the drug and / or changes in the treating physician's judgment.

[0148] The compositions of the present invention may also include one or more adjuvants. Adjuvants are substances that nonspecifically enhance or confer an immune response (e.g., an immune response to an antigen induced by CD8-positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the present invention. Suitable adjuvants include (but are not limited to) 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived from flagellin, FLT3 ligands, GM-CSF, IC30, IC31, imiquimod (ALDARA®), rasimod, ImuFact IMP321, interleukins such as IL-2, IL-13, IL-21, interferon α or interferon β or their pegylated derivatives, IS Patch, ISS, ISCMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophospholipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® carrier system, poly(lactide co-glycolide) [PLG]-based and polydextrose microparticles, lactoferrin SRL172, virions and other virus-like particles, YF-17D, VEGF scavengers, R848, β-glucan, Pam3Cys, Aquila QS21 stimulator (derived from saponins, *Bacillus subtilis* extracts and synthetic bacterial cell wall mimics), and other proprietary adjuvants (such as Ribi's Detox, Quil, or Superfos). Adjuvants such as Freund or GM-CSF are preferred. Several immunoadjuvants specifically for dendritic cells (e.g., MF59) and their preparation have been previously described (Allison and Krummel, 1995). Cytokines can also be used. A variety of cytokines are directly linked to influence dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into effective antigen-presenting cells against T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is specifically incorporated herein by reference in its entirety) and acting as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-α, IFN-β) (Gabrilovich et al., 1996).

[0149] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine formulation. Unbound by theory, CpG oligonucleotides act by activating the innate (adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide range of antigens, including peptide or protein antigens in both prophylactic and therapeutic vaccines, live or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates. More importantly, this activation enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and the production of cytotoxic T-lymphocytes (CTLs), even in the absence of CD4 T cell assistance. The TLR9-induced TH1 bias is maintained even in the presence of vaccine adjuvants that normally promote TH2 bias, such as alum or incomplete Freund's adjuvant (IFA). CpG oligonucleotides exhibit enhanced adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations. CpG oligonucleotides are particularly essential for inducing strong responses when the antigen is relatively weak. CpG oligonucleotides also accelerate immune responses and allow for reductions in antigen dosage by approximately two orders of magnitude, exhibiting significant antibody responses to some experimental full-dose vaccines without CpG (Krieg, 2006). US 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens for inducing antigen-specific immune responses. The CpG TLR9 antagonist is Mologen's (Berlin, Germany) double-stem-loop immunomodulator (dSLIM), a preferred component of the pharmaceutical compositions of this invention. Other TLR-binding molecules, such as RNA-binding TLR 7, TLR 8, and / or TLR 9, may also be used.

[0150] Other examples of useful adjuvants include (but are not limited to) chemically modified CpGs (e.g., CpR, Idera), dsRNA analogs such as poly(I:C) and its derivatives (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab, Bevacizumab®, celecoxib. x), NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF scavengers, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (e.g., anti-CD40, anti-TGFβ, anti-TNFα receptor), and SC58175, which may have therapeutic effects and / or act as adjuvants. In the context of this invention, the amount and concentration of useful adjuvants and additives can be readily determined by those skilled in the art without excessive experimentation.

[0151] Preferred adjuvants include anti-CD40, imiquimod, ralsimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon α, interferon β, CpG oligonucleotides and derivatives, poly-(I:C) and derivatives, RNA, sildenafil and particle formulations containing poly(lactide co-glycolide); PLG, virions and / or interleukins (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21 and IL-23.

[0152] In a preferred embodiment, the adjuvant in the pharmaceutical composition according to the invention is selected from the group consisting of: colony-stimulating factor such as granulocyte macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, rasimod, and interferon α.

[0153] In a preferred embodiment, the adjuvant in the pharmaceutical composition according to the invention is selected from the group consisting of: a colony-stimulating factor such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and ralsimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod, or ralsimod. More preferred adjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, polyICLC (Hiltonol®), and anti-CD40 mAB, or combinations thereof.

[0154] The engineered γδ T cells of this invention can be used to treat subjects with conditions requiring treatment (e.g., cancers described herein).

[0155] Methods of treating a subject's condition (e.g., pain) with γδ T cells may include administering a therapeutically effective amount of engineered γδ T cells to the subject. The γδ T cells of this invention can be administered in various ways (e.g., timing, concentration, dosage, treatment intervals, and / or formulations). Subjects may also undergo pretreatment, such as chemotherapy, radiation, or a combination of both, before receiving the engineered γδ T cells of this invention. Engineered γδ T cell populations may also be frozen or cryopreserved before administration to the subject. Engineered γδ T cell populations may include two or more cells exhibiting the same, different tumor-recognizing regions, or combinations of the same and different tumor-recognizing regions. For example, an engineered γδ T cell population may include several different engineered γδ T cells designed to recognize different antigens or different epitopes within the same antigen.

[0156] The γδ T cells of this invention can be used to treat various conditions. In one instance, the engineered γδ T cells of this invention can be used to treat cancer, including solid tumors and hematologic malignancies. Non-limiting examples of cancer include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt's lymphoma, tumor of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma), and cervical cancer. Childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, pharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer (such as non-small cell and small cell lung cancers). (e.g., lung cancer), lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma of osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma with occult primary lesions, oral cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal cavity carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, pancreatic cancer, pancreatic cancer islet cells, paranasal and nasal cavity carcinoma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma. Pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary germ cell tumor, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy-related), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0157] In one embodiment, the engineered γδ T cells of this invention can be used to treat infectious diseases. In another embodiment, the engineered γδ T cells of this invention can be used to treat infectious diseases caused by viruses. In yet another embodiment, the engineered γδ T cells of this invention can be used to treat immune diseases, such as autoimmune diseases.

[0158] Treatment using the γδ T cells of this invention can be provided to subjects before, during, and after the clinical onset of the disease. Treatment can be provided to subjects 1 day, 1 week, 6 months, 12 months, or 2 years after the clinical onset of the disease. Treatment can be provided to subjects after the clinical onset of the disease for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer. Treatment can be provided to subjects after the clinical onset of the disease for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years. Treatment may also include treating humans in clinical trials. Treatment may include administering a pharmaceutical composition comprising the engineered γδ T cells of this invention to subjects.

[0159] In another embodiment, administration of the engineered γδ T cells of the present invention to a subject can modulate the activity of intrinsic lymphocytes in the subject's body. In another embodiment, administration of engineered γδ T cells to a subject can provide antigens to intrinsic T cells and enhance the immune response. In another embodiment, the memory T cells can be CD4+ T cells. In another embodiment, the memory T cells can be CD8+ T cells. In another embodiment, administration of the engineered γδ T cells of the present invention to a subject can activate the cytotoxicity of another immune cell. In another embodiment, the other immune cell can be CD8+ T cells. In another embodiment, the other immune cell can be a natural killer T cell. In another embodiment, administration of the engineered γδ T cells of the present invention to a subject can inhibit regulatory T cells. In another embodiment, the regulatory T cells can be FOX3+ Treg cells. In another embodiment, the regulatory T cells can be FOX3- Treg cells. Non-limiting examples of cells whose activity can be regulated by the engineered γδ T cells of the present invention may include: hematopoietic stem cells; B cells; CD4; CD8; erythrocytes; leukocytes; dendritic cells, including dendritic antigen-presenting cells; leukocytes; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophilic granulocytes; T helper cells; and T killer cells.

[0160] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation is routinely used to prevent the recipient's immune system from rejecting the hematopoietic stem cells (HSCs) in the graft. In one case, donor bone marrow can be cultured in vitro using interleukin-2 (IL-2) to enhance the production of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be essential for the growth, proliferation, and differentiation of wild-type lymphocytes. Current research on the receptive transfer of γδ T cells to humans may require co-administration of γδ T cells with interleukin-2. However, both low and high doses of IL-2 can have highly toxic side effects. IL-2 toxicity is observed in multiple organs / systems, with the most pronounced effects in the heart, lungs, kidneys, and central nervous system. In another embodiment, the present invention provides a method for administering engineered γδ T cells to a subject without co-administering an initial cytokine or a modified form thereof (such as IL-2, IL-15, IL-12, IL-21). In another embodiment, engineered γδ T cells can be administered to a subject without co-administering IL-2. In yet another embodiment, engineered γδ T cells can be administered to a subject during a procedure (such as bone marrow transplantation without co-administering IL-2).

[0161] Drug administration method

[0162] One or more engineered γδ T cell populations can be administered to a subject in any order or simultaneously. If administered simultaneously, the multiple engineered γδ T cells can be provided in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., multiple intravenous infusions, subcutaneous injections (SC), injectables, or tablets). Engineered γδ T cells can be packaged together or separately in a single package or multiple packages. One or all of the engineered γδ T cells can be administered in multiple doses. If not administered simultaneously, the timing between multiple doses can be varied up to about one week, one month, two months, three months, four months, five months, six months, or about one year. In another embodiment, after administration to a subject, the engineered γδ T cells can be expanded in the subject's body or in vitro. Engineered γδ T cells can be cryopreserved to provide cells for multiple treatments using the same cell preparation. The engineered γδ T cells of the present invention and the pharmaceutical compositions comprising them can be packaged as kits. The kits may include instructions (e.g., written instructions) regarding the use of the engineered γδ T cells and the compositions comprising them.

[0163] In another embodiment, a method of treating cancer includes administering a therapeutically effective amount of engineered γδ T cells to a subject, wherein the drug administration treats cancer. In another embodiment, the therapeutically effective amount of engineered γδ T cells is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In another embodiment, the therapeutically effective amount of engineered γδ T cells is administered for at least one week. In another embodiment, the therapeutically effective amount of engineered γδ T cells is administered for at least two weeks.

[0164] The engineered γδ T cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the pharmaceutical composition containing engineered γδ T cells can be varied. For example, engineered γδ T cells can be used for prophylaxis and can be administered continuously to the subject as the condition or disease trend progresses to reduce the likelihood of the disease or condition occurring. Engineered γδ T cells can be administered to the subject as quickly as possible during or after the onset of symptoms. Administration of engineered γδ T cells can begin immediately at the onset of symptoms, within 3 hours before the onset of symptoms, within 6 hours before the onset of symptoms, within 24 hours before the onset of symptoms, within 48 hours before the onset of symptoms, or at any time during the onset of symptoms. Initial administration can be via any practical route, such as using any formulation described herein via any route described herein. In another embodiment, administration of the engineered γδ T cells of the present invention can be intravenous. One or more doses of engineered γδ T cells can be administered whenever feasible, following the onset of cancer, infectious diseases, immune disorders, sepsis, or during bone marrow transplantation, for the duration required to treat immune diseases, for example, approximately 24 to 48 hours, approximately 48 hours to 1 week, approximately 1 week to 2 weeks, approximately 2 weeks to 1 month, or approximately 1 month to 3 months. For cancer treatment, one or more doses of engineered γδ T cells can be administered many years after cancer onset and before or after other treatments. In another variant, engineered γδ T cells can be administered for at least approximately 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment can be varied for each subject.

[0165] save

[0166] In one state, γδ T cells can be prepared in a cryogenic medium and placed in a cryogenic storage unit such as a liquid nitrogen cryostat (-196°C) or an ultra-low temperature cryostat (-65°C, -80°C, -120°C, or -150°C) for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The cryogenic medium may contain dimethyl sulfoxide (DMSO) and / or sodium chloride (NaCl) and / or glucose and / or polydextrose sulfate and / or hydroyethyl starch (HES), and have a physiological pH buffer to maintain the pH between about 6.0 and about 6.5, about 6.5 and about 7.0, about 7.0 and about 7.5, about 7.5 and about 8.0, or about 6.5 and about 7.5. Cryopreserved γδ T cells can be thawed and further processed using stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. Cryopreserved γδ T cells can be thawed and genetically modified using viral vectors (including retroviruses, adeno-associated viruses (AAVs), and lentiviral vectors) or non-viral methods (including RNA, such as transposable DNA, and proteins) as described herein. Modified γδ T cells can be further cryopreserved to form cell banks in cryopreserved media with cell numbers of at least approximately 1, 5, 10, 100, 150, 200, or 500 vials per ml of at least 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or at least approximately 10¹⁰ cells per ml. Cryopreserved cell banks retain their functionality and can be thawed, further stimulated, and expanded. In another formulation, thawed cells can be stimulated and expanded in suitable sealed containers (such as cell culture bags and / or bioreactors) to produce large quantities of cells as an allogeneic cell product. Cryopreserved γδ T cells can maintain their biological function under cryopreservation conditions for at least approximately 6, 7, 8, 9, 10, 11, 12, 13, 15, 18, 20, 24, 30, 36, 40, 50 months, or at least approximately 60 months. In this formulation, no preservatives may be used in the formulation. Cryopreserved γδ T cells can be thawed and infused as an allogeneic off-the-shelf cell product into multiple patients.

[0167] In one state, the engineered γδ T cells described herein may be present in the composition in the following amounts: at least 1 × 10³ cells / ml, at least 2 × 10³ cells / ml, at least 3 × 10³ cells / ml, at least 4 × 10³ cells / ml, at least 5 × 10³ cells / ml, at least 6 × 10³ cells / ml, at least 7 × 10³ cells / ml, at least 8 × 10³ cells / ml, at least 9 × 10³ cells / ml, at least 1 × 10⁴ cells / ml, at least 2 × 10⁴ cells / ml, at least 3 × 10⁴ cells / ml, at least 4 × 10⁴ cells / ml, at least 5 × 10⁴ cells / ml, at least 6 × 10³ cells / ml, at least 1 × 10³ ... 0.4 cells / ml, at least 7×10⁴ cells / ml, at least 8×10⁴ cells / ml, at least 9×10⁴ cells / ml, at least 1×10⁵ cells / ml, at least 2×10⁵ cells / ml, at least 3×10⁵ cells / ml, at least 4×10⁵ cells / ml, at least 5×10⁵ cells / ml, at least 6×10⁵ cells / ml, at least 7×10⁵ cells / ml, at least 8×10⁵ cells / ml, at least 9×10⁵ cells / ml, at least 1×10⁶ cells / ml, at least 2×10⁶ cells / ml, at least 3×10⁶ cells / ml. At least 4 × 10⁶ cells / ml, at least 5 × 10⁶ cells / ml, at least 6 × 10⁶ cells / ml, at least 7 × 10⁶ cells / ml, at least 8 × 10⁶ cells / ml, at least 9 × 10⁶ cells / ml, at least 1 × 10⁷ cells / ml, at least 2 × 10⁷ cells / ml, at least 3 × 10⁷ cells / ml, at least 4 × 10⁷ cells / ml, at least 5 × 10⁷ cells / ml, at least 6 × 10⁷ cells / ml, at least 7 × 10⁷ cells / ml, at least 8 × 10⁷ cells / ml, at least 9 × 10⁷ cells / ml, at least 1 × 10⁸ cells / ml Cells / ml, at least 2×10⁸ cells / ml, at least 3×10⁸ cells / ml, at least 4×10⁸ cells / ml, at least 5×10⁸ cells / ml, at least 6×10⁸ cells / ml, at least 7×10⁸ cells / ml, at least 8×10⁸ cells / ml, at least 9×10⁸ cells / ml, at least 1×10⁹ cells / ml, or more, about 1×10³ cells / ml to about at least 1×10⁸ cells / ml, about 1×10⁵ cells / ml to about at least 1×10⁸ cells / ml, or about 1×10⁶ cells / ml to about at least 1×10⁸ cells / ml.

[0168] In one embodiment, the methods described herein can be used to produce autologous or exogenous products according to one embodiment of the invention.

[0169] In one instance, the carrier, construct, or sequence described herein may contain approximately 80%, approximately 85%, approximately 90%, approximately 85%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% of any of SEQ ID NO: 1 to 97 and 265 to 266. A sequence “having at least 85% identity with a reference sequence” is a sequence that has 85% or higher, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the entire length of the reference sequence.

[0170] In the context of this application, global alignment (i.e., comparing two sequences over their entire length) is used to calculate the "percentage of consistency". Methods for comparing the consistency of two or more sequences are well known in the art. For example, the "needle" procedure can be used, which employs the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences when considering the overall length. The needle procedure is available, for example, from the ebi.ac.uk World Wide Web website and is described in the subsequent publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). According to the present invention, the percentage of consistency between two peptides is calculated using the EMBOSS:Needle (Global) program with a "Gap Open" parameter of 10.0, a "Gap Extension" parameter of 0.5, and a Blosum62 matrix.

[0171] Proteins composed of amino acid sequences that are "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to the reference sequence may contain mutations, such as deletions, insertions, and / or substitutions compared to the reference sequence. In the case of substitution, proteins composed of amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the reference sequence may correspond to homologous sequences derived from a species different from the reference sequence.

[0172] "Amino acid substitution" can be conservative or non-conservative. Preferably, the substitution is conservative, in which one amino acid is replaced by another amino acid having similar structure and / or chemical properties.

[0173] In one embodiment, the conserved substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5" (Natl. Biomedical Research), the contents of which are incorporated herein by reference in their entirety. For example, in a single sample, amino acids belonging to one of the following groups can exchange with each other, thus constituting a conservative exchange: Group 1: alanine (A), proline (P), glycine (G), aspartic acid (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In a single-state sample, the conservative amino acid substitutions can be selected from the following: T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0174] In another embodiment, conservative amino acid substitution may include the substitution of an amino acid with another amino acid of the same class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be carried out as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

[0175] In another embodiment, conservative substitution can be performed according to Table 1. Methods for predicting tolerance to protein modification can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated herein by reference in their entirety.

[0176] [surface] [A]: Conservative amino acid substitution Conservative amino acid substitution amino acids Substitution (other substitutions are known in this technology) Ala Ser, Gly, Cys Arg Lys,Gln,His Asn Gln,His,Glu,Asp Asp Glu,Asn,Gln Cys Ser,Met,Thr Gln Asn, Lys, Glu, Asp, Arg Glu Asp,Asn,Gln Gly Pro,Ala,Ser His Asn, Gln, Lys Ile Leu,Val,Met,Ala Leu Ile, Val, Met, Ala Lys Arg,Gln,His Met Leu,Ile,Val,Ala,Phe Phe Met,Leu,Tyr,Trp,His Ser Thr, Cys, Ala Thr Ser,Val,Ala Trp Tyr,Phe Tyr Trp,Phe,His Val Ile,Leu,Met,Ala,Thr

[0177] In one state, the sequence described herein may include 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions, or deletions. In another state, any one of SEQ ID NO: 1 to 97 and 265 to 266 may include 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In yet another state, the mutation or substitution is a conserved amino acid substitution.

[0178] In another embodiment, conservative substitution may be the substitutions shown in Table B under the heading "Conservative Substitution". If such substitutions result in changes in biological activity, further substantial changes may be introduced (displayed as "Exemplary Substitutions" in Table B) and products may be screened if necessary.

[0179] Table B: Amino Acid Substitution Amino acid substitution Original residues (naturally occurring amino acids) Conservative replacement Exemplary substitution Ala (A) Val Val;Leu;Ile Arg (R) Lys Lys;Gln;Asn Asn (N) Gln Gln;His;Asp;Lys;Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser;Ala Gln (Q) Asn Asn;Glu Glu (E) Asp Asp;Gln Gly (G) Ala Ala His (H) Arg Asn;Gln;Lys;Arg Ile (I) Leu Leu;Val;Met;Ala;Phe;leucine Leu (L) Ile Leucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg;Gln;Asn Met (M) Leu Leu;Phe;Ile Phe (F) Tyr Leu;Val;Ile;Ala;Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr;Phe Tyr(Y) Phe Trp;Phe;Thr;Ser Val(V) Leu Ile;Leu;Met;Phe;Ala;leucine

[0180] Example 1

[0181] Table 2. DNA and protein sequences SEQ ID NO: describe Sequence 1 MSCV promoter Tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcact 2 Furin RAKR 3 P2A ATNFSLLKQAGDVEENPGP 4 T2A EGRGSLLTCGDVEENPGP 5 E2A QCTNYALLKLAGDVESNPGP 6 F2A VKQTLNFDLLKLAGDVESNPGP 7 Furin consensus sequence RXXR 8 Linker SGSG 9 ​​cagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc 10 X protein promoter Ggggaagctgacgtcctttcc 11 CD8 α chain MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV 12 CD8 β chain MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT 13 R11KE alpha chain MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 14 R11KE beta chain MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 15 R20P1H7 alpha chain[[ID=1MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQGENSGYSTLTFGKGTMLLVSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 16 R20P1H7 β-chain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLGPGLAAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 17 R7P1D5 α-chain MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEYSSASKIIFGSGTRLSIRPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 18 R7P1D5 β-chain MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRANTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 19 R10P2G12 α-chain MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEGNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 20 R10P2G12 β-chain MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSSGSHQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 21 R10P1A7 alpha chain MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESKETRLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 22 R10P1A7 beta chain MLLLLLLLGPGISLLLPGSLAGSGLGAWSQHPSVWICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARAGGHEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWVWNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 23 R4P1D10 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFHDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 24 R4P1D10 beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVASAYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 25 R4P3F9 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 26 R4P3F9 beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 27 R4P3H3 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKAGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 28 R4P3H3 beta chain MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLLTSGGDNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 29 R36P3F9 alpha chain METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATVSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 30 R36P3F9 β-chain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSSTSGGLSGETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 31 R52P2G11 α-chain MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSAYGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 32 R52P2G11 β-chain MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLGSPDGNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 33 R53P2A9 α-chain MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYNSYAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 34 R53P2A9 β-chain MGPGLLCWVLLCLLGAGPVDAGVTQSPTHLIKTRGQQVTLRCSPISGHKSVSWYQQVLGQGPQFIFQYYEKEERGRGNFPDRFSARQFPNYSSELNVNALLLGDSALYLCASSLDGTSEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 35 R26P1A9 alpha chain METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCLIGASGSRLTFGEGTQLTVNPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 36 R26P1A9 beta chain MGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSYFGWNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 37 R26P2A6 α-chain MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDVSGGYNKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 38 R26P2A6 β-chain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASTTPDGTDEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 39 R26P3H1 α-chain MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMNRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 40 R26P3H1 β-chain MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRAEGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 41 R35P3A4 α-chain MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASPTGGYNKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 42 R35P3A4 β-chain MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSLGGASQEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 43 R37P1C9 α-chain MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILFNFNKFYFGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 44 R37P1C9 β-chain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSSGETNEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 45 R37P1H1 alpha chain MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFGYSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 46 R37P1H1 beta chain MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSNEGQGWEAEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 47 R42P3A9 alpha chain MKRILGALLGLLSAQVCCVRGIQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYISSSQTTDSGVYFCAVHNFNKFYFGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 48 R42P3A9 beta chain MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLLGQGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 49 R43P3F2 alpha chain MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSNNNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 50 R43P3F2 beta chain MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPTGTSGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 51 R43P3G5 alpha chain MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALNRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 52 R43P3G5 beta chain MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRLPSRTYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 53 R59P2E7 alpha chain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVNSDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54 R59P2E7 beta chain MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGLGTGDYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 55 R11P3D3 alpha chain MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 56 R11P3D3 beta chain MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATlLYEILLGKATLYAVLVSALVLMAMVKRKDSRG 57 R16P1C10 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 58 R16P1C10 beta chain MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 59 R16P1E8 alpha chain MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGGGTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 60 R16P1E8 beta chain MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 61 R17P1A9 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTTLSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 62 R17P1A9 beta chain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 63 R17P1D7 alpha chain MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLVFGKGTKLTVNPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 64 R17P1D7 beta chain MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 65 R17P1G3 alpha chain IMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 66 R17P1G3 beta chain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 67 R17P2B6 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKGTHLIIQPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 68 R17P2B6 beta chain MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGRGGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 69 R11P3D3KE alpha chain MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 70 R11P3D3KE β-chain NNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATlLYEILLGKATLYAVLVSALVLMAMVKRKDSRG 71 R39P1C12 α-chain TYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIDNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 72 R39P1C12 β-chain MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSQLNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF ​​​​MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 74 R39P1F5 β-chain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGANEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 75 R40P1C2 α-chain MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYLNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 76 R40P1C2 β-chain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEMTAVGQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 77 R41P3E6 α-chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFT AQLNKASQYVSLLIRDSQPSDSATYLCAAFSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 78 R41P3E6 β-chain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSQYTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 79 R43P3G4 alpha chain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNGGDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 80 R43P3G4 beta chain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGQGALEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 81 R44P3B3 α-chain MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGLYNQGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 82 R44P3B3 β-chain MGCRLLCCVVFCLLQAGPLDTAVSQTPKYLVTQMGNDKSIKCEQNLGHDTMYWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSLGDRGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 83 R44P3E7 α-chain MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEINNNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 84 R44P3E7 β-chain MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSPPDQNTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 85 R49P2B7 α-chain MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSEGALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTTGATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVRIFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 86 R49P2B7 β-chain MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLMGELTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 87 R55P1G7 α-chain MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMMGDTGTASKLTFGTGTRLQVTLDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 88 R55P1G7 β-chain MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSFGGYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 89 R59P2A7 alpha chain VKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 90 R59P2A7 beta chain MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSGLVAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 91 PTE WPRE 92 TPE WPRE 93 PTE fn WPRE 94 PTE CD8 TCR WPRE 95 R11KE WPRE 96 CD8 WPRE 97 RD114TR MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMTNQKWKCRVTPKISPSGGELQNCPCNTFQDSMHSSCYTEYRQCRRINKTYYTATLLKIRSGSLNEVQILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMTPELQYHPLALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANASCQIIPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTYLPQNWTRLCVQASLLPDIDINPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKSGIVRNKIRTLQEELQKRRESLASNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFNRLVQFVKDRISVVQALVLTQQYHQLKPL 265 WPREmut1 cagtctgacgtacgcgtaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcc 266 WPREmut2 gagcatcttaccgccatttatacccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacattttttgggatatgtaattactagttcaggtgtattgccacaagacaaacttgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactttgttgctccttttacgctgtgtggatttgctgctttattgcctctgtatcttgctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttttgaggagttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgtc Table 3. TAA peptide sequence SEQ ID NO: Amino acid sequence SEQ ID NO: Amino acid sequence SEQ ID NO: Amino acid sequence 98 YLYDSETKNA 151 LLWGHPRVALA 204 SLLNQPKAV 99 HLMDQPLSV 152 VLDGKVAVV 205 KMSELQTYV 100 GLLKKINSV 153 GLLGKVTSV 206 ALLQTGDMSL 101 FLVDGSSAL 154 KMISAIPTL 207 VIIKGLEEITV 102 FLFDGSANLV 155 GLLETTGLLAT 208 KQFEGTVEI 103 FLYKIIDEAL 156 TLNTLDINL 209 KLQEEIPVL 104 FILDSAETTL 157 2019-08-11 210 GLAEEFQENV 105 SVDVSPPKV 158 YLEDGFAYV 211 NVAEIVIHI 106 VADKIHSV 159 KIWEELSVLEV 212 ALAGIVTNV 107 IVDDLTINL 160 LLIPFTIFM 213 NLLIDDKGTIKL 108 GLLEELVTV 161 ISLDEVAVSL 214 VLMQDSRLYL 109 TLDGAAVNQV 162 KISDFGLATV 215 KVLEHVVRV 110 SVLEKEIYSI 163 KLIGNIHGNEV 216 LLWGNLPEI 111 LLDPKTIFL 164 ILLSVLHQL 217 SLMEKNQSL 112 YTFSGDVQL 165 LDSEALLTL 218 KLLAVIHEL 113 YLMDDFSSL 166 VLQENSSDYQSNL 219 ALGDKFLLRV 114 KVWSDVTPL 167 HLLGEGAFAQV 220 FLMKNSDLYGA 115 LLWGHPRVALA 168 SLVENIHVL 221 KLIDHQGLYL 116 KIWEELSVLEV 169 YTFSGDVQL 222 GPGIFPPPPPQP 117 LLIPFTIFM 170 SLSEKSPEV 223 ALNESLVEC 118 FLIENLLAA 171 AMFPDTIPRV 224 GLAALAVHL 119 LLWGHPRVALA 172 FLIENLLAA 225 LLLEAVWHL 120 FLLEREQLL 173 FTAEFLEKV 226 SIIEYLPTL 121 SLAETIFIV 174 ALYGNVQQV 227 TLHDQVHLL 122 TLLEGISRA 175 LFQSRIAGV 228 SLLMWITQC 123 KIQEILTQV 176 ILAEEPIYIRV 229 FLLDKPQDLSI 124 VIFEGEPMYL 177 FLLEREQLL 230 YLLDMPLWYL 125 SLFESLEYL 178 LLLPLELSLA 231 GLLDCPIFL 126 SLLNQPKAV 179 SLAETIFIV 232 VLIEYNFSI 127 GLAEFQENV 180 AILNVDEKNQV 233 TLYNPERTITV 128 KLLAVIHEL 181 RLFEEVLGV 234 AVPPPPSSV 129 TLHDQVHLL 182 YLDEVAFML 235 KLQEELNKV 130 TLYNPERTITV 183 KLIDEDEPLFL 236 KLMDPGSLPPL 131 KLQEKIQEL 184 KLFEKSTGL 237 ALIVSLPYL 132 SVLEKEIYSI 185 SLLEVNEASSV 238 FLLDGSANV 133 RVIDDSLVVGV 186 GVYDGREHTV 239 ALDPSGNQLI 134 VLFGELPAL 187 GLYPVTLVGV 240 ILIKHLVKV 135 GLVDIMVHL 188 ALLSSVAEA 241 VLLDTILQL 136 FLNAIETAL 189 TLLEGISRA 242 HLIAEIHTA 137 ALLQALMEL 190 SLIEESEEL 243 SMNGGVFAV 138 ALSSSQAEV 191 ALYVQAPTV 244 MLAEKLLQA 139 SLITGQDLLSV 192 KLIYKDLVSV 245 YMLDIFHEV 140 QLIEKNWLL 193 ILQDGQFLV 246 ALWLPTDSATV 141 LLDPKTIFL 194 SLLDYEVSI 247 GLASS RIDGE 142 RLHDENILL 195 LLGDSSFFL 248 ALSVLRAL 143 YTFSGDVQL 196 VIFEGEPMYL 249 SYVKVLHHL 144 GLPSATTTV 197 ALSYILPYL 250 VYLPKIPSW 145 GLLPSAESIKL 198 FLVDPELV 251 NEWDHPLL 146 KTASINQNV 199 SEWGSPHAAVP 252 VYIAELEKI 147 SLLQHLIGL 200 ALSELERVL 253 VHFEDTGKTLLF 148 YLMDDFSSL 201 SLFESLEYL 254 VLSPFILTL 149 LMYPYIYHV 202 KVLEYVIKV 255 HLLEGSVGV 150 KVWSDVTPL 203 VLLNEILEQV

[0182] Example 2

[0183] γδ T cell production

[0184] To isolate γδ T cells, γδ T cells can be isolated from the subject or from a complex sample of the subject in a single-state sample. A complex sample can be a peripheral blood sample, umbilical cord blood sample, tumor, stem cell precursor, tumor biopsy, tissue, lymph, or epithelial site of the subject derived from stem precursor cells that has directly contacted the external environment. γδ T cells can be isolated directly from a complex sample of the subject, for example, by classifying γδ T cells exhibiting one or more cell surface markers using flow cytometry. Wild-type γδ T cells can exhibit numerous antigen recognition, antigen presentation, co-stimulatory, and adhesion molecules that can be associated with γδ T cells. One or more cell surface markers (such as specific γδ TCRs, antigen recognition, antigen presentation, ligands, adhesion molecules, or co-stimulatory molecules) can be used to isolate wild-type γδ T cells from complex samples. Various molecules associated with or expressed by γδ T cells can be used to isolate γδ T cells from complex samples, for example, to isolate Vδ1+, Vδ2+, Vδ3+ cells or any combination of them.

[0185] For example, peripheral blood mononuclear cells can be collected from the subject using a blood cell separator (including the Ficoll-Paque™ PLUS (GE Healthcare) system or another suitable device / system). γδ T cells or desired subsets of γδ T cells can be purified from the collected sample using (e.g., using flow cytometry). Umbilical cord blood cells can also be obtained from the umbilical cord blood during the subject's birth.

[0186] Positive and / or negative selection of cell surface markers on collected γδ T cells can be used to directly isolate γδ T cells or populations of γδ T cells exhibiting similar cell surface markers from peripheral blood samples, umbilical cord blood samples, tumors, tumor biopsy specimens, tissues, lymph, or epithelial samples from the subject. For example, γδ T cells can be isolated from complex samples based on the positive or negative expression of the following: CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCR α, TCR β, TCR α, TCR δ, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other suitable cell surface markers.

[0187] Figure 1 illustrates the manufacture of γδ T cells according to one embodiment of the present invention. This procedure may include collecting or obtaining white blood cells or PBMCs from a leukocyte ablation product. Leukocyte ablation may include collecting whole blood from a donor and using a hematology separator to separate components. The hematology separator separates the desired blood components and returns the remainder to the donor's circulation. For example, a hematology separator may be used to collect white blood cells, plasma, and platelets, and return red blood cells and neutrophils to the donor's circulation. Commercially available leukocyte ablation products may be used in this procedure. Another method of obtaining white blood cells is from a buffy coat. To separate the buffy coat, anticoagulated whole blood is obtained from the donor and centrifuged. After centrifugation, the blood separates into plasma, red blood cells, and the buffy coat. The buffy coat is the layer located between the plasma and the red blood cell layer. Collecting blood cells separately can result in higher purity and a significantly increased monocyte count compared to collecting by the buffy coat method. The amount of monocytes that can be achieved using leukocyte ablation is typically 20 times higher than that obtained from skin-colored blood cells. Further ablation may require the use of a Ficoll gradient to enrich the monocyte count.

[0188] To deplete αβ T cells from PBMCs, αβ TCR-expressing cells can be separated from PBMCs by magnetic separation (e.g., using CliniMACS® magnetic beads coated with anti-αβ TCR antibodies), followed by cryopreservation of PBMCs depleted of αβ TCR-T cells. To manufacture "off-the-shelf" T-cell products, depleted PBMCs of αβ TCR-T cells stored at low temperature can be thawed and activated for 1 to 10 days (e.g., 2 to 7 days) in the presence of amino bisphosphonates (e.g., zoledronic acid) and / or isopentenyl pyrophosphate (IPP) and / or interleukins (e.g., interleukin 2 (IL-2), interleukin 15 (IL-15) and / or interleukin 18 (IL-18)) and / or other activators (e.g., Toll-like receptor (TLR2) ligands).

[0189] Figure 1 illustrates that activated T cells can be engineered by transduction using viral vectors (such as lentiviral vectors) that express exogenous genes of interest (such as αβ TCRs targeting specific cancer antigens and CD8) into isolated γδ T cells. Transduction can be performed once or multiple times to achieve stable transgenic expression lasting ½ to 5 days (e.g., 1 day) on a small (e.g., 24 to 4 to 6-well discs) or medium / large scale.

[0190] Figure 1 further illustrates that the expansion of transduced or engineered γδ T cells can be carried out on a small / medium scale (e.g., flasks / G-Rex) or a large scale (e.g., 50 ml to 100 liter bags) for 7 to 35 days (e.g., 7 to 28 days) in the presence of intercytokines (e.g., IL-2, IL-15, IL-18, and other intercytokines). The expanded transduced T cell product can then be cryopreserved as an "off-the-shelf" T cell product for infusion into patients.

[0191] Example 3

[0192] Lentiviral viral vector

[0193] The lentiviral vectors used in this paper contain several elements previously shown to enhance vector function, including a central polypurine tract (cPPT) for improved replication and nuclear importation, a promoter from murine stem cell virus (MSCV) (SEQ ID NO: 1) (which has been shown to reduce vector silencing in certain cell types), a woodchuck hepatitis virus posttranscriptional responsive element (WPRE) (SEQ ID NO: 9) for improved transcription termination, and a backbone line that can have improved safety, sustained gene expression, and anti-silencing properties with a 3'-LTR deletion self-inactivating (SIN) vector design (Yang et al., Gene Therapy (2008) 15, 1411–1423, the contents of which are incorporated herein by reference in their entirety).

[0194] In one state, the vector, construct, or sequence described herein contains a mutated form of WPRE. In another state, the sequence or vector described herein contains a mutation of WPRE type 1 (e.g., WPREmut1 (SEQ ID NO: 265)) or a mutation of WPRE type 2 (e.g., WPREmut2 (SEQ ID NO: 266)). In one state, the WPRE mutant contains at most one mutation, at most two mutations, at most three mutations, at least four mutations, or at most five mutations. In one state, the vector, construct, or sequence described herein does not contain WPRE. In another state, the present invention provides one, two, three, four, five, ten, or 20 substitutions of any one of SEQ ID NO: 91 to 96.

[0195] In another variant, the vectors, constructs, or sequences described herein do not include the X protein promoter.

[0196] To achieve optimal co-expression levels of TCRαβ and CD8αβ in transduced γδ T cells, lentiviral vectors with various designs have been developed. Figure 2 illustrates that T cells can be transduced using two separate lentiviral vectors expressing either TCRαβ or CD8αβ (2-in-1) and a single lentiviral vector co-expressing both TCRαβ and CD8αβ (4-in-1). In the 4-in-1 vector, nucleotides encoding the TCRα, TCRβ, CD8α, and CD8β chains can be mixed in various orders. These various 4-in-1 vectors can be used to transduce γδ T cells, and the TCR / CD8 co-expression levels of these transduced cells can then be measured using techniques known in this field (e.g., flow cytometry).

[0197] To generate lentiviral vectors that co-express TCRαβ and CD8αβ, the nucleotide encoding the furin linker (GSG or SGSG (SEQ ID NO: 8))-2A peptide can be positioned between the TCRα and TCRβ chains, between the CD8α and CD8β chains, and between the TCR and CD8 chains to achieve efficient gene expression. The 2A peptide can be selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5), or F2A (SEQ ID NO: 6).

[0198] Lentiviral vectors may also contain post-transcriptional regulatory elements (PREs), such as the woodchuck post-transcriptional regulatory element (WPRE) (SEQ ID NO: 9), to enhance transgene expression by increasing both nuclear and cytoplasmic mRNA levels. One or more of the following regulatory elements may also be used and / or combined with WPREs to enhance transgene expression: mouse RNA transport element (RTE), constitutive transport element (CTE) of simian retrovirus type 1 (SRV-1), and the 5′ untranslated region of human heat shock protein 70 (Hsp70 5′UTR).

[0199] Lentiviral vectors can be pseudotyped using RD114TR (SEQ ID NO: 97), a chimeric glycoprotein of feline endogenous virus (RD114) containing the extracellular and transmembrane domains fused to the cytoplasmic tail (TR) of murine leukemia virus. Other viral envelope proteins (such as VSV-G env, MLV 4070A env, RD114 env, chimeric envelope protein RD114pro, baculovirus GP64 env, or GALV env) or their derivatives can also be used.

[0200] Figure 3 shows four different 4-in-1 vectors (i.e., PTE WPRE (SEQ ID NO: 91), TPE WPRE (SEQ ID NO: 92), PTE fn WPRE (SEQ ID NO: 93), and PTE CD8 TCR WPRE (SEQ ID NO: 94)) that express both TCRαβ (R11KE) and CD8αβ, and two 2-in-1 vectors (i.e., R11KE WPRE (SEQ ID NO: 95) that expresses TCRαβ (R11KE) and CD8 WPRE (SEQ ID NO: 96) that expresses CD8αβ. TCRαβ (R11KE)) that bind to PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) that is complexed with the MHC molecule.

[0201] Example 4

[0202] Co-expression of TCR and CD8

[0203] γδ T cell lines obtained from donors 1 and 2 were manufactured using the procedure shown in Figure 1. On day 3 or 6 after activation with zoledronic acid, IL2, and IL15, γδ T cells were transduced with RD114TR pseudotyped lentiviruses (e.g., PTE WPRE (SEQ ID NO: 91), TPE WPRE (SEQ ID NO: 92), PTE fn WPRE (SEQ ID NO: 93), and PTE CD8 TCR WPRE (SEQ ID NO: 94). The co-expression levels of R11KE and CD8 were then measured using flow cytometry. Transduction efficiency was assessed using antibodies specific to TCR (Vβ8) and CD8 (CD8α) via flow cytometry.

[0204] Figure 4 shows that in γδ T cells from donor 1, the co-expression levels of R11KE and CD8 generated by transduction using PTE CD8 TCR WPRE (i.e., 40.5% (day 3) and 18.5% (day 6)) were higher than those generated by transduction using PTE WPRE (29.6% (day 3), 16.2% (day 6)), TPE WPRE (30.8% (day 3), 11.0% (day 6)) and PTE fn WPRE (33.0% (day 3), 15.0% (day 6)). In γδ T cells from donor 2, the co-expression level of R11KE and CD8 on day 6 post-activation, generated by transduction using PTE WPRE (i.e., 18.8%), was higher than that generated by transduction using TPE WPRE (14.2%), PTE fn WPRE (14.7%), and PTE CD8 TCR WPRE (17.2%). As a control, background levels of R11KE and CD8 were detected in γδ T cells transduced separately using a 2-in-1 vector (i.e., TCRαβ (R11KE) or CD8 WPRE).

[0205] Example 5

[0206] Effects on transgenic expression and functionality of 4-in-1 viral vectors (e.g., lentiviral vectors), which contain sequences encoding the CD8αβ chain and the TCRαβ chain located at different positions within the vector.

[0207] WO 2019 / 204662 describes CD4+ cells exhibiting an exogenous CD8αβ co-receptor and one or more exogenous engineered antigen receptors (e.g., TCRs). Table 4 shows a comparison between the 4-in-1 construct described in WO 2019 / 204662 and the 4-in-1 construct according to the present invention. Table 4 WO 2019 / 204662 The state described in this article Orientation of genetic modification (from the 5' end to the 3' end) TCRβ-TCRα-CD8α-CD8β CD8β-CD8α-TCRβ-TCRα Origin of CD8αβ sequence GenBank Codon optimization (for performance enhancement) 2A connector 2A 2A+ furin connector (used to facilitate efficient cleavage of the residual 2A sequence of the gene of interest) Cellular type CD4+ cells and CD8+ cells γδ T cells (low (0-20%) CD8 expression and no CD4 expression) Viruses and pseudotypes Retroviruses and RD114 Lentiviral viruses, RD114TR, and VSV-G

[0208] The open reading frame (ORF) of the nucleic acid molecule of the present invention can be at least partially codon-optimized. Codon optimization is based on the finding that translation efficiency can be determined by the different frequencies of transfer RNA (tRNAs) appearing in the cell. Therefore, the ORF of the nucleic acid molecule of the present invention can be modified compared to the corresponding wild-type coding region so that at least one codon of the wild-type sequence encoding a relatively rare tRNA in the cell can be used to exchange for a codon encoding a tRNA that is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA. Through this modification, the ORF of the nucleic acid molecule of the present invention can be modified so that codons (for tRNAs with frequent occurrences of such codons) can replace codons corresponding to rare tRNAs. In other words, according to the present invention, through this modification, all codons of the wild-type ORF encoding a rare tRNA can be used to exchange for codons encoding a tRNA that is more frequent in the cell and carries the same amino acid as the rare tRNA. It is known to those skilled in the art which tRNAs appear relatively frequently in cells and which appear relatively rarely; for example, Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the open reading frame of the nucleic acid molecule of the present invention may preferably be codon-optimized relative to the system in which the nucleic acid molecule of the present invention is to be expressed, and preferably relative to the system in which the nucleic acid molecule of the present invention is to be translated. Preferably, the codon usage of the open reading frame of the nucleic acid molecule of the present invention may be codon-optimized according to the use of mammalian codons, and more preferably according to the use of human codons. Preferably, the open reading frame may be codon-optimized and modified with G / C inclusions.

[0209] To determine which transgene direction provides better transgene expression and functionality, three 4-in-1 viral vectors (e.g., PTE.WPRE (SEQ ID NO: 91), TPE.WPRE (SEQ ID NO: 92), and PTE.fn.WPRE (SEQ ID NO: 93)) each containing a sequence encoding the TCRαβ chain upstream of the sequence encoding the CD8αβ chain, and one 4-in-1 viral vector (e.g., PTE.CD8.TCR.WPRE (SEQ ID NO: 94)) containing a sequence encoding the CD8αβ chain upstream of the sequence encoding the TCRαβ chain, were transduced into γδ T cells. Fluorescence-activated cell sorting (FACS) analysis was then performed using fluorescently labeled anti-CD8 antibody and fluorescently labeled anti-TCR Vβ8 (Vb8) antibody to detect the expression of CD8 and TCR on the cell surface.

[0210] Figures 10 and 11 show the percentage of CD8+Vb8+ double positive cells (Figure 10) and the MFI of CD8 or Vb8 (Figure 11). Compared with donors transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE or PTE.fn.WPRE, γδ T cells obtained from donors 3 and 4 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE showed the highest expression of both CD8 and TCR on the cell surface on day 14 of manufacturing.

[0211] The high expression of CD8 and TCR on the cell surface of γδ T cells transduced with a 4-in-1 viral vector containing PTE, CD8, TCR, and WPRE is well correlated with the in vitro cytotoxic activity of these cells. For example, Figure 12 shows that γδ T cells obtained from donor 3 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE exhibited the best killing activity against both the high-target peptide presenting cell line UACC257 (top detection combination) and the low-target peptide presenting cell line U2OS (bottom detection combination) compared to donors transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE, PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE.

[0212] Figure 14 shows that γδ T cells obtained from donor 4 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE also exhibited the best killing activity against both UACC257 (top detection combination) and U2OS (bottom detection combination) compared to donors transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE.

[0213] Figure 16 illustrates that, similar to donors transduced with PTE.WPRE, TPE.WPRE, and PTE.fn.WPRE, γδ T cells obtained from donors 3 and 4 transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE produced less than 0.6 copies of the integrated vector per cell. This low copy number of the integrated vector per cell is within the safety limits, i.e., less than 5 copies of the integrated vector per cell.

[0214] Figures 17A and 17B show that the γδ T cells obtained from donors 3 and 4, transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE, achieved a level of cell expansion comparable to that of donors transduced with a 4-in-1 viral vector containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE on day 14 of manufacturing.

[0215] To determine the memory cell phenotype of transduced γδ T cells, cells were stained with allophycocyanin (APC)-Cy7-labeled anti-CD45RA antibody and BV421-labeled anti-CCR7 antibody, followed by FACS analysis to determine the percentages of Tcm, naive T cells, TemRA, and Teff present in transduced γδ T cells. Figure 18A shows an example of this analysis.

[0216] Figure 18B shows that the γδ T cells obtained from donors 3 and 4, transduced with a 4-in-1 viral vector containing PTE.CD8.TCR.WPRE, achieved a level of memory T cell phenotype comparable to that of donors transduced with a 4-in-1 viral vector containing PTE.WPRE, TPE.WPRE, or PTE.fn.WPRE on day 14 of manufacturing.

[0217] Example 6

[0218] Effects of transgenic expression on cells transduced with one 4-in-1 viral vector versus cells transduced with two 2-in-1 viral vectors

[0219] Figure 19 shows that the number of CD8+TCR+γδ T cells generated by transduction using a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (120 µl) (test combination B, 20.9%) was higher than that generated by transduction using a mixture of a 2-in-1 lentiviral vector containing CD8.WPRE (120 µl) and a 2-in-1 lentiviral vector containing R11KE.WPRE (120 µl) (test combination D, 15.5%). On the other hand, the number of CD8+TCR-γδ T cells transduced by a mixture of a 2-in-1 lentiviral vector containing CD8.WPRE (120 µl) and a 2-in-1 lentiviral vector containing R11KE.WPRE (120 µl) (test combination D, 28.3%) was higher than that of CD8+TCR-γδ T cells transduced by a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (test combination B, 21.2%). Similarly, the number of CD8+TCR+γδ T cells transduced with a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (240 µl) (test combination C, 27.7%) was higher than that of CD8+TCR-γδ T cells transduced with a mixture of a 2-in-1 lentiviral vector containing CD8.WPRE (240 µl) and a 2-in-1 lentiviral vector containing R11KE.WPRE (240 µl) (test combination E, 21.1%). On the other hand, CD8+TCR-γδ T cells transduced using a mixture of a 2-in-1 lentiviral vector containing CD8.WPRE (240 µl) and a 2-in-1 lentiviral vector containing R11KE.WPRE (240 µl) (test combination E, 40.2%) were more abundant than those transduced using a 4-in-1 lentiviral vector containing PTE.CD8.TCR.WPRE (test combination C, 24.4%). Non-transduced (NT) γδ T cells served as a control (test combination A). Two-color staining was performed using APC-labeled anti-CD8β antibody and phycoerythrin (PE)-labeled target peptide / MHC tetramer. These results suggest that transduction using a 4-in-1 lentiviral vector containing sequences encoding CD8αβ and TCRαβ (e.g., PTE.CD8.TCR.WPRE) can result in a greater number of CD8+TCR+ T cells than transduced using a mixture of a 2-in-1 lentiviral vector containing a sequence encoding CD8αβ (e.g., CD8.WPRE) and a 2-in-1 lentiviral vector containing a sequence encoding TCRαβ (e.g., R11KE.WPRE).On the other hand, transduction using a mixture of a 2-in-1 lentiviral vector encoding CD8αβ (e.g., CD8.WPRE) and a 2-in-1 lentiviral vector encoding TCRαβ (e.g., R11KE.WPRE) resulted in a greater number of CD8+TCR-T cells than those transduced using a 4-in-1 lentiviral vector encoding both CD8αβ and TCRαβ (e.g., PTE.CD8.TCR.WPRE).

[0220] Figure 20 illustrates that increased amounts of the 4-in-1 viral vector containing PTE, CD8, TCR, and WPRE (e.g., 30 µl, 120 µl, and 240 µl) used for transduction enhance transduction efficiency, e.g., increasing the percentage of CD8+TCR+ γδ T cells, e.g., 9.6% at 30 µl, 20.9% at 120 µl, and 27.7% at 240 µl. Untransduced γδ T cells serve as a control. Two-color staining was performed using APC-labeled anti-CD8β antibody and PE-labeled target peptide / MHC complex tetramer.

[0221] Example 7

[0222] The manifestation of the 4-in-1 construct in αβ T cells

[0223] Engineered lymphocytes, including engineered αβ T cells expressing recombinant proteins (e.g., CD8αβ and / or TCRαβ), can be manufactured according to the methods disclosed in US 2019 / 0247433, the contents of which are incorporated herein by reference in their entirety. For example, Figure 37 illustrates the T cell manufacturing process.

[0370] The T cell manufacturing process may include the following steps: isolating PBMCs (

[0371] PBMCs can be used fresh or frozen until ready for use, or can be used as a product for leukocyte apheresis (e.g., leukopak), or as a starting material for T cell production and as an option for lymphocyte populations (e.g., αβ TCR+ T cells, CD8+, CD4+, or both); thaw the lymphocytes and let them stand overnight (e.g., about 16 hours or about 4 to 6 hours).

[0372] This allows apoptotic cells to die and restores T cell functionality (this step may be optional if fresh material is used); activates lymphocytes (

[0373] ), this activation can be carried out using anti-CD3 and anti-CD28 antibodies (soluble or surface-bound, e.g., magnetic or biodegradable beads, antibodies immobilized on culture dishes); transduction with a viral vector containing a sequence encoding a recombinant protein (e.g., CD8αβ and / or TCRαβ polypeptides)

[0374] ), where the viral vector can be a lentiviral vector or a retroviral vector, or transfection can be performed by non-viral methods; and expanding lymphocytes, harvesting and cryopreserving

[0375] ), this step can be carried out in the presence of cytokines (e.g., IL-7 and IL-15), serum (ABS or FBS) and / or cryopreservation medium.

[0224] Exogenous CD8 expression

[0225] To determine the exogenous CD8 expression in αβ T cells transduced with a viral vector containing a 4-in-1 construct encoding sequences for CD8 and TCR, T cells obtained from donors 5 and 6 were transduced with increasing amounts of LV-PTE.CD8.TCR.WPRE, and then FACS was performed on lymphocytes<monomorph<live cells<CD3+ population gating to detect the % CD8α+ cells in CD4+ cells. Figure 21 shows the following: an increase in % CD8α+CD4+ cells from donor 5, from 2.87% (untransduced) to 14.7% (2.5 μl / 1x106 cells), 19.5% (5 μl / 1x106 cells), 21.7% (7.5 μl / 1x106 cells), and 24.1% (10 μl / 1x106 cells); and an increase in % CD8α+CD4+ cells from donor 6, from 1.93% (untransduced) to 12.5% (2.5 μl / 1x106 cells), 17.2% (5 μl / 1x106 cells), 19.6% (7.5 μl / 1x106 cells), and 20.8% (10 μl / 1x106 cells).

[0226] Exogenous TCR expression [[ID=​To determine exogenous TCR expression in αβ T cells transduced with a viral vector containing a 4-in-1 construct encoding sequences for CD8 and TCR, T cells obtained from donors 5 and 6 were transduced with increasing amounts of LV-PTE.CD8.TCR.WPRE, and then FACS was performed with lymphocyte <monomorphic <live cell <CD3+ <CD4+CD8+ population gating to detect % target peptide / MHC complex Dextramer203+ (i.e., TCR+) cells in the CD4+CD8+ cell population. Figure 22 shows that the % Dextramer203+ cells from donor 5 increased from 0.32% (untransduced) to 41.9% (2.5 μl / 1x106 cells), 48.3% (5 μl / 1x106 cells), 54.5% (7.5 μl / 1x106 cells), and 49.5% (10 μl / 1x106 cells); and the % Dextramer203+ cells from donor 6 increased from 0.19% (untransduced) to 35.5% (2.5 μl / 1x106 cells), 41.2% (5 μl / 1x106 cells), 44.6% (7.5 μl / 1x106 cells), and 44.0% (10 μl / 1x106 cells).

[0228] To detect TCR expression in various αβ T cell populations, αβ T cells transduced with LV-PTE.CD8.TCR.WPRE were analyzed by performing FACS with lymphocyte <monomorphic <live cell <CD3+ <CD4+ / -CD8+ / - gating. Figure 23 shows that the % Dextramer203 (Dex203)+ (i.e., TCR+) cells obtained from donor 5 (top panel) and donor 6 (bottom panel) were generally higher in the CD4+CD8α+ cell population than in the CD4-CD8α+ cell population. In contrast, the % Dex203+ (i.e., TCR+) cells were the lowest in the CD4+CD8α- cell population. Similarly, Figure 24 shows that the % Dextramer203 (Dex203) MFI obtained from donor 5 (top panel) and donor 6 (bottom panel) was generally higher in the CD4+CD8α+ cell population than in the CD4-CD8α+ cell population. In contrast, the % Dex203 MFI was the lowest in the CD4+CD8α- cell population. These results suggest that the exogenous TCR and CD8 encoded by LV-PTE.CD8.TCR.WPRE can be co-expressed in both CD4+ T cells and CD4- T cells.

[0229] Example 8

[0230] Functional analysis of αβ T cells exhibiting the 4-in-1 construct or the TCR construct alone

[0231] Figure 25 illustrates the experimental design for testing the functionality of αβ T cells transduced with a lentiviral vector (LV) containing a 4-in-1 construct (e.g., PTE.CD8.TCR.WPRE (LV-CD8.TCR)) or a TCR-only construct (e.g., R11KE.WPRE (LV-TCR)). Briefly, on day -1, target cells (e.g., high antigen-expressing UACC257+RFP cell line (positive control) and antigen-negative MCF7+GFP cell line (negative control)) were seeded in 96-well plates. Donor cell products (e.g., PBMCs transduced with LV-CD8.TCR (5 µl / 1 x 10⁶ cells) or LV-TCR (2.5 µl / 1 x 10⁶ cells) (obtained from donors 5, 6, 7, and 8)) were thawed and incubated overnight in 24-well G-Rex® gas-permeable rapid amplification devices. On day 0, donor cell products (effect cells) were co-cultured with target cells (e.g., UACC257+RFP and MCF7+GFP) at an effector cell to target cell (E / T) ratio of 2:1 (e.g., 200,000 effector cells : 100,000 target cells). After culturing at 37°C for 5 hours, a protein transport inhibitor (e.g., GolgiStop™ (BD Biosciences)) was added to each well at 0.5 µl / well, followed by culturing at 37°C for 4 hours. Cells were then centrifuged to collect the supernatant for ELISA to detect IFN-γ expression, and T cells for staining (e.g., CD3, CD4, CD8, IFN-γ, granzyme B, and live / dead cells) were harvested using intracellular cytokine staining (ICS) plates.

[0232] CD4-CD8+ T cell population

[0233] Figure 26 illustrates that co-culturing CD4-CD8α+ T cells obtained from grouped donors transduced with autologous LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in higher % IFN-γ-positive cells (top detection group) and IFN-γ MFI (bottom detection group) compared to the untransduced (NT) group. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and untransduced cells in terms of % IFN-γ-positive cells and IFN-γ MFI. FACS was performed on CD4-CD8α+IFN-γ+ T cell gate control. Untransduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor group N = 4). These results suggest that CD4-CD8α+ T cells transduced with LV-TCR or LV-CD8.TCR become functionally active, for example, by expressing IFN-γ, when exposed to high antigen expression target cells (e.g., UACC257 cells), and that the transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0234] Figure 27 illustrates that co-culturing CD4-CD8α+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in a higher percentage of granzyme B-positive cells (top detection group) and granzyme B MFI (bottom detection group) compared to the untransduced (NT) group. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and untransduced cells in terms of percentage of granzyme B-positive cells and granzyme B MFI. FACS was performed on CD4-CD8α+ granzyme B+ T cell gate control. Untransduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor group N = 3). These results suggest that CD4-CD8α+ T cells transduced with LV-TCR or LV-CD8.TCR become functionally active, for example, by expressing granzyme B, when exposed to high antigen expression target cells (e.g., UACC257 cells), and that transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0235] CD4+CD8+ T cell population

[0236] Figure 28 illustrates that co-culturing CD4+CD8α+ T cells obtained from grouped donors transduced with LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in higher % IFN-γ-positive cells (top detection group) and IFN-γ MFI (bottom detection group) compared to the non-transduced (NT) group. In contrast, when CD4+CD8α+ T cells transduced with LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and non-transduced cells in terms of % IFN-γ-positive cells and IFN-γ MFI. FACS were performed to assess CD4+CD8α-IFN-γ+ gated targeting NT cells and CD4+CD8α+IFN-γ+ gated targeting LV-CD8.TCR transduced cells. Non-transduced (NT) cells served as a control. (Effectant to target cell ratio = 2:1 and donor N = 4 in the grouping). These results suggest that CD4+CD8α+ T cells transduced with LV-CD8.TCR become functionally active, for example, by expressing IFN-γ, when exposed to target cells with high antigen expression (e.g., UACC257 cells), and that the transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0237] Figure 29 illustrates that co-culturing CD4+CD8α+ T cells obtained from grouped donors transduced with LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in a higher percentage of granzyme B-positive cells (top detection group) and granzyme B MFI (bottom detection group) compared to the non-transduced (NT) condition. In contrast, when CD4+CD8α+ T cells transduced with LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and non-transduced cells in terms of percentage of granzyme B-positive cells and granzyme B MFI. FACS were performed to assess CD4+CD8α-granzyme B+ gated on NT cells and CD4+CD8α+granzyme B+ gated on LV-CD8.TCR transduced T cells. Non-transduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor group N = 3). These results suggest that CD4+CD8α+ T cells transduced with LV-CD8.TCR become functionally active, for example, by expressing granzyme B, when exposed to high antigen expression target cells (e.g., UACC257 cells), and that the transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0238] CD3+ T cells

[0239] Figure 30 illustrates that co-culturing CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in higher % IFN-γ-positive cells (top detection group) and IFN-γ MFI (bottom detection group) compared to the untransduced (NT) group. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and untransduced cells in terms of % IFN-γ-positive cells and IFN-γ MFI. FACS was performed on CD3+ T cell gate control. Untransduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor group N = 4). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR become functionally active, for example, by expressing IFN-γ, when exposed to high antigen expression target cells (e.g., UACC257 cells), and that transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0240] Figure 31 illustrates that co-culturing CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in a higher percentage of granzyme B-positive cells (top detection group) and granzyme B MFI (bottom detection group) compared to the untransduced (NT) group. In contrast, when CD3+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference was observed between transduced and untransduced cells in terms of percentage of granzyme B-positive cells and granzyme B MFI. FACS was performed on CD3+ T cell gate control. Untransduced (NT) cells served as controls. (Effective cell to target cell ratio = 2:1 and donor group N = 3). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR become functionally active, for example, by expressing granzyme B, when exposed to high antigen expression target cells (e.g., UACC257 cells), and that transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0241] Figure 32 illustrates that co-culturing CD3+ T cells obtained from grouped donors transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) with high-target-performance UACC257 cells resulted in higher levels of IFN-γ crypticity compared to untransduced (NT), MCF7 cells only, and UACC257 cells only. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference in IFN-γ crypticity levels was observed between transduced and untransduced cells (effectant to target cell ratio = 2:1 and grouped donor N = 4). These results suggest that CD3+ T cells transduced with LV-TCR or LV-CD8.TCR become functionally active upon contact with high antigen-expressing target cells (e.g., UACC257 cells), for example by cryptic IFN-γ, and that transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0242] Figure 33 shows that co-culturing CD3+ T cells obtained from individual donors 5, 6, 7, and 8, transduced with autologous LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8), with high-target-performance UACC257 cells resulted in higher levels of IFN-γ crypticity compared to untransduced (NT), MCF7 cells only, and UACC257 cells only. In contrast, when CD4-CD8α+ T cells transduced with LV-TCR (TCR) or LV-CD8.TCR (TCR+CD8) were co-cultured with antigen-negative MCF7 cells, no significant difference in IFN-γ crypticity levels was observed between transduced and untransduced cells (effectant to target cell ratio = 2:1). These results suggest that CD3+ T cells derived from individual donors using LV-TCR or LV-CD8.TCR transduction become functionally active upon contact with high antigen-expressing target cells (e.g., UACC257 cells), for example, by cryptic IFN-γ, and that transduced cells have little effect on antigen-negative cells (e.g., MCF7 cells).

[0243] Example 9

[0244] Effects of statin on the expression of T cell activation markers

[0245] To determine the effect of statins on the expression of T cell activation markers, T cells were treated with statins (e.g., atorvastatin, pravastatin, or rosuvastatin), followed by FACS analysis to measure the expression of T cell activation markers (e.g., CD25, CD69, and hLDLR).

[0246] CD4+ T cell population

[0247] Figure 34 shows the percentages of CD25+ cells (top assay), CD69+ cells (middle assay), and hLDLR+ cells (bottom assay) in CD3+CD4+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Pre-activated cells, cells activated without statin or DMSO (controls), and DMSO served as controls. These results demonstrate that while atorvastatin, pravastatin, and rosuvastatin have little effect on percentages of CD4+CD25+ and CD4+CD69+ cells, statins (e.g., atorvastatin) can increase the percentage of CD4+hLDLR+ cells. FACS was performed regarding the lymphocyte > monomorphic > live / dead > CD3+ > CD4+ gate.

[0248] CD8+ T cell population

[0249] Figure 35 shows the percentages of CD25+ cells (top assay), CD69+ cells (middle assay), and hLDLR+ cells (bottom assay) in CD3+CD8+ T cells treated with atorvastatin, pravastatin, or rosuvastatin. Pre-activated cells, cells activated without statin or DMSO (controls), and DMSO served as controls. These results demonstrate that while atorvastatin, pravastatin, and rosuvastatin have little effect on the percentages of CD8+CD25+ and CD8+CD69+ cells, statins (e.g., atorvastatin) can increase the percentage of CD8+hLDLR+ cells. FACS was performed regarding the lymphocyte > monomorphic > live / dead > CD3+ > CD8+ gate.

[0250] Example 10

[0251] The effect of WPRE on lentiviral titration

[0252] To determine the effect of WPRE on lentiviral titration, lentiviral vectors (LVs) containing wild-type (wt) WPRE (SEQ ID NO: 9) (LV-A), WPRE-free (LV-B), WPREmut1 (SEQ ID NO: 265) (LV-C), or WPREmut2 (SEQ ID NO: 266) (LV-D) were generated. HEK293T cells were transfected with LV-A, LV-B, LV-C, or LV-D, and titration was then determined using methods known in this technique. Figure 36 shows the titration order of these lentiviral vectors as follows: LV-C > LV-D ≥ LV-A > LV-B. These results suggest that WPREmut1 and WPREmut2 may be useful for improving lentiviral vector production.

[0253] Advantages of the present invention may include generating viral vectors that co-express multiple transgenes (e.g., four polypeptides) in a single vector, and generating γδ T cells that co-express TCRαβ and CD8αβ as safe and target-specific "off-the-shelf" T cell products for receptive cell therapy.

[0254] All references listed in this specification are incorporated herein by reference as if each reference were specifically and individually indicated as incorporated by reference. Any reference cited is for disclosure prior to the filing date and should not be construed as granting any right to an invention prior to this reference.

[0255] 370: T-cell manufacturing process 371, 372, 373, 374, 375: Steps

[0256] Domestic storage information (please note in order of storage institution, date, and number) none Overseas storage information (please note in the order of storage country, institution, date, and number) none

[0257] Sequence List <![CDATA[ <110> Immatics US, Inc. <![CDATA[ <120> Viral vectors and their application in recipient-receptor cell therapy]]> <![CDATA[ <130> 3000011-013977]]> <![CDATA[ <140> TW 109117632]]> <![CDATA[ <141> 2020-05-27 <![CDATA[ <150> US 62 / 853,123 <![CDATA[ <151> 2019-05-27 <![CDATA[ <160> 266 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 367]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Mouse stem cell virus promoter]]> <![CDATA[ <400> 1]]> tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 60 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 120 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 180 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 240 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt 300 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc 360 cctcact 367 <![CDATA[<210> 2]]> <![CDATA[<211> 4]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Furin]]> <![CDATA[<400> 2]]> Arg Ala Lys Arg 1 <![CDATA[ <210> 3]]> <![CDATA[ <211> 19]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> P2A peptide]]> <![CDATA[ <400> 3]]> Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <![CDATA[ <210> 4]]> <![CDATA[ <211> 18]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> T2A peptide]]> <![CDATA[ <400> 4]]> Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <![CDATA[ <210> 5]]> <![CDATA[ <211> 20]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> E2A peptide]]> <![CDATA[ <400> 5]]> Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <![CDATA[ <210> 6]]> <![CDATA[ <211> 22]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> F2A peptide]]> <![CDATA[ <400> 6]]> Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <![CDATA[ <210> 7]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Flynn Consistent Sequence <![CDATA[ <220> ]]> <![CDATA[ <221> misc_feature]]> <![CDATA[ <222> (2)..(3)]]> <![CDATA[ <223> Xaa can be any naturally occurring amino acid. <![CDATA[ <400> 7]]> Arg Xaa Xaa Arg 1 <![CDATA[ <210> 8]]> <![CDATA[ <211> 4]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Linking peptides <![CDATA[ <400> 8]]> Ser Gly Ser Gly 1 <![CDATA[ <210> 9]]> <![CDATA[ <211> 607]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> WPRE]]> <![CDATA[ <400> 9]]> cagtctgacg tacgcgtaat caacctctgg attacaaaat ttgtgaaaga ttgactggta 60 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 120 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 180 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 240 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 300 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 360 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt 420 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct 480 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 540 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 600 ccccgcc 607 <![CDATA[ <210> 10]]> <![CDATA[ <211> 21]]> <![CDATA[ <212> DNA <![CDATA[ <213> Artificial Sequence <![CDATA[ <220> ]]> <![CDATA[ <223> X protein promoter <![CDATA[ <400> 10]]> ggggaagctg acgtcctttc c 21 <![CDATA[ <210> 11]]> <![CDATA[ <211> 235]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Homo sapiens <![CDATA[ <400> 11]]> Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr 20 25 30 Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser 35 40 45 Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala 50 55 60 Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala 65 70 75 80 Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp 85 90 95 Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr 100 105 110 Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe 115 120 125 Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg 130 135 140 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 145 150 155 160 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 165 170 175 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 180 185 190 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His 195 200 205 Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser 210 215 220 Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val 225 230 235 <![CDATA[<21]]>0> 12]]> <![CDATA[<211> 243]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 12]]> <![CDATA[Met Arg Pro Arg Leu Trp Leu Leu Leu Ala Ala Gln Leu Thr Val Leu 1 5 10 15 His Gly Asn Ser Val Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln 20 25 30 Thr Asn Lys Met Val Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser 35 40 45 Asn Met Arg Ile Tyr Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp 50 55 60 Ser His His Glu Phe Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile 65 70 75 80 His Gly Glu Glu Val Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala 85 90 95 Ser Arg Phe Ile Leu Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly 100 105 110 Ile Tyr Phe Cys Met Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys 115 120 125 Gly Thr Gln Leu Ser Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro 130 135 140 Thr Lys Lys Ser Thr Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro 145 150 155 160 Glu Thr Gln Lys Gly Pro Leu Cys Ser Pro Ile Thr Leu Gly Leu Leu 165 170 175 Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly Val Ala Ile His 180 185 190 Leu Cys Cys Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Pro 195 200 205 Gln Gly Glu Gly Ile Ser Gly Thr Phe Val Pro Gln Cys Leu His Gly 210 215 220 Tyr Tyr Ser Asn Thr Thr Thr Ser Gln Lys Leu Leu Asn Pro Trp Ile 225 230 235 240 Leu Lys Thr <![CDATA[ <210> 13]]> <![CDATA[ <211> 273]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> R11KEA α-chain]]> <![CDATA[<400> 13]]> Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Lys Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Leu Tyr Asn Asn Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu 115 120 125 Thr Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Serum <![CDATA[ <210> 14]]> <![CDATA[ <211> 311]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence(Artificial Sequence)]]> <![CDATA[ <220> ]]> <![CDATA[ <223> R11KE βẹọs]]> <![CDATA[ <400> 14]]> Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asn Ser Leu Phe Trp Tyr Arg Glu Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Pro Gly Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210>]]> 15 <![CDATA[<211> 274]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 15]]> Met Glu Lys Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu 1 5 10 15 Gly Trp Leu Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu 20 25 30 Arg Leu Gln Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val 35 40 45 Ser Gly Leu Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly 50 55 60 Pro Glu Phe Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys 65 70 75 80 Glu Arg Leu Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile 85 90 95 Thr Ala Pro Lys Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln 100 105 110 Gly Glu Asn Ser Gly Tyr Ser Thr Leu Thr Phe Gly Lys Gly Thr Met 115 120 125 Leu Leu Val Ser Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <![CDATA[<210> 16]]> <![CDATA[<211> 314]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 16]]> Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 <![CDATA[ ]]> Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Leu Gly Pro Gly Leu Ala Ala Tyr Asn Glu Gln Phe Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 17]]> <![CDATA[<211> 272]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 17]]> Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu 1 5 10 15 Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser 20 25 30 Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser 35 40 45 Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu 50 55 60 Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln 65 70 75 80 Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg 85 90 95 Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu 100 105 110 Tyr Ser Ser Ala Ser Lys Ile Ile Phe Gly Ser Gly Thr Arg Leu Ser 115 120 125 Ile Arg Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 <![CDATA[ ]]>Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[<210> 18]]> <![CDATA[<211> 310]]> <![CDATA[<​​​​​​ <![CDATA[<400> 18]]> Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Arg Ala Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu 115 120 125 Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 19]]> <![CDATA[<211> 277]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 19]]> Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Gly Asn Ser Gly Asn Thr Pro Leu Val Phe Gly Lys 115 120 125 Gly Thr Arg Leu Ser Val Ile Ala Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275 <![CDATA[<210> 20]]> <![CDATA[<211> 313]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 20]]> Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val 1 5 10 15 Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys 20 25 30 Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His 35 40 45 Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile 85 90 95 Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Ser Ser Gly Ser His Gln Glu Thr Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 21]]> <![CDATA[<211> 271]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 21]]> Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu 1 5 10 15 Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser 20 25 30 Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser 35 40 45 Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu 50 55 60 Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln 65 70 75 80 Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg 85 90 95 Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu 100 105 110 Ser Lys Glu Thr Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val 115 120 125 Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135 140 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 180 185 190 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 195 200 205 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 210 215 220 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[ <210> 22]]> <![CDATA[ <211> 317]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 22]]> Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Gly Ile Ser Leu Leu Leu 1 5 10 15 Pro Gly Ser Leu Ala Gly Ser Gly Leu Gly Ala Trp Ser Gln His Pro 20 25 30 Ser Val Trp Ile Cys Lys Ser Gly Thr Ser Val Lys Ile Glu Cys Arg 35 40 45 Ser Leu Asp Phe Gln Ala Thr Thr Met Phe Trp Tyr Arg Gln Phe Pro 50 55 60 Lys Gln Ser Leu Met Leu Met Ala Thr Ser Asn Glu Gly Ser Lys Ala 65 70 75 80 Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys Phe Leu Ile Asn His Ala 85 90 95 Ser Leu Thr Leu Ser Thr Leu Thr Val Thr Ser Ala His Pro Glu Asp 100 105 110 Ser Ser Phe Tyr Ile Cys Ser Ala Arg Ala Gly Gly His Glu Gln Phe 115 120 125 Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val 130 135 140 Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser 145 150 155 160 His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro 165 170 175 Asp His Val Glu Leu Ser Trp Val Trp Asn Gly Lys Glu Val His Ser 180 185 190 Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn 195 200 205 Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe 210 215 220 Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly 225 230 235 240 Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr 245 250 255 Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr 260 265 270 Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu 275 280 285 Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu 290 295 300 Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 315 <![CDATA[ <210> 23]]> <![CDATA[ <211> 271]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 23]]> Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 <![CDATA[ ]]><![CDATA[ ]]><![CDATA[ ]]><![CDATA[ ]]><![CDATA[ ]]><![CDATA[ ]]>Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Asn Phe His Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile 115 120 125 Leu Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135 140 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 180 185 190 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 195 200 205 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 210 215 220 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[<210> 24]]> <![CDATA[<211> 308]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 24]]> Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile His Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Val Ala Ser Ala Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu 115 120 125 Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Phe 305 <![CDATA[<210>]]> 25 <![CDATA[<211> 274]]> <![CDATA[<212> PRT]]> <![CDATA[<HR>]]> <![CDATA[<400>]]> 25 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 <000P4254>Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 It should be noted that there seems to be an error in the original text where "000P4254" in line 52 should probably be "0004254". I've translated it as it is for now, but this might need to be corrected in the original source. Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala 100 105 110 Tyr Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys 115 120 125 Leu Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Serum Serum <![CDATA[ <210> 26]]> <![CDATA[<211> 308]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 26]]> Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Val Glu Ser Ser Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu 115 120 125 Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Phe 305 <![CDATA[ <210> 27]]> <![CDATA[ <211> 271]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 27]]> Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Lys Ala Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val 115 120 125 Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135 140 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 180 185 190 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 195 200 205 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 210 215 220 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[<210> 28]]> <00047​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80 Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95 Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Leu Thr Ser Gly Gly Asp Asn Glu Gln Phe Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 29]]> <![CDATA[<211> 270]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 29]]> Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser 20 25 30 Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser 35 40 45 Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val 50 55 60 His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg 65 70 75 80 Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile 85 90 95 Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Val 100 105 110 Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile Ile Lys 115 120 125 Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser 130 135 140 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 145 150 155 160 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 165 170 175 Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 180 185 190 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn 195 200 205 Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 210 215 220 Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 225 230 235 240 Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 245 250 255 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[ <210> 30]]> <![CDATA[ <211> 314]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 30]]> Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln [[ID=...]] 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Ser Thr Ser Gly Gly Leu Ser Gly Glu Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Leu Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[ <210> 31]]> <![CDATA[ <211> 272]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 31]]> Met Lys Lys His Leu Thr Thr Phe Leu Val Ile Leu Trp Leu Tyr Phe 1 5 10 15 Tyr Arg Gly Asn Gly Lys Asn Gln Val Glu Gln Ser Pro Gln Ser Leu 20 25 30 Ile Ile Leu Glu Gly Lys Asn Cys Thr Leu Gln Cys Asn Tyr Thr Val 35 40 45 Ser Pro Phe Ser Asn Leu Arg Trp Tyr Lys Gln Asp Thr Gly Arg Gly 50 55 60 Pro Val Ser Leu Thr Ile Met Thr Phe Ser Glu Asn Thr Lys Ser Asn 65 70 75 80 Gly Arg Tyr Thr Ala Thr Leu Asp Ala Asp Thr Lys Gln Ser Ser Leu 85 90 95 His Ile Thr Ala Ser Gln Leu Ser Asp Ser Ala Ser Tyr Ile Cys Val 100 105 110 Val Ser Ala Tyr Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val 115 120 125 Val Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[<210> 32]]> <![CDATA[<211> 312]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 32]]> Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Leu Gly Ser Pro Asp Gly Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Phe 305 310 <![CDATA[<210> 33]]> <![CDATA[<211> 279]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 33]]> Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Tyr Asn Ser Tyr Ala Gly Gly Thr Ser Tyr Gly Lys Leu Thr Phe 115 120 125 Gly Gln Gly Thr Ile Leu Thr Val His Pro Asn Ile Gln Asn Pro Asp 130 135 140 Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val 145 150 155 160 Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys 165 170 175 Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser 180 185 190 Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp 195 200 205 Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr 210 215 220 Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys 225 230 235 240 Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile 245 250 255 Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met 260 265 270 Thr Leu Arg Leu Trp Ser Ser 275 <![CDATA[<210> 34]]> <![CDATA[<211> 310]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 34]]> Met Gly Pro Gly Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Ile Phe Gln Tyr Tyr Glu Lys Glu Glu Arg Gly Arg Gly Asn Phe Pro 65 70 75 80 Asp Arg Phe Ser Ala Arg Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn 85 90 95 Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Asp Gly Thr Ser Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 35]]> <![CDATA[<211> 271]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 35]]> Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser 20 25 30 Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser 35 40 45 Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val 50 55 60 His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg 65 70 75 80 Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile 85 90 95 Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Leu Ile Gly 100 105 110 Ala Ser Gly Ser Arg Leu Thr Phe Gly Glu Gly Thr Gln Leu Thr Val 115 120 125 Asn Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135 140 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 165 170 175 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 180 185 190 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 195 200 205 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 210 215 220 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[<210> 36]]> <![CDATA[<211> 310]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400>]]> 36 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Tyr Phe Gly Trp Asn Glu Lys Leu Phe Phe Gly Ser Gly Thr 115 120 125 Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val 130 135 140 Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu 165 170 175 Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp 180 185 190 Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys 195 200 205 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg 210 215 220 Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp 225 230 235 240 Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala 245 250 255 Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln 260 265 270 Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 275 280 285 Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met 290 295 300 Val Lys Arg Lys Asp Phe 305 310 <![CDATA[<210> 37]]> <![CDATA[<211> 276]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 37]]> Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro 20 25 30 Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser 35 40 45 Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys 50 55 60 Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp 65 70 75 80 Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu 85 90 95 Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala 100 105 110 Met Ser Asp Val Ser Gly Gly Tyr Asn Lys Leu Ile Phe Gly Ala Gly 115 120 125 Thr Arg Leu Ala Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leo Trp Serum Serum 275 <![CDATA[<210> 38]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 38]]> Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Thr Thr Pro Asp Gly Thr Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 39]]> <![CDATA[<211> 274]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 39]]> Met Ala Ser Ala Pro Ile Ser Met Leu Ala Met Leu Phe Thr Leu Ser 1 5 10 15 Gly Leu Arg Ala Gln Ser Val Ala Gln Pro Glu Asp Gln Val Asn Val 20 25 30 Ala Glu Gly Asn Pro Leu Thr Val Lys Cys Thr Tyr Ser Val Ser Gly 35 40 45 Asn Pro Tyr Leu Phe Trp Tyr Val Gln Tyr Pro Asn Arg Gly Leu Gln 50 55 60 Phe Leu Leu Lys Tyr Ile Thr Gly Asp Asn Leu Val Lys Gly Ser Tyr 65 70 75 80 Gly Phe Glu Ala Glu Phe Asn Lys Ser Gln Thr Ser Phe His Leu Lys 85 90 95 Lys Pro Ser Ala Leu Val Ser Asp Ser Ala Leu Tyr Phe Cys Ala Val 100 105 110 Arg Asp Met Asn Arg Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg 115 120 125 Leu His Ile Leu Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <![CDATA[<210> 40]]> <![CDATA[<211> 310]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 40]]> Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Arg Ala Glu Gly Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly 305 310 <00068五十一> <![CDATA[<210> 41]]> <![CDATA[<211> 273]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 41]]> It should be noted that there seems to be an error in the translation of " " in the original text you provided. It is translated as "<00068五十一>" which is incorrect. It should probably be something like " ". Please check and correct if necessary.Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Pro Thr Gly Gly Tyr Asn Lys Leu Ile Phe Gly Ala Gly Thr Arg Leu 115 120 125 Ala Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Ser <![CDATA[<210> 42]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 42]]> Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Leu Gly Gly Ala Ser Gln Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 43]]> <![CDATA[<211> 266]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 43]]> Met Lys Leu Val Thr Ser Ile Thr Val Leu Leu Ser Leu Gly Ile Met 1 5 10 15 Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu 20 25 30 Glu Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp 35 40 45 Tyr Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val 50 55 60 Ile His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala 65 70 75 80 Ile Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr 85 90 95 Leu Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Phe Asn Phe Asn Lys 100 105 110 Phe Tyr Phe Gly Ser Gly Thr Lys Leu Asn Val Lys Pro Asn Ile Gln 115 120 125 Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp 130 135 140 Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser 145 150 155 160 Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp 165 170 175 Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn 180 185 190 Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro 195 200 205 Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu 210 215 220 Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu 225 230 235 240 Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn 245 250 255 Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <![CDATA[<210> 44]]> <![CDATA[<211> 309]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <000<![CDATA[<400> 44]]> Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80 Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85 90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr 100 105 110 Ser Ser Gly Glu Thr Asn Glu Lys Leu Phe Phe Gly Ser Gly Thr Gln 115 120 125 Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Phe 305 <![CDATA[<210> 45]]> <![CDATA[<211> 277]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 45]]> Met Thr Arg Val Ser Leu Leu Trp Ala Val Val Val Ser Thr Cys Leu 1 5 10 15 Glu Ser Gly Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asn Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Thr Ala Met Tyr Phe Cys 100 105 110 Ala Phe Gly Tyr Ser Gly Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys 115 120 125 Gly Thr His Leu Ile Ile Gln Pro Tyr Ile Gln Asn Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser (此处可能是编号,原文为275,推测与序列位置等有关,可根据具体情况调整翻译)275 <![CDATA[<210> 46]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 46]]> Met Gly Pro Gly Leu Leu Cys Trp Ala Leu Leu Cys Leu Leu Gly Ala [[ID=(此处可能是编号,原文为45,推测与序列位置等有关,可根据具体情况调整翻译)45]]1 5 10 15 Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His 35 40 45 Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe 50 55 60 Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro 65 70 75 80 Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn 85 90 95 Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Asn Glu Gly Gln Gly Trp Glu Ala Glu Ala Phe Phe Gly Gln Gly 115 120 125 Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe 305 310 <![CDATA[<210> 47]]> <![CDATA[<211> 268]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 47]]> Met Lys Arg Ile Leu Gly Ala Leu Leu Gly Leu Leu Ser Ala Gln Val 1 5 10 15 <000跟我一起读7863> <00078跟我一起读66>Cys Cys Val Arg Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile 20 25 30 It should be noted that in the original text, the tag and seem to have incorrect display in the provided format. They are presented as "跟我一起读7863" and "跟我一起读66" respectively. This might be a display issue during input. The translation is done based on the original content as accurately as possible while maintaining the original tags. Leu Gln Glu Gly Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser 35 40 45 Val Asn Asn Leu Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile 50 55 60 Asn Leu Phe Tyr Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser 65 70 75 80 Ala Thr Thr Val Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser 85 90 95 Ser Gln Thr Thr Asp Ser Gly Val Tyr Phe Cys Ala Val His Asn Phe 100 105 110 Asn Lys Phe Tyr Phe Gly Ser Gly Thr Lys Leu Asn Val Lys Pro Asn 115 120 125 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 130 135 140 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 145 150 155 160 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val 165 170 175 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 180 185 190 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 195 200 205 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 210 215 220 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 225 230 235 240 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <![CDATA[ <210> 48]]> <![CDATA[ <211> 322]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 48]]> Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Leu Gly Gln Gly 115 120 125 Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu 130 135 140 Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 145 150 155 160 Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 165 170 175 Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 180 185 190 Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 195 200 205 Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 210 215 220 Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 225 230 235 240 Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 245 250 255 Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 260 265 270 Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala 275 280 285 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 290 295 300 Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser 305 310 315 320 Arg Gly <![CDATA[<210> 49]]> <![CDATA[<211> 276]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 49]]> Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Asn Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly 115 120 125 Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala Val 130 135 140 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 145 150 155 160 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 165 170 175 Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe 180 185 190 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 195 200 205 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 210 215 220 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 225 230 235 240 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 245 250 255 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 260 265 270 Leu Trp Ser Ser 275 <![CDATA[<210> 50]]> <![CDATA[<211> 323]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 50]]> Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Pro Thr Gly Thr Ser 115 120 125 Gly Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 130 135 140 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro 145 150 155 160 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 165 170 175 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 180 185 190 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 195 200 205 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu 210 215 220 Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 225 230 235 240 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 245 250 255 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 260 265 270 Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser 275 280 285 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 290 295 300 Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 305 310 315 320 Ser Arg Gly <![CDATA[ <210> 51]]> <![CDATA[ <211> 273]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 51]]> Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 <000854​​​​​​​​​​​​​​​​​​​​​​​​​​​65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Leu Asn Arg Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu 115 120 125 His Ile Leu Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Ser <![CDATA[<210> 52]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 52]]> Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val 1 5 10 15 Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys 20 25 30 Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His 35 40 45 Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile 85 90 95 Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser 100 105 110 Arg Leu Pro Ser Arg Thr Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 [[ID=二十八]] <![CDATA[<210> 53]]> <![CDATA[<211> 270]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 53]]> Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 [[ID=五四]] Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn 100 105 110 Ser Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg 115 120 125 Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser 130 135 140 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 145 150 155 160 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 165 170 175 Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 180 185 190 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn 195 200 205 Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 210 215 220 Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 225 230 235 240 Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 245 250 255 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <![CDATA[ <210> 54]]> <![CDATA[<211> 321]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 54]]> Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Gly Leu Gly Thr 115 120 125 Gly Asp Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 130 135 140 Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro 145 150 155 160 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 165 170 175 Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 180 185 190 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 195 200 205 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu 210 215 220 Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 225 230 235 240 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 245 250 255 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 260 265 270 Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser 275 280 285 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 290 295 300 Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 305 310 315 320 Phew <![CDATA[<210> 55]]> <![CDATA[<211> 273]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 55]]> Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Leu Tyr Asn Asn Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu 115 120 125 Thr Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Ser <![CDATA[<210> 56]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 56]]> Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Pro Gly Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Leu Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 57]]> <![CDATA[<211> 275]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 57]]> Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala 100 105 110 Val Ile Ser Asn Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr 115 120 125 Arg Leu Thr Ile Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr 130 135 140 Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr 145 150 155 160 Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 165 170 175 Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys 180 185 190 Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala 195 200 205 Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser 210 215 220 Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr 225 230 235 240 Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile 245 250 255 Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu 260 265 270 Trp Ser Ser 275 [[ID=I5]] <![CDATA[<210> 58]]> <![CDATA[<211> 311]]> <![CDATA[<212> PRT]]> / <ID=29><![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 58]]> Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 / <ID=43> Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys 20 25 30 It should be noted that there may be some inaccuracies in the translation due to the complexity and specific nature of patent - related biological sequence texts. It is recommended to double - check with relevant experts or in - depth reference materials for highly accurate translation in the context of patent applications.Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His 35 40 45 Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe 50 55 60 Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro 65 70 75 80 Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn 85 90 95 Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Pro Trp Asp Ser Pro Asn Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 59]]> <![CDATA[<211> 274]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 59]]> Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro 20 25 30 Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser 35 40 45 Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys 50 55 60 Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp 65 70 75 80 Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu 85 90 95 Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala 100 105 110 Met Ser Glu Ala Ala Gly Asn Lys Leu Thr Phe Gly Gly Gly Thr Arg 115 120 125 Val Leu Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Serum Serum <![CDATA[ <210> 60]]> <![CDATA[ <211> 309]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 60]]> Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Thr Glu Ala Gly Val Ala Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu 50 55 60 ​​Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Tyr Thr Asn Gln Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg 115 120 125 Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Light Arg Light Asp Phe 305 <![CDATA[ <210> 61]]> <![CDATA[ <211> 273]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 61]]> Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 [[ID=5​Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Leu Asn Gln Ala Gly Thr Ala Leu Ile Phe Gly Lys Gly Thr Thr Leu 115 120 125 Ser Val Ser Ser Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Ser <![CDATA[<210> 62]]> <![CDATA[<211> 314]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 62]]> Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Ala Glu Thr Gly Pro Trp Leu Gly Asn Glu Gln Phe Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285 Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300 Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 63]]> <![CDATA[<211> 277]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 63]]> [[ID=***]]Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 It should be noted that there seems to be an error in the ID numbering in the original text. The "***" in ID=49 is likely incorrect. Please check and correct it if possible for a more accurate translation. Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Tyr Arg Trp Ala Gln Gly Gly Ser Glu Lys Leu Val Phe Gly Lys 115 120 125 Gly Thr Lys Leu Thr Val Asn Pro Tyr Ile Gln Lys Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Serum Serum 275 <![CDATA[<210> 64]]> <![CDATA[<211> 313]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 64]]> Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe Tyr Phe Leu Gly Ala 1 5 10 15 Gly Leu Met Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile 0 20 25 30 Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His 35 40 45 <00107?1> Note: There seems to be an error in the original text where "?1" appears in the translation of line 53. It should be "01" to match the original tag format. If this is a formatting issue in the original, the translation should be adjusted accordingly.Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu 50 55 60 Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser 65 70 75 80 Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr 85 90 95 Leu Glu Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Thr 100 105 110 Glu Leu Trp Ser Ser Gly Gly Thr Gly Glu Leu Phe Phe Gly Glu Gly 115 120 125 Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[<210> 65]]> <![CDATA[<211> 206]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 65]]> <![CDATA[<400> 65]]>Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr 1 5 10 15 Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp 20 25 30 Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gly Pro Ser 35 40 45 Gly Thr Tyr Lys Tyr Ile Phe Gly Thr Gly Thr Arg Leu Lys Val Leu 50 55 60 Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser 65 70 75 80 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 85 90 95 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 100 105 110 Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 115 120 125 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn 130 135 140 Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 145 150 155 160 Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 165 170 175 Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 180 185 190 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 195 200 205 <![CDATA[ <210> 66]]> <![CDATA[ <211> 311]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 66]]> Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 [[ID=1,7]] Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His <![CDATA[ ]]> 35 40 45 [[ID=4,0]]Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Pro Gly Gly Ser Gly Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <![CDATA[ <210> 67]]> <![CDATA[ <211> 274]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Homo sapiens]]> The original text to be translated is as below which wraped by :<![CDATA[<400> 67]]> <00Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 [[ID=Id=31]] Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser <00Id=45]] 50 55 60 <00111Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly ​​​​​​​​ 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Val Ser Gly Gly Gly Ala Asp Gly Leu Thr Phe Gly Lys Gly Thr His 115 120 125 Leu Ile Ile Gln Pro Tyr Ile Gln Lys Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <![CDATA[<210> 68]]> <![CDATA[<211> 319]]> <![CDATA[<212> PRT]]><![CDATA[<![CDATA[<213> Homo sapiens]]>]]> <![CDATA[<400> 68]]> Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 (注:原文中的翻译存在一处错误,原文为<![CDATA[<213> 智人(Homo sapiens)]]>,翻译应为<![CDATA[<213> Homo sapiens]]>,我在翻译中已修正。)​Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Gly Arg Gly Gly 115 120 125 Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu Glu Asp 130 135 140 Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu 145 150 155 160 Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr 165 170 175 Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys 180 185 190 Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln 195 200 205 Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val 210 215 220 Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val 225 230 235 240 Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala 245 250 255 Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp 260 265 270 Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr 275 280 285 Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 290 295 300 Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe 305 310 315 <![CDATA[<210> 69]]> <![CDATA[<211> 273]]> <![CDATA[<212> PRT]]> [[ID=​​​​​​​​​​​​​​​​​Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Lys Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Leu Tyr Asn Asn Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu 115 120 125 Thr Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Ser <![CDATA[<210> 70]]> ' <![CDATA[<211> 243]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> '<![CDATA[<220>]]> <![CDATA[<223> R11P3D3KE β-chain]]> <![CDATA[<400> 70]]> Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg Phe 1 5 10 15 Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln Pro It should be noted that there may be some inaccuracies in the translation due to the complexity and some unclear parts of the original text. For example, the handling of some special tags and the overall context understanding might need further improvement. If possible, it would be beneficial to have more background information or clarify some aspects of the text for a more accurate translation. 20 25 30 Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Pro Gly 35 40 45 Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 50 55 60 Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro 65 70 75 80 Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 85 90 95 Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 100 105 110 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 115 120 125 Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu 130 135 140 Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 145 150 155 160 Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 165 170 175 Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 180 185 190 Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser 195 200 205 Ala Thr Leu Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 210 215 220 Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp 225 230 235 240 Ser Arg Gly <![CDATA[<210> 71]]> <![CDATA[<211> 223]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 71]]> Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu 1 5 10 15 Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu 20 25 30 Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala 35 40 45 Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Ile Asp 50 55 60 Asn Gln Gly Gly Lys Leu Ile Phe Gly Gln Gly Thr Glu Leu Ser Val 65 70 75 80 Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 85 90 95 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 100 105 110 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 115 120 125 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 130 135 140 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 145 150 155 160 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 165 170 175 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 180 185 190 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 195 200 205 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 210 215 220 <![CDATA[<210> 72]]> <![CDATA[<211> 307]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> [[ID=​​​​​​​​​​ Gly Leu Val Asp Ala Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Pro Lys Ser Gly His 35 40 45 Asp Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe 50 55 60 Ile Phe Gln Tyr Tyr Glu Glu Glu Glu Arg Gln Arg Gly Asn Phe Pro 65 70 75 80 Asp Arg Phe Ser Gly His Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn 85 90 95 Val Asn Ala Leu Leu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Gln Leu Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr 115 120 125 Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe 130 135 140 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val 145 150 155 160 Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 165 170 175 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro 180 185 190 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 225 230 235 240 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val 260 265 270 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg 290 295 300 Lys Asp Phe 305 <![CDATA[<210> 73]]> <![CDATA[<211> 270]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 73]]> Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 ...

Claims

1. A vector comprising a nucleotide sequence S1 encoding a CD8α polypeptide, a nucleotide sequence S2 encoding a CD8β polypeptide, a nucleotide sequence S3 encoding a T-cell receptor (TCR) α polypeptide, and a nucleotide sequence S4 encoding a TCRβ polypeptide, wherein the nucleotide sequences S1, S2, S3, and S4 are arranged in a 5' to 3' orientation of S2-S1-S4-S3; and wherein: (i) The CD8α polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, and the CD8β polypeptide contains an amino acid sequence that is up to 90% identical to SEQ ID NO: 12; and (ii) The TCRα polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 13, and the TCRβ polypeptide contains an amino acid sequence that is up to 90% identical to SEQ ID NO:

14.

2. The vector as claimed in claim 1 further comprises a nucleotide sequence S5 encoding a 2A peptide and a nucleotide sequence S6 encoding a linker peptide, wherein S5 and S6 are located between S1 and S2, between S1 and S4, and / or between S3 and S4.

3. The vector as claimed in claim 2, wherein the 2A peptide is selected from P2A (SEQ ID NO: 3), T2A (SEQ ID NO: 4), E2A (SEQ ID NO: 5) and F2A (SEQ ID NO: 6).

4. The vector as described in claim 2, wherein the linker peptide is GSG or SGSG (SEQ ID NO: 8).

5. The vector as claimed in claim 1 further comprises a nucleotide sequence S7 encoding a furin peptide (SEQ ID NO: 2), wherein S7 is located between S1 and S2, between S1 and S4 and / or between S3 and S4.

6. The vector as claimed in claim 1 further comprises a post-transcriptional regulatory element (PRE) sequence selected from a marmot PRE (WPRE) and a hepatitis B virus (HBV) PRE (HPRE).

7. The vector as described in claim 1 further comprises a promoter sequence that controls transcription of S1 to S7, wherein the promoter sequence is selected from cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR (MNDU3) containing myeloproliferative sarcoma virus enhancer, ubiquitin C promoter, EF-1 α promoter, and murine stem cell virus (MSCV) promoter.

8. The vector as described in claim 1, comprising the nucleotide sequence PTE CD8 TCR WPRE (SEQ ID NO: 94).

9. The carrier as described in claim 1, comprising a groundhog PRE (WPRE) of SEQ ID NO: 265 or SEQ ID NO:

266.

10. The vector as claimed in claim 1, wherein the vector is a viral vector selected from the following: adenovirus, poxvirus, alpha virus, arenavirus, flavivirus, rod-shaped virus, retrovirus, lentivirus, herpesvirus, paramyxovirus, and microRNA virus.

11. The vector as claimed in claim 10, wherein the vector is pseudotyped with an envelope protein selected from the following viruses: native feline endogenous virus (RD114), a chimeric form of RD114 (RD114TR), gibberish leukemia virus (GALV), a chimeric form of GALV (GALV-TR), bitropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl cholera virus (FPV), Ebola virus (EboV), lymphocytic choriomeningitis virus (LCMV), or baboon retrovirus envelope glycoprotein (BaEV).

12. A method for preparing T cells for immunotherapy, comprising the steps of: activating the T cells, transducing the activated T cells with a vector as described in any one of claims 1 to 11, and expanding the transduced T cells.

13. The method as described in claim 12, wherein the T cells comprise αβ T cells, γδ T cells and / or natural killer T cells.

14. The method as described in claim 12 or 13, wherein the T cells comprise αβ T cells.

15. The method as described in claim 14, wherein the activation is performed in the presence of anti-CD3 antibody and anti-CD28 antibody.

16. The method as described in claim 12 or 13, wherein the amplification is performed in the presence of IL-7 and IL-15.

17. An expanded population of T cells prepared by the method described in any one of claims 12 to 16.

18. Use of an expanded population of T cells as described in claim 17 or a composition comprising thereto for the manufacture of a medicament for the treatment of a patient with cancer.

19. Use of a carrier or composition comprising any one of claims 1 to 11 for manufacturing a medicament for treating a patient with cancer.

20. The use as described in claim 18 or 19, wherein the cancer is selected from the group consisting of: melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, stomach cancer, prostate cancer, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, neuroblastoma, basal cell carcinoma, bone cancer, brain tumor, bronchial adenoma, Burkitt's disease. Kassym-Jolly lymphoma, tumor of unknown primary origin, central nervous system lymphoma, cervical cancer, childhood cancer, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, connective tissue proliferative small round cell tumor, endometrial cancer, ependymoma, Ewing's sarcoma, germ cell tumors, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gliomas, head and neck cancer, heart cancer, Hodgkin's lymphoma, pharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, lip and oral cancer, liposarcoma, lung cancer, lymphoma Macroglobulinemia, osteosarcoma with malignant fibrous histiocytoma, medulloblastoma, mesothelioma, metastatic squamous neck cancer with occult primary lesions, oral cancer, multiple endocrine tumor syndrome, myelodyplasia syndrome, nasal and paranasal cavity cancer, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer (islet cells), parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleura Pulmonary germ cell tumor, plasma cell neoplasm, primary central nervous system lymphoma, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor, cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

21. The use as described in claim 20, wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.

22. The use as described in claim 20, wherein the brain tumor is selected from the group consisting of: cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, and visual pathway and hypothalamic glioma.

23. The use as described in claim 20, wherein the trophoblastic tumor is a gestational trophoblastic tumor.

24. The use as described in claim 20, wherein the leukemia is selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, and myeloid leukemia.

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