Two types of gene vectors for generating CAR-T cells and their uses

Abstract

This invention provides a two-gene retroviral vector composition comprising a polynucleotide encoding an anti-CD7 chimeric activation receptor (CAR) and a polynucleotide encoding an anti-CD7 protein expression blocker. Methods for producing this composition and methods of use in cancer therapy are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 767,069, filed on November 14, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0003] References to sequence listings

[0004] This application contains a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The sequence listing was created on November 14, 2019, is titled "119419-5007-WO-Sequence-Listing_ST25.txt" and is 139,264 bytes in size. Background Art

[0005] Chimeric antigen receptors (CARs) can redirect immune cells to specifically recognize and kill tumor cells. CARs are artificial multimolecular proteins composed of an antibody single-chain variable region (scFv) linked to a signaling molecule via a transmembrane domain. When the scFv binds to its cognate antigen, signal transduction is triggered, resulting in the killing of tumor cells by CAR-expressing cytotoxic T lymphocytes (Eshhar Z, Waks T et al., PNAS USA, 90(2):720-724, 1993; Geiger TL et al., J Immunol., 162(10):5931-5939, 1999; Brentjens RJ et al., Nat Med. 9(3):279-286, 2003; Cooper LJ et al., Blood 101(4):1637-1644, 2003; Imai C et al., Leukemia, 18:676-684, 2004). Clinical trials with autologous T lymphocytes expressing CARs have shown positive responses in patients with B-cell refractory leukemias and lymphomas (see, e.g., Till BG et al., Blood 119(17):3940-3950, 2012; Maude SL et al., N Engl J Med. 371(16):1507-1517, 2014).

[0006] The development of CAR technology targeting T-cell malignancies has lagged far behind the progress made in B cells. Novel therapies for T-cell malignancies are needed, but progress has been slow to date. In particular, there is a lack of effective immunotherapy options, and the treatment of T-cell acute lymphoblastic leukemia (T-ALL) relies on intensive chemotherapy and hematopoietic stem cell transplantation. Although aggressive treatment regimens are associated with significant morbidity, the results of these approaches are far from satisfactory.

[0007] CAR-T cells have recently been developed in which the target antigen of the CAR-T is itself expressed in the CAR-T cell (Png et al., Blood, 2017, 1(25): 2348-2360, WO 2018 / 098306). To avoid suicide (e.g., killing), the CAR-T cell also expresses a PEBL that can reduce the expression of the target antigen on the surface of the CAR-T cell. To generate viable CAR-T cells, a protein expression blocker (PEBL) protein is first expressed to bind and sequester the target protein, and then the CAR is expressed. Since the target antigen is pre-existing on the cell surface of the resulting engineered T cell, the simultaneous expression of CAR and PEBL results in a killer molecule. In particular, the pre-existing cell surface target antigen is not easily sequestered by the newly expressed PEBL protein and is recognized and targeted by the newly expressed CAR protein.

[0008] An alternative to simultaneous expression could be sequential expression. However, expressing PEBL first and then CAR in T cells raises some questions about the clinical implementation of PEBLCAR-T cells. First, the sequential engineering of T cells requires the separate manufacture and administration of different viral vectors, one for PEBL and the other for CAR. This increases cost and time, and increases the complexity of experimental operations to produce engineered CAR-T cells. In addition, the sequential engineering of T cells leads to a complex mix of engineered cells in the final clinical product, posing challenges in product characterization, uniformity, and efficacy. Since only a portion of the T cells integrate the introduced genes in each engineering step, the final product (engineered T cells) will contain some cells that only receive the PEBL gene, some cells that only receive the CAR gene, and some cells that receive both genes at the same time.

[0009] In summary, there is a clear unmet need for new treatment options for patients with B-cell malignancies. Additionally, methods are needed to generate engineered CAR-T cells and eliminate CAR-mediated T-cell suicide or killing. Summary of the Invention

[0010] In some aspects, the present invention provides a bicistronic retroviral vector comprising: (a) a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR), the first polynucleotide having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOS: 28-31; (b) a second polynucleotide encoding an internal ribosome entry site (IRES) or a ribosomal codon skipping site; (c) a third polynucleotide encoding an anti-CD7 protein expression blocker (PEBL), which has at least 90% sequence identity to the amino acid sequence of SEQ ID NOS: 24-27, wherein the first polynucleotide is operably linked to the second polynucleotide, and the second polynucleotide is operably linked to the third polynucleotide.

[0011] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of any one of SEQ ID NOS: 28-31.

[0012] In some embodiments, the anti-CD7 PEBL comprises the amino acid sequence of any one of SEQ ID NOS: 24-27.

[0013] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO: 29, and the anti-CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 25.

[0014] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO: 31, and the anti-CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 27.

[0015] In some embodiments, the IRES is derived from encephalomyocarditis virus (EMCV) or an enterovirus.

[0016] In some embodiments, the ribosomal codon skipping site comprises a self-cleaving 2A peptide. In some embodiments, the self-cleaving 2A peptide is selected from the group consisting of a F2A peptide (foot-and-mouth disease virus 2A peptide), an E2A peptide (equine rhinitis A virus 2A peptide), a P2A peptide (porcine tetanus virus-1 2A peptide), and a T2A peptide (TaV 2A).

[0017] In some embodiments, the bicistronic retroviral vector has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0018] In some embodiments, the bicistronic retroviral vector has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the bicistronic retroviral vector comprises the nucleic acid sequence of SEQ ID NO:13.

[0019] In some embodiments, the bicistronic retroviral vector further comprises a promoter element.

[0020] In some embodiments, the promoter element is selected from the group consisting of a CMV promoter, an EF1α promoter, an EFS promoter, an MSCV promoter, and a PGK promoter.

[0021] In some embodiments, the promoter element has at least 90% sequence identity to a nucleic acid sequence selected from any one of SEQ ID NOS: 6-10.

[0022] In some embodiments, the promoter element comprises a nucleic acid sequence selected from any one of SEQ ID NOS: 6-10.

[0023] In some embodiments, the bicistronic retroviral vector has at least 90% sequence identity to any one nucleic acid sequence selected from SEQ ID NOs: 14-16.

[0024] In some embodiments, the bicistronic retroviral vector comprises a nucleic acid sequence selected from any one of SEQ ID NOS: 14-16.

[0025] In some embodiments, the retroviral vector is a lentiviral vector.

[0026] In some aspects, provided herein are engineered immune cells comprising any of the bicistronic retroviral vectors outlined herein.

[0027] In some embodiments, the engineered immune cells are allogeneic T cells. In some embodiments, the engineered immune cells are autologous T cells.

[0028] In some embodiments, the engineered immune cells have reduced surface expression of CD7 and express an anti-CD7 CAR compared to corresponding immune cells.

[0029] In some aspects, provided herein is a pharmaceutical composition comprising any of the engineered immune cells described herein and a pharmaceutically effective carrier.

[0030] In some aspects, provided herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of any of the engineered immune cells described herein or a pharmaceutical composition thereof.

[0031] In some aspects, provided herein is a method of producing an engineered immune cell, the method comprising transducing an immune cell with any one of the bicistronic retroviral vectors described herein and recovering the engineered immune cell.

[0032] In some embodiments, the immune cells are selected from peripheral blood mononuclear cells, isolated CD4+ T cells, isolated CD8+ T cells, and isolated CD3+ T cells.

[0033] In some embodiments, the engineered immune cells have reduced surface expression of CD7 and express an anti-CD7 CAR compared to corresponding immune cells.

[0034] In some aspects, provided herein is a recombinant retroviral vector comprising: (a) a first promoter element operably linked to a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR), the first polynucleotide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30; and (b) a second promoter element operably linked to a second polynucleotide encoding an anti-CD7 protein expression blocker (PEBL), the second polynucleotide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26.

[0035] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

[0036] In some embodiments, the anti-CD7 PEBL comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26.

[0037] In some embodiments, the first promoter element and / or the second promoter element is selected from the group consisting of a CMV promoter, an EF1α promoter, an EFS promoter, an MSCV promoter, and a PGK promoter.

[0038] In some embodiments, the first promoter element and / or the second promoter element has at least 90% sequence identity to a nucleic acid sequence selected from any one of SEQ ID NOs: 6-10.

[0039] In some embodiments, the first promoter element and / or the second promoter element comprises a nucleic acid sequence selected from any one of the group consisting of SEQ ID NOS: 6-10.

[0040] In some embodiments, the first promoter and the second promoter share less than 95% sequence identity.

[0041] In some embodiments, a first promoter element operably linked to a first polynucleotide is 5' to a second promoter element operably linked to a second polynucleotide.

[0042] In some embodiments, the second promoter element operably linked to the second polynucleotide is 5' to the first promoter element operably linked to the first polynucleotide.

[0043] In some embodiments, the recombinant retroviral vector has at least 90% sequence identity to any one nucleic acid sequence selected from SEQ ID NOs: 18-23.

[0044] In some embodiments, the retroviral vector is a lentiviral vector.

[0045] Also provided are engineered immune cells comprising any of the recombinant retroviral vectors described herein.

[0046] In some embodiments, the engineered immune cells are engineered T cells. In some embodiments, the engineered immune cells are autologous T cells.

[0047] In some embodiments, the engineered immune cells have reduced surface expression of CD7 and express an anti-CD7 CAR compared to corresponding immune cells.

[0048] In some aspects, provided herein is a pharmaceutical composition comprising any of the engineered immune cells described herein and a pharmaceutically effective carrier.

[0049] In some aspects, provided herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of any of the engineered immune cells described herein or a pharmaceutical composition thereof.

[0050] In some aspects, provided herein are methods of producing engineered immune cells comprising transducing an immune cell with any of the recombinant retroviral vectors described herein and recovering the engineered immune cell.

[0051] In some embodiments, the immune cells are selected from peripheral blood mononuclear cells, isolated CD4+ T cells, isolated CD8+ T cells, and isolated CD3+ T cells.

[0052] In some embodiments, the engineered immune cells have reduced surface expression of CD7 and express an anti-CD7 CAR compared to corresponding immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1A and Figure 1B The results of flow cytometry ( Figure 1A ) and Western blot ( Figure 1B) CAR and PEBL expressed by transduced primary T cells. Primary T cells were transduced with the indicated retroviruses (e.g., PEBL, CAR, PEBL, and CAR in sequence; PEBL-IRES-CAR and CAR-P2A-PEBL) and analyzed by flow cytometry for CD7 and CAR expression. Cell lysates of primary T cells transduced with the indicated retroviruses were analyzed by Western blot for β-actin, Myc-tagged PEBL, CAR, and endogenous CD3ζ expression.

[0054] Figure 2A Figure 2F A schematic diagram of the bicistronic promoter 1-CAR-promoter 2-PEBL lentiviral construct is provided. Figure 2A A schematic diagram of an exemplary dual promoter construct comprising MSCV-promoter-anti-human CD7 (TH69) CAR-EFS promoter-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 19, is depicted. Figure 2B A schematic diagram of an exemplary dual promoter construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 18, is depicted. Figure 2C A schematic diagram of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (TH69) CAR-EFS promoter-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 23, is depicted. Figure 2D A schematic diagram of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 22, is depicted. Figure 2E A schematic diagram of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (3A1F) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 20, is depicted. Figure 2F A schematic diagram of an exemplary dual promoter construct is depicted, comprising PGK promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (3A1F) PEBL, such as one of SEQ ID NO: 21.

[0055] Figure 3 CAR and CD7 expression by transduced primary T cells according to flow cytometry is shown. Primary T cells were transduced with the indicated dual promoter lentiviruses and analyzed by flow cytometry 5 and 14 days after transduction.

[0056] Figure 4A Figure 4C A schematic diagram of the bicistronic CAR-P2A-PEBL lentiviral construct is provided. Figure 4A A schematic diagram of an exemplary bicistronic construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 14, is depicted. Figure 4B A schematic diagram of an exemplary bicistronic construct comprising EF1a promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 15, is depicted. Figure 4C A schematic diagram of an exemplary bicistronic construct comprising EFS promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 16, is depicted.

[0057] Figure 5 Shown are the expressions of CAR and CD7 in primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed by flow cytometry 3, 6, and 9 days after transduction.

[0058] Figure 6 A graph showing expression of CAR and CD7 by transduced primary T cells according to flow cytometry is shown. Primary T cells were transduced with the indicated bicistronic CD7CAR-P2A-CD7PEBL and CD19CAR lentiviruses and analyzed by flow cytometry 5 and 14 days after transduction.

[0059] Figure 7A and Figure 7B The results of flow cytometry ( Figure 7A ) and Western blotting ( Figure 7B ) Expression of CAR and PEBL expressed by transduced primary T cells. Primary T cells were transduced with two independently generated large quantities of MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed by flow cytometry for CD7 and CAR expression. Cell lysates from transduced cells were analyzed by Western blotting for expression of β-actin, Myc-tagged PEBL, CAR, and endogenous CD3ζ.

[0060] Figure 8 CAR and CD7 expression by transduced primary T cells according to flow cytometry is shown. + and CD8 + Positively selected T cells and CD3 +Positively selected T cells were transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus. Cells were analyzed by flow cytometry 4, 7, and 10 days after transduction.

[0061] Figure 9A Primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus and cultured in serum-free TexMACS medium or TexMACS medium supplemented with 3% human AB serum express CAR and CD7. Figure 9B Shown are the total fold expansions of transduced cells 11 days after activation (biological mean ± SEM).

[0062] Figure 10A and Figure 10B The percentage of CAR+ T cells 11 days after activation is shown ( Figure 10A ) and the total expansion fold of transduced cells ( Figure 10B ) (Biological mean ± SEM). Primary T cells were cultured in serum-free TexMACS medium and transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus on day 1, 2, 3, or 4 after activation.

[0063] Figure 11 CAR and CD7 expression by transduced primary T cells as determined by flow cytometry. + and CD8 + Positively selected T cells were transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus at the indicated multiplicity of infection (MOI). Cells were analyzed by flow cytometry 3 and 9 days after transduction.

[0064] Figure 12A and Figure 12B The percentage of CAR+ T cells 11 days after activation is shown ( Figure 12A ) and the transgenic vector copy number (VCN) of transduced cells ( Figure 12B (Biological mean values). Primary T cells were cultured in serum-free TexMACS medium and transduced with the MSCV-CD7CAR-P2A-CD7PEBL lentivirus at an MOI of 3, 5, or 10. T cells were analyzed for CAR expression by flow cytometry. Genomic DNA was extracted from the transduced cells, and transgenic VCN was confirmed by RT-qPCR analysis.

[0065] Figures 13A-13E The expression of various surface markers on primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus 11 days after activation is shown. CAR and CD7 in transduced cells were analyzed by flow cytometry. Figure 13A ), CD3 and CD14 / CD19 / CD56 ( Figure 13B ), CD4 and CD8 ( Figure 13C ), CD45RO and CCR7 ( Figure 13D ) and PD-1 and Tim-3 ( Figure 13E ). Primary T cells from 3 unique donors transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus at MOI 10 were analyzed in triplicate.

[0066] Figure 14A and Figure 14B showed that the secretion of IFNγ Figure 14A ) and cytotoxicity ( Figure 14B ) Functional responses of PEBL-CAR-T cells generated with the MSCV-CD7CAR-P2A-CD7PEBL lentivirus against CD7+ Jurkat cells and CD7- Nalm6 cells. IFNγ secretion was measured in the culture supernatant of PEBL-CAR-T cells co-cultured with Jurkat or Nalm6 cells at the indicated E:T ratios for 24 hours (technical mean ± SD). Cytolytic activity of PEBL-CAR T cells was measured after 4 hours of co-culture with Jurkat or Nalm6 cells at the indicated E:T ratios (technical mean ± SD).

[0067] Figure 15 The nucleic acid sequence of CPPT-CMV-MCS-PGK-GFP-WPRE is depicted (SEQ ID NO: 1). CPPT is bold, CMV promoter is single underlined, PGK promoter is double underlined, GFP is bold / single underlined, and WPRE element is bold / double underlined.

[0068] Figure 16 The nucleic acid sequence of an exemplary anti-human CD7 PEBL based on antibody TH69 is depicted (SEQ ID NO: 2). The CD8 signal peptide begins at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the myc-KDEL peptide is double underlined, and the stop codon terminates the sequence.

[0069] Figure 17 The nucleic acid sequence of an exemplary anti-human CD7 PEBL based on antibody 3A1F is depicted (SEQ ID NO: 3). The CD8 signal peptide begins at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the myc-KDEL peptide is double underlined, and the stop codon terminates the sequence.

[0070] Figure 18 The nucleic acid sequence of an exemplary anti-human CD7 CAR based on antibody TH69 is depicted (SEQ ID NO: 4). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the CD8a hinge and transmembrane domain are double underlined, the 4-1BB signaling domain is located between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, the CD3ζ signaling domain is represented by bold / double underlined, and the stop codon terminates the sequence.

[0071] Figure 19 The nucleic acid sequence of an exemplary anti-human CD7 CAR based on antibody 3A1F is depicted (SEQ ID NO: 5). The CD8 signal peptide begins at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the CD8a hinge and transmembrane domain are double underlined, the 4-1BB signaling domain is located between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, which is indicated in bold / double underlined, and the stop codon terminates the sequence.

[0072] Figure 20 The nucleic acid sequence of an exemplary CMV promoter is depicted (SEQ ID NO: 6).

[0073] Figure 21 The nucleic acid sequence of an exemplary EF1α promoter is depicted (SEQ ID NO: 7).

[0074] Figure 22 The nucleic acid sequence of an exemplary EFS promoter is depicted (SEQ ID NO: 8).

[0075] Figure 23 The nucleic acid sequence of an exemplary MSCV promoter is depicted (SEQ ID NO: 9).

[0076] Figure 24 The nucleic acid sequence of an exemplary PGK promoter is depicted (SEQ ID NO: 10).

[0077] Figure 25 Depicted is the nucleic acid sequence of an exemplary bicistronic construct comprising anti-human CD7 (TH69), PEBL-IRES-anti-human CD7 (TH69) CAR (SEQ ID NO: 11). Anti-human CD7 (TH69) PEBL is in regular font, IRES is in bold, and anti-human CD7 (TH69) CAR is double underlined.

[0078] Figure 26Depicted is the nucleic acid sequence of an exemplary bicistronic construct comprising anti-human CD7 (TH69) CAR-IRES-anti-human CD7 (TH69) PEBL (SEQ ID NO: 12). Anti-human CD7 (TH69) CAR is shown in normal font, IRES is shown in bold, and anti-human CD7 (TH69) PEBL is marked with a single underline.

[0079] Figure 27 Depicted is the nucleic acid sequence of an exemplary bicistronic construct comprising anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL (SEQ ID NO: 13). Anti-human CD7 (TH69) CAR is in regular font, P2A is in bold, and anti-human CD7 (TH69) PEBL is single underlined.

[0080] Figure 28A and Figure 28B The nucleic acid sequence of an exemplary bicistronic construct comprising an MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL is depicted (SEQ ID NO: 14). The MSCV promoter is double-underlined, the restriction enzyme site and Kozak sequence are located between the MSCV promoter and the CAR, the anti-human CD7 (TH69) CAR is in bold, P2A is in normal font, and the anti-human CD7 (TH69) PEBL is single-underlined.

[0081] Figure 29A and Figure 29B The nucleic acid sequence of an exemplary bicistronic construct comprising EF1a promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL is depicted (SEQ ID NO: 15). The EF1α promoter is double-underlined, the restriction enzyme site and Kozak sequence are located between the EF1α promoter and the CAR, the anti-human CD7 (TH69) CAR is in bold, P2A is in regular font, and the anti-human CD7 (TH69) PEBL is single-underlined.

[0082] Figure 30A and Figure 30B Depicted is the nucleic acid sequence of an exemplary bicistronic construct comprising EFS promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL (SEQ ID NO: 16). The EFS promoter is double-underlined, the restriction enzyme site and Kozak sequence are between the EFS promoter and CAR, the anti-human CD7 (TH69) CAR is in bold, P2A is in regular font, and the anti-human CD7 (TH69) PEBL is single-underlined.

[0083] Figure 31A and Figure 31B Described is the nucleic acid sequence of the exemplary dual promoter construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-PGK promoter-anti-human CD7 (TH69) PEBL (SEQ ID NO: 17). The MSCV promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the MSCV promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the PGK promoter, the PGK promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are located between the PGK promoter and the PEBL, the anti-human CD7 (TH69) PEBL is bolded / single-underlined, and the restriction enzyme site (italics) terminates the sequence.

[0084] Figure 32A and Figure 32B Described is the nucleic acid sequence of the exemplary dual promoter construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL (SEQ ID NO: 18). The MSCV promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the MSCV promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EF1a promoter, the EF1a promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EF1a promoter and the PEBL, and the anti-human CD7 (TH69) PEBL is bolded / single-underlined.

[0085] Figure 33A and Figure 33B Described is the nucleic acid sequence of the exemplary dual promoter construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-EFS promoter-anti-human CD7 (TH69) PEBL (SEQ ID NO: 19). The MSCV promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the MSCV promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EFS promoter, the EFS promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EFS promoter and the PEBL, and the anti-human CD7 (TH69) PEBL is bolded / single-underlined.

[0086] Figure 34A and Figure 34BDepicted is the nucleic acid sequence of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (3A1F) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL (SEQ ID NO: 20). The PGK promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the PGK promoter and the CAR, the anti-human CD7 (3A1F) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EF1a promoter, the EF1a promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EF1a promoter and the PEBL, and the anti-human CD7 (TH69) PEBL is bolded / single-underlined.

[0087] Figure 35A and Figure 35B The nucleic acid sequence of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (3A1F) PEBL is depicted (SEQ ID NO: 21). The PGK promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the PGK promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EF1a promoter, the EF1a promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EF1a promoter and the PEBL, and the anti-human CD7 (3A1F) PEBL is bolded / single-underlined.

[0088] Figures 36A-36C Depicted is the nucleic acid sequence of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (TH69) CAR-EF1a promoter-anti-human CD7 (TH69) PEBL (SEQ ID NO: 22). The PGK promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the PGK promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EF1a promoter, the EF1a promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EF1a promoter and the PEBL, and the anti-human CD7 (TH69) PEBL is bolded / single-underlined.

[0089] Figure 37A and Figure 37BDescribed is the nucleic acid sequence (SEQ ID NO: 23) of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7 (TH69) CAR-EFS promoter-anti-human CD7 (TH69) PEBL. The PGK promoter is double-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the PGK promoter and the CAR, the anti-human CD7 (TH69) CAR is bolded, the restriction enzyme site (italics) is between the CAR and the EFS promoter, the EFS promoter is single-underlined, the restriction enzyme site (italics) and the Kozak sequence are between the EFS promoter and the PEBL, and the anti-human CD7 (TH69) PEBL is bolded / single-underlined.

[0090] Figure 38 The amino acid sequence of an exemplary anti-human CD7 PEBL based on antibody TH69 is depicted (SEQ ID NO: 24). The CD8 signal peptide begins at position 1 and is of normal type, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, and the myc-KDEL peptide is double underlined.

[0091] Figure 39 The amino acid sequence of an exemplary anti-human CD7 PEBL variant based on antibody TH69 is depicted (SEQ ID NO: 25). The N-terminal proline is italicized, the CD8 signal peptide starts at position 2 and is of normal type, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is underlined, the anti-CD7 VH domain is bold / single underlined, and the myc-KDEL peptide is double underlined.

[0092] Figure 40 The amino acid sequence of an exemplary anti-human CD7 PEBL based on antibody 3A1F is depicted (SEQ ID NO: 26). The CD8 signal peptide begins at position 1 and is of normal type, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, and the myc-KDEL peptide is double underlined.

[0093] Figure 41 The amino acid sequence of an exemplary anti-human CD7 PEBL variant based on antibody 3A1F is depicted (SEQ ID NO: 27). The N-terminal proline is italicized, the CD8 signal peptide starts at position 2 and is of normal type, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is underlined, the anti-CD7 VH domain is bold / single underlined, and the myc-KDEL peptide is double underlined.

[0094] Figure 42 The amino acid sequence of an exemplary anti-human CD7 CAR based on antibody TH69 is depicted (SEQ ID NO: 28). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the CD8a hinge and transmembrane domain are double underlined, and the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, which is of normal type. The CD3ζ signaling domain is bold / double underlined.

[0095] Figure 43 The amino acid sequence of an exemplary anti-human CD7 CAR variant based on antibody TH69 is depicted (SEQ ID NO: 29). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, and the CD8a hinge and transmembrane domain are double underlined. The 4-1BB signaling domain is located between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, which is of normal type. The CD3ζ signaling domain is bold / double underlined, and the amino acids at the C-terminus of the CD3ζ signaling domain are generated by skipping the ribosome at the P2A site.

[0096] Figure 44 The amino acid sequence of an exemplary anti-human CD7 CAR based on antibody 3A1F is depicted (SEQ ID NO: 30). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, the CD8a hinge and transmembrane domain are double underlined, and the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, which is of normal type and is bold / double underlined.

[0097] Figure 45 The amino acid sequence of an exemplary anti-human CD7 CAR variant based on antibody 3A1F is depicted (SEQ ID NO: 31). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is bold / single underlined, and the CD8a hinge and transmembrane domain are double underlined. The 4-1BB signaling domain is located between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain and is of normal type. The CD3ζ signaling domain is bold / double underlined, and the amino acids at the C-terminus of the CD3ζ signaling domain are generated by skipping the ribosome at the P2A site.

[0098] Figure 46Amino acid sequence of an exemplary anti-human CD7 CAR based on antibody TH69 - P2A based on antibody TH69 - anti-human CD7 PEBL (SEQ ID NO: 95). CD7 CAR is in normal font, P2A is double underlined, and CD7 PEBL is in bold / single underlined. DETAILED DESCRIPTION

[0099] Introduction

[0100] The present invention provides methods for simultaneously expressing an estrogen-killing chimeric antigen receptor (e.g., CAR) and an estrogen-killing protein (e.g., PEBL) in T cells, which results in the survival of CAR-expressing cytotoxic T lymphocytes (CAR-T) targeting T cell antigens.

[0101] Viral vectors have been produced, in which two or more genes can be expressed from a single construct. Typically, these vectors use bicistronic elements or dual promoter configurations. In the case of a bicistronic vector, a sequence element is introduced between the two genes, which enables two proteins to be translated from a single messenger RNA. Examples include internal ribosome entry site sequences (IRES) and virus-derived " codon skipping " peptide sequences, such as P2A, T2A, F2A, E2A, etc. In the case of a vector with two promoter designs, an independent promoter element is configured upstream of each gene to allow each promoter to transcribe the mRNA of its proximal connection gene. In some embodiments, an expression vector (e.g., construct) includes a first promoter operably connected to CAR and a second promoter operably connected to PEBL.

[0102] This article describes methods for producing and testing various bicistronic vectors and two promoter-designed vectors for expressing two different genes (e.g., a gene encoding a CAR and a gene encoding a PEBL). Unexpectedly, it was found that certain two gene vectors were able to direct the expression of PEBL and CAR proteins in T cells in such a way that the resulting engineered T cells were able to survive, expand, and kill target cells. The relative expression time and expression level of each gene in the two gene vectors identified enabled CAR to downregulate the target antigen before causing unnecessary lethal expression to the engineered T cells.

[0103] Described herein are anti-killer CAR-T cells expressing CAR for CD7, and such CAR-T cells have reduced CD7 expression or no surface expression of CD7. The present invention is based in part on the use of bicistronic constructs such as bicistronic viral vectors to co-express chimeric antigen receptors (CAR) for CD7 and protein expression blockers (PEBL) for CD7 in immune cells (such as T cells). On the one hand, the present invention relates to engineered immune cells (for example, engineered T cells), which include bicistronic constructs that include polynucleotide sequences encoding anti-CD7 CAR and polynucleotide sequences encoding anti-CD7 PEBL. In some embodiments, CAR includes intracellular signaling domains of 4-1BB and CD3ζ, and antibodies (for example, single-chain variable fragments or scFv) that specifically bind to CD7. The CD7 CAR of the present invention is sometimes referred to herein as "anti-CD7-41BB-CD3". In some embodiments, CAR also includes CD8α hinges and transmembrane domains. In some embodiments, the anti-CD7 PEBL comprises an antibody (e.g., scFv) that specifically binds CD7 and an intracellular localization sequence. In certain embodiments, the anti-CD7 PEBL comprises an antibody (e.g., scFv) that specifically binds CD7, the CD8 α hinge and transmembrane domains, and an intracellular localization sequence.

[0104] CD7 is a 40kDa type I transmembrane glycoprotein that is a major marker of T cell malignancies and is highly expressed in all cases of T cell ALL, including early T cell progenitor acute lymphoblastic leukemia (ETP-ALL). Anti-CD7 CARs can induce T cells to exert specific cytotoxicity against T cell malignancies. In addition, when anti-CD7 CARs are used in combination with downregulation of CD7 expression on effector T cells, T cell cytotoxicity has been shown to be significantly increased. Downregulation (e.g., elimination, reduction, and / or relocalization) of CD7 in T cells through the expression of anti-CD7 PEBL prevents the killing effect produced by the corresponding anti-CD7 CAR. CAR expression can lead to greater T cell recovery and more effective cytotoxicity against T leukemia / lymphoma cells compared to cells that retain the target antigen (e.g., CD7).

[0105] definition

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in practice to test the present invention, preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0107] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0108] The articles "a" and "an" are used herein to refer to one or more (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0109] As used herein, the term "about" and its grammatical equivalents relative to a reference value and its grammatical equivalents may include values ​​within plus or minus 10% of that value. For example, the number "about 10" includes numbers from 9 to 11. The term "about" relative to a reference value may also include plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value.

[0110] As used herein, the term "nucleic acid" refers to a polymer comprising a plurality of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In some embodiments, nucleic acid molecules can be modified. In the case of double-stranded polymers, "nucleic acid" can refer to any one or both chains of the molecule. Nucleic acid and polynucleotide used herein are interchangeable.

[0111] The term "nucleotide sequence" with respect to nucleic acids refers to a series of contiguous nucleotides linked by covalent linkages, such as phospho linkages (e.g., phosphodiester, alkyl and aryl phosphonates, phosphorothioate, phosphotriester bonds) and / or non-phospho linkages (e.g., peptide and / or sulfamate bonds). In certain embodiments, the nucleotide sequence encoding a target binding molecule, e.g., linked to a localization domain, is a heterologous sequence (e.g., a gene having an origin in a different species or cell type).

[0112] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotides or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Thus, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0113] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, in the case of two protein coding regions, are in the same reading frame.

[0114] The term "sequence identity" refers to when two nucleotide sequences or two amino acid sequences are optimally aligned, for example, by using GAP or the BESTFIT program using default gap weights, at least sharing for example 70% sequence identity or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. In order to compare sequences, usually a sequence serves as a reference sequence (for example, parental sequence) compared with a test sequence. When using a sequence comparison algorithm, test sequence and reference sequence are input to a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percentage sequence identity of one or more test sequences relative to the reference sequence based on specified program parameters.

[0115] Optimal alignment of sequences for comparison can be determined, for example, by the method of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the method of finding similarity of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Genetics Computer Group, Wisconsin Genetics Software Package, 575, Sci. Doctor, Madison, Wisconsin), or by visual inspection (see generally, Ausubel et al., Current Protocols in Molecular Biology). An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly available through the NCBI National Institutes of Health (NCBI) internet server). Typically, sequence comparisons can be performed using the default program parameters, but custom parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proceedings of the National Academy of Sciences of the United States of America, 89: 10915 (1989)).

[0116] As will be appreciated by those skilled in the art, in some aspects, the nucleic acid further comprises a plasmid sequence. The plasmid sequence may comprise one or more sequences such as a promoter sequence, a selectable marker sequence, or a locus targeting sequence.

[0117] As used herein, the term "promoter" or "promoter element" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

[0118] The term "retroviral vector" may refer to a gammaretroviral vector. A retroviral vector may include, for example, a promoter, a packaging signal, a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a polynucleotide of interest, such as a polynucleotide encoding a CAR or a polynucleotide encoding a PEBL. A retroviral vector may lack viral structural genes, such as gag, pol, and env. Exemplary retroviral (e.g., gammaretroviral) vectors include murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), murine leukemia virus (MLV), spleen forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Maetzig et al., Viruses, 2011; 3(6): 677-713.

[0119] The term "bicistronic expression" is typically achieved by operably linking a polynucleotide as described herein to a promoter and incorporating the bicistronic construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic organisms. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0120] "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all known vectors in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), into which the recombinant polynucleotide is incorporated.

[0121] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Vol. 1-4, Cold Spring Harbor Press, New York, described, and in other virology and molecular biology handbooks. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Typically, suitable vectors are included in at least one biologically active origin of replication, promoter sequences, convenient restriction endonuclease sites and one or more selective markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0122] Other promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, they are located in the region 30-110bp upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. The interval between promoter elements is normally flexible, so when elements are reversed or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the interval between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, it seems that a single element can work in a coordinated or independent manner to activate transcription. Exemplary promoters include immediate early cytomegalovirus (CMV), EF-1α, ubiquitin C or phosphoglycerate kinase (PGK) promoters. A strong constitutive promoter sequence that can drive the high-level expression of any polynucleotide sequence effectively connected thereto can be used. Other constitutive promoter sequences can be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, elongation factor 1 avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, etc. In some embodiments, the promoter is an inducible promoter, which provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked to it when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include but are not limited to metallothioneine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0123] As used herein, "antibody" means a complete antibody or an antigen-binding fragment of an antibody, including a complete antibody or antigen-binding fragment that has been modified or engineered, or a human antibody. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single-chain antibodies (e.g., scFv), miniantibodies, and diabodies.

[0124] When referring to a protein or peptide, the term "specifically (or selectively) binds" or "specifically (or selectively) immunoreacts with" refers to a binding reaction that determines the presence of a protein, typically in a heterogeneous population of proteins and other biological agents. Thus, under specified immunoassay conditions, the binding of a specified antibody to a specific protein is at least twice the background, more typically 10 to 100 times greater than the background. Specific binding to an antibody under such conditions requires selection of an antibody that is specific for a particular protein. For example, polyclonal antibodies can be selected to obtain those that specifically immunoreact only with a selected antigen and not with other proteins. This selection can be achieved by subtracting antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0125] In a specific embodiment, the antibody that binds to CD7 is a single-chain variable fragment antibody ("scFv antibody"). scFv refers to an antibody fragment comprising the VH domain and VL domain of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker located between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994), The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., New York, Springer-Verlag, pp. 269-315. See also PCT Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. As will be appreciated by those skilled in the art, various suitable linkers can be designed and tested to achieve optimal function, as provided in the art and as disclosed herein.

[0126] As used herein, "engineered" immune cells include immune cells that have been genetically modified compared to naturally occurring immune cells. For example, engineered T cells produced according to the present methods carry nucleic acids that include nucleotide sequences that do not naturally occur in the T cells from which they are derived.

[0127] As used herein, "substantially purified" cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous population of cells. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0128] As used herein, "CD7 CAR+ / CD7 negative" T cells refer to T cells that express a chimeric antigen receptor for human CD7 and have low or no endogenous CD7 surface expression. In some embodiments, the low or no surface expression of endogenous CD7 is due to the expression of PEBL for human CD7, which prevents or blocks the transfer of endogenous CD7 protein to the surface of T cells. In some cases, standard methods known to those skilled in the art, such as, but not limited to, immunocytochemistry, flow cytometry, or FACS, can be used to determine the surface expression of CD7.

[0129] As used herein, the term "autologous" and its grammatical equivalents can refer to originating from the same entity. For example, a sample (e.g., cells) can be removed at a later time, processed, and returned to the same subject (e.g., patient). Autologous procedures differ from allogeneic procedures in that the donor and recipient are different subjects.

[0130] "Allogeneic" refers to a transplant derived from a different animal of the same species.

[0131] As used herein, the terms "treat," "treating," or "treatment" refer to counteracting a medical condition (e.g., a condition associated with a T-cell malignancy) to the extent that the medical condition is improved according to clinically accepted standards.

[0132] As used herein, "subject" refers to a mammal (e.g., a human, non-human primate, cattle, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In certain embodiments, the subject is a human. A "subject in need thereof" refers to a subject (e.g., a patient) who has or is at risk of developing a disease or condition that can be treated (e.g., improved, ameliorated, prevented) by inducing T cells to exert a specific effect on malignant T cell cytotoxicity.

[0133] As defined herein, a "therapeutic amount" refers to an amount that, when administered to a subject, is sufficient to achieve the desired therapeutic effect (treatment of a condition associated with a T-cell malignancy) in the subject under the conditions of administration. The effective amount of the agent to be administered can be determined by a clinician of ordinary skill in the art using the guidance provided herein and other methods known in the art, and depends on several factors, including, for example, the specific agent selected, the subject's age, sensitivity, tolerance to the drug, and overall health. DETAILED DESCRIPTION

[0135] As described in detail below, anti-CD7 CAR (also referred to as "CD7 CAR") may comprise an antigen binding domain targeting CD7 based on a TH69 antibody. In some embodiments, the antigen binding domain of CD7 CAR is based on the 3A1F antibody. In some embodiments, the antigen binding domain of CD7 CAR is based on the T3-3A1 antibody. In some embodiments, the CD7 CAR of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. In some embodiments, the CD7 CAR comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31. In some cases, the engineered immune cells of the present invention comprise a CD7 CAR of SEQ ID NO: 28. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some cases, the engineered immune cells comprise a CD7 CAR of SEQ ID NO: 29. In some cases, the engineered immune cell comprises a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some cases, the engineered immune cell comprises a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some cases, the engineered immune cell comprises a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31. In some cases, the engineered immune cell comprises a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0136] In some embodiments, the CD7 PEBL of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In some embodiments, the CD7 PEBL of the invention comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In some cases, the engineered immune cells of the invention comprise the CD7 PEBL of SEQ ID NO: 24. In some cases, the engineered immune cells of the invention comprise a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25. In some cases, the engineered immune cells comprise the CD7 PEBL of SEQ ID NO: 24. In some cases, the engineered immune cells comprise a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25. In some cases, the engineered immune cells comprise the CD7 PEBL of SEQ ID NO: 26. In some cases, the engineered immune cell comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 26. In some cases, the engineered immune cell comprises a CD7 PEBL of SEQ ID NO: 27. In some cases, the engineered immune cell comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27.

[0137] In some embodiments, the engineered immune cells or populations of engineered immune cells of the present invention comprise a CD7 PEBL of SEQ ID NO: 24 and a CD7 CAR of SEQ ID NO: 28. In some embodiments, the engineered immune cells or populations of engineered immune cells comprise a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 24 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the engineered immune cells or populations of engineered immune cells comprise a CD7 PEBL of SEQ ID NO: 24 and a CD7 CAR of SEQ ID NO: 30. In some embodiments, the engineered immune cells or populations of engineered immune cells comprise a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 24 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the engineered immune cells or populations of engineered immune cells comprise a CD7 PEBL of SEQ ID NO: 26 and a CD7 CAR of SEQ ID NO: 28. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 26 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO: 26 and a CD7 CAR of SEQ ID NO: 308. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 26 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO: 25 and a CD7 CAR of SEQ ID NO: 29. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO: 25 and a CD7 CAR of SEQ ID NO: 31. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO: 27 and a CD7 CAR of SEQ ID NO: 29. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO: 27 and a CD7 CAR of SEQ ID NO: 31. In some embodiments, the engineered immune cell or population of engineered immune cells comprises a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0138] In certain embodiments, the engineered immune cells are engineered T cells. In some embodiments, the engineered immune cells are engineered CD4+T cells. In some embodiments, the engineered immune cells are engineered CD8+T cells. In some embodiments, the engineered immune cells with a bicistronic construct or a dual promoter construct are produced from PBMC. In some embodiments, the engineered immune cells with a bicistronic construct or a dual promoter construct are produced from purified CD4+T cells. In some embodiments, the engineered immune cells with a bicistronic construct or a dual promoter construct are produced from purified CD8+T cells. In some embodiments, the engineered immune cells with a bicistronic construct or a dual promoter construct are produced from a cell population comprising purified CD4+T cells and purified CD8+T cells. In some embodiments, the engineered immune cells with a bicistronic construct or a dual promoter construct are produced from a cell population comprising purified CD3+T cells.

[0139] Bicistronic expression construct

[0140] Provided herein is a recombinant bicistronic virus construct or vector, comprising a polynucleotide encoding CAR as described herein and a polynucleotide encoding PEBL. In some embodiments, the recombinant bicistronic virus construct is included in an internal ribosome entry site (IRES) sequence between the nucleic acid sequence of CAR and the nucleic acid sequence of PEBL. In some embodiments, the recombinant bicistronic virus construct includes a ribosomal codon skipping site sequence (also referred to as a sequence encoding 2A self-cleaving peptide) between the nucleic acid sequence of CAR and the nucleic acid sequence of PEBL. In some embodiments of the bicistronic construct, the polynucleotide encoding CAR is located upstream (5' end) of the IRES sequence, and the polynucleotide encoding PEBL is located downstream (3' end) of the IRES sequence. In some cases, the nucleic acid sequence encoding CAR is operably connected to the IRES sequence, and the IRES sequence is operably connected to the nucleic acid sequence encoding PEBL. In some cases, the nucleic acid sequence encoding PEBL is operably connected to the IRES sequence, and the IRES sequence is operably connected to the nucleic acid sequence encoding CAR.

[0141] In some embodiments of the bicistronic construct, the polynucleotide encoding CAR is located upstream (5' end) of the polynucleotide encoding the self-cleavage 2A peptide, and the polynucleotide encoding PEBL is located downstream (3' end) of the polynucleotide encoding the self-cleavage 2A peptide. In some cases, the nucleic acid sequence encoding CAR is operably connected to the nucleic acid sequence encoding the self-cleavage 2A peptide, and the self-cleavage 2A peptide is operably connected to the nucleic acid sequence encoding PEBL. In some cases, the nucleic acid sequence encoding PEBL is operably connected to the nucleic acid sequence encoding the self-cleavage 2A peptide, and the self-cleavage 2A peptide is operably connected to the nucleic acid sequence encoding CAR.

[0142] The mechanism of skipping ribosomal codons by 2A peptide sequences can be used to generate two proteins from a single transcript; the normal peptide bond is damaged at the 2A sequence, resulting in two discrete protein fragments produced from a single translation event. Self-cleaving 2A peptides (e.g., 2A cleavage sites) are described in Kim et al., PLoS One, 2011, 6(4):e18556.

[0143] In some embodiments, the IRES is from an encephalomyocarditis virus. In some embodiments, the IRES is from an enterovirus. In some embodiments, the nucleic acid sequence of the IRES sequence is set forth in SEQ ID NO: 62 (see, e.g., Table 1).

[0144] In some embodiments, the ribosomal codon skipping site is based on a self-cleaving 2A peptide (see, e.g., Table 2). In some embodiments, the self-cleaving 2A peptide is selected from P2A, E2A, F2A, and T2A. In some cases, the amino acid sequence of the P2A peptide comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 90% sequence identity thereto. In some cases, the amino acid sequence of the E2A peptide comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90% sequence identity thereto. In some cases, the amino acid sequence of the F2A peptide comprises the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 90% sequence identity thereto. In some cases, the amino acid sequence of the T2A peptide comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% sequence identity thereto.

[0145] In some embodiments, the viral construct (e.g., a retroviral construct) comprises a nucleic acid sequence encoding a self-cleaving 2A peptide (e.g., a 2A peptide cleavage site) selected from P2A, E2A, F2A, and T2A, wherein the nucleotide encoding the self-cleaving 2A peptide is connected to the nucleic acid sequence encoding CAR and the nucleic acid sequence encoding PEBL. In other words, the polynucleotide encoding the self-cleaving 2A peptide is between the nucleic acid sequence encoding CAR and the nucleic acid sequence encoding PEBL. As described above, in some embodiments, the construct comprises 5' to 3' end or consists of: a nucleic acid sequence encoding CAR, a nucleic acid sequence encoding a self-cleaving P2A peptide, and a nucleic acid sequence encoding PEBL. In some embodiments, the construct comprises 5' to 3' end or consists of: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a self-cleaving P2A peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. As described above, in some embodiments, the construct comprises from the 5' end to the 3' end or consists of: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a self-cleaving E2A peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises from the 5' end to the 3' end or consists of: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a self-cleaving F2A peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises from the 5' end to the 3' end or consists of: a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a self-cleaving T2A peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein.

[0146] In some embodiments, the construct comprises or consists of from the 5′ end to the 3′ end: a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding a self-cleaving P2A peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from the 5′ end to the 3′ end: a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding a self-cleaving E2A peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from the 5′ end to the 3′ end: a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding a self-cleaving F2A peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of from the 5′ end to the 3′ end: a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding a self-cleaving T2A peptide, and a nucleic acid sequence encoding a CAR.

[0147] In some embodiments, the nucleic acid sequence encoding P2A comprises or consists of a nucleic acid having at least 90% sequence identity to SEQ ID NO: 63. In some embodiments, the nucleic acid sequence encoding P2A comprises or consists of a nucleic acid of SEQ ID NO: 63. In some embodiments, the nucleic acid sequence encoding E2A comprises or consists of a nucleic acid having at least 90% sequence identity to SEQ ID NO: 64. In some embodiments, the nucleic acid sequence encoding E2A comprises or consists of a nucleic acid of SEQ ID NO: 64. In some embodiments, the nucleic acid sequence encoding F2A comprises or consists of a nucleic acid having at least 90% sequence identity to SEQ ID NO: 65. In some embodiments, the nucleic acid sequence encoding F2A comprises or consists of a nucleic acid of SEQ ID NO: 65. In some embodiments, the nucleic acid sequence encoding T2A comprises or consists of a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 66. In some embodiments, the nucleic acid sequence encoding T2A comprises or consists of a nucleic acid of SEQ ID NO: 66.

[0148] In some embodiments, the nucleic acid sequence encoding PEBL is arranged (eg, located) 5' to the nucleic acid sequence encoding CAR. In some embodiments, the nucleic acid sequence encoding CAR is located 5' to the nucleic acid sequence encoding PEBL. In some embodiments, the bicistronic construct comprises or consists of, from 5′ to 3′ ends: (a1) SEQ ID NO: 4, SEQ ID NO: 63, and SEQ ID NO: 2; and (a2) SEQ ID NO: 4, SEQ ID NO: 63, and SEQ ID NO: 3; (a3) ​​SEQ ID NO: 5, SEQ ID NO: 63, and SEQ ID NO: 2; (a4) SEQ ID NO: 5, SEQ ID NO: 63, and SEQ ID NO: 3; (b1) SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 2; (b2) SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 3; (b3) SEQ ID NO: 5, SEQ ID NO: 64, and SEQ ID NO: 2; (b4) SEQ ID NO: 5, SEQ ID NO: 64, and SEQ ID NO: 3; (c1) SEQ ID NO: 4, SEQ ID NO: 65, and SEQ ID NO: 2; (c2) SEQ ID NO: 4, SEQ ID NO: 65 and SEQ ID NO: 3; (c3) SEQ ID NO: 5, SEQ ID NO: 65 and SEQ ID NO: 2; (c4) SEQ ID NO: 5, SEQ ID NO: 65 and SEQ ID NO: 3; (d1) SEQ ID NO: 4, SEQ ID NO: 66 and SEQ ID NO: 2; (d2) SEQ ID NO: 4, SEQ ID NO: 66 and SEQ ID NO: 3; (d3) SEQ ID NO: 5, SEQ ID NO: 66 and SEQ ID NO: 2; (d4) SEQ ID NO: 5, SEQ ID NO: 66 and SEQ ID NO: 3; (e1) SEQ ID NO: 2, SEQ ID NO: 63 and SEQ ID NO: 4; (e2) SEQ ID NO: 3, SEQ ID NO: 63 and SEQ ID NO: 4; (e3) SEQ ID NO: 2, SEQ ID NO: 63 and SEQ ID NO: 5; (e4) SEQ ID NO: 3, SEQ ID NO: 63 and SEQ ID NO: 5; (f1) SEQ ID NO: 2, SEQ ID NO: 64 and SEQ ID NO: 4;(f2) SEQ ID NO: 3, SEQ ID NO: 64 and SEQ ID NO: 4; (f3) SEQ ID NO: 2, SEQ ID NO: 63 and SEQ ID NO: 5; (f4) SEQ ID NO: 3, SEQ ID NO: 64 and SEQ ID NO: 5; (g1) SEQ ID NO: 2, SEQ ID NO: 65 and SEQ ID NO: 4; (g2) SEQ ID NO: 3, SEQ ID NO: 65 and SEQ ID NO: 4; (g3) SEQ ID NO: 2, SEQ ID NO: 65 and SEQ ID NO: 5; (g4) SEQ ID NO: 3, SEQ ID NO: 65 and SEQ ID NO: 5; (h1) SEQ ID NO: 2, SEQ ID NO: 66 and SEQ ID NO: 4; (h2) SEQ ID NO: 3, SEQ ID NO: 66 and SEQ ID NO: 5 NO: 4; (h3) SEQ ID NO: 2, SEQ ID NO: 66 and SEQ ID NO: 5; or (h4) SEQ ID NO: 3, SEQ ID NO: 66 and SEQ ID NO: 5. ;

[0149] In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends: Figure 16 The sequence of SEQ ID NO: 63 and Figure 18 In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 16 A sequence of any one of SEQ ID NOS: 63-66 and Figure 19 In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 17 A sequence of any one of SEQ ID NOS: 63-66 and Figure 18 In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 17 A sequence of any one of SEQ ID NOS: 63-66 and Figure 19 sequence.

[0150] In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends: Figure 18 A sequence of any one of SEQ ID NOS: 63-66 and Figure 16 In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 19 A sequence of any one of SEQ ID NOS: 63-66 and Figure 16In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 18 A sequence of any one of SEQ ID NOS: 63-66 and Figure 17 In some embodiments, the bicistronic construct comprises or consists of the 5′ to 3′ ends of: Figure 19 SEQ ID NOS: 63-66, and Figure 17 sequence.

[0151] In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(TH67) CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(TH67) PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(TH67) CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(TH67) PEBL of SEQ ID NO: 24. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding Figure 38 In some embodiments, the bicistronic construct comprises from 5′ to 3′ or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding Figure 40 In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 44 A polynucleotide encoding the CD7(3A1F)CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the Figure 38In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 44 A polynucleotide encoding the CD7(3A1F)CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the Figure 40 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding Figure 38 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding Figure 40 In some embodiments, the bicistronic construct comprises or consists of, from 5′ to 3′ ends, a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises or consists of, from 5′ to 3′ ends, a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 44 A polynucleotide encoding the CD7(3A1F) CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding Figure 38In some embodiments, the bicistronic construct comprises from 5′ to 3′ or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 44 A polynucleotide encoding the CD7(3A1F) CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding Figure 40 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding Figure 38 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding Figure 40 In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 44 A polynucleotide encoding the CD7(3A1F)CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the Figure 38In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 44 A polynucleotide encoding the CD7(3A1F)CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the Figure 40 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding Figure 38 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 42 A polynucleotide encoding a CD7 (TH67) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding Figure 40 In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises from 5′ to 3′ ends or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of SEQ ID NO: 24. Figure 44 A polynucleotide encoding the CD7(3A1F)CAR, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding Figure 38In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises, from 5′ to 3′, or consists of: a polynucleotide encoding the CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of SEQ ID NO: 26. Figure 44 A polynucleotide encoding a CD7(3A1F) CAR, a polynucleotide encoding a P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding Figure 40 CD7(3A1F)PEBL polynucleotide.

[0152] In some embodiments, the polynucleotide sequence encoding PEBL is located at the 5' (upstream) end of the IRES site, and the IRES site is located at the 5' end of the polynucleotide sequence encoding CAR. In some embodiments, the polynucleotide sequence encoding CAR is located at the 5' end of the IRES site, and the IRES site is located at the 5' end of the polynucleotide sequence encoding PEBL.

[0153] In some embodiments, the polynucleotide sequence encoding PEBL is located at the 5' (upstream) end of the ribosomal codon skipping site, and the ribosomal codon skipping site is located at the 5' end of the polynucleotide sequence encoding CAR. In some embodiments, the polynucleotide sequence encoding CAR is located at the 5' end of the ribosomal codon skipping site, and the ribosomal codon skipping site is located at the 5' end of the polynucleotide sequence encoding PEBL.

[0154] In some aspects, provided herein are recombinant bicistronic constructs that have at least 90% sequence identity to one or more nucleic acid sequences selected from SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66. In some embodiments, the recombinant bicistronic construct has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the recombinant bicistronic construct has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the recombinant bicistronic construct has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the recombinant bicistronic construct has at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the recombinant bicistronic construct comprises a nucleic acid sequence selected from SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.

[0155] Table 1. Nucleic acid sequences of ribosomal codon skipping peptides and IRES

[0156]

[0157]

[0158] Table 2. Amino acid sequences of ribosomal codon skipping sites

[0159] name SEQ ID NO Amino acid sequence P2A SEQ ID NO: 67 ATNFSLLKQAGDVEENPGP E2A SEQ ID NO: 68 QCTNYALLKLAGDVESNPGP F2A SEQ ID NO: 69 VKQTLNFDLLKLAGDVESNPGP T2A SEQ ID NO: 70 EGRGSLLTCGDVEENPGP

[0160] The present invention provides vectors, such as expression vectors, into which any polynucleotide described herein is inserted. In some embodiments, the vector is derived from a retrovirus, such as a lentivirus. Such vectors are suitable for realizing a tool for long-term gene transfer because they allow long-term, stable integration of exogenous polynucleotides (e.g., transgenes) and their propagation in daughter cells. Different from vectors derived from oncorretroviruses (e.g., murine leukemia viruses), lentiviral vectors can transduce non-proliferative cells. Slow viral vectors also have low immunogenicity. In other embodiments, the vector is an adenoviral vector. In certain embodiments, the vector is a plasmid.

[0161] In some embodiments, the promoter has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to the CMV promoter. In some embodiments, the vector comprises a CMV promoter. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 6. In some embodiments, any construct described herein comprises or consists of the CMV promoter of SEQ ID NO: 6.

[0162] In some embodiments, the promoter has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the EF1α promoter. In some embodiments, the promoter comprises the EF1α promoter. In some embodiments, the EF1α promoter comprises the sequence of SEQ ID NO: 7. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 6. In some embodiments, any of the constructs described herein comprises or consists of the EF1α promoter of SEQ ID NO: 7.

[0163] In some embodiments, the promoter has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to the EFS promoter. In some embodiments, the promoter comprises the EFS promoter. In some embodiments, the EFS promoter comprises the sequence of SEQ ID NO: 8. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 6. In some embodiments, any of the constructs described herein comprises or consists of the EFS promoter of SEQ ID NO: 8.

[0164] In some embodiments, the promoter has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to the murine stem cell virus (MSCV) promoter. In some embodiments, the promoter comprises the MSCV promoter. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO: 9. In some embodiments, any construct described herein comprises or consists of the MSCV promoter of SEQ ID NO: 9.

[0165] In some embodiments, the promoter has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the phosphoglycerate kinase (PGK) promoter. In some embodiments, the promoter comprises the PGK promoter. In some embodiments, the PGK promoter comprises the sequence of SEQ ID NO: 10. In some embodiments, any of the constructs described herein comprises or consists of the PGK promoter of SEQ ID NO: 10.

[0166] In some embodiments, the bicistronic vector comprises or consists of the nucleic acid sequence of SEQ ID NO: 11. An exemplary embodiment of such a sequence is Figure 25 In some embodiments, the bicistronic vector has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to SEQ ID NO: 11. The bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: a nucleic acid sequence encoding CD7 PEBL, an IRES sequence, a nucleic acid sequence encoding CD7 CAR, and optionally at the 5′ end, a promoter selected from a CMV promoter (e.g., SEQ ID NO: 6 or Figure 20 ), EF1a promoter (e.g., SEQ ID NO: 7 or Figure 21), EFS promoter (e.g., SEQ ID NO: 8 or Figure 22 ), MSCV promoter (e.g., SEQ ID NO: 9 or Figure 23 ) and PGK promoter (e.g., SEQ ID NO: 10 or Figure 24 ).

[0167] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the bicistronic vector has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 12. Exemplary embodiments of such sequences are described in Figure 26 The bicistronic vector includes a nucleic acid sequence from the 5′ end to the 3′ end: a nucleic acid sequence encoding CD7 CAR, an IRES sequence, and a nucleic acid sequence encoding CD7 PEBL, and optionally, at the 5′ end, a promoter selected from the group consisting of CMV promoter, EF1a promoter, EFS promoter, MSCV promoter, and PGK promoter.

[0168] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 13. An exemplary embodiment of such a sequence is Figure 27 In some embodiments, the bicistronic vector has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to SEQ ID NO: 13. The bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL, optionally at the 5′ end, a promoter selected from a CMV promoter, an EF1a promoter, an EFS promoter, an MSCV promoter, and a PGK promoter.

[0169] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the bicistronic vector has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more high sequence identity with SEQ ID NO: 14. The bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: a promoter, a nucleic acid sequence encoding CD7 CAR, a P2A sequence, and a nucleic acid sequence encoding CD7 PEBL. In some cases, the bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: an MSCV promoter, a nucleic acid sequence encoding CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding CD7 PEBL. Exemplary embodiments of such sequences are described in Figures 28A-28B Shown in.

[0170] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the bicistronic vector has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to SEQ ID NO: 15. In some cases, the bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: an EF1α promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL. Exemplary embodiments of such sequences are described in Figure 29A-29B Shown in.

[0171] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the bicistronic vector has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity to SEQ ID NO: 16. In some cases, the bicistronic vector comprises a nucleic acid sequence comprising from the 5′ end to the 3′ end: an EFS promoter, a nucleic acid sequence encoding a CD7 CAR, a nucleic acid sequence encoding a P2A peptide, and a nucleic acid sequence encoding a CD7 PEBL. Exemplary embodiments of such sequences are described in Figure 30A-Figure 30B Shown in.

[0172] Dual promoter retroviral constructs

[0173] Provided herein is a recombinant retroviral construct (or vector) for expressing CAR and PEBL simultaneously in a cell, such as a T cell. In some embodiments, the retroviral construct includes a promoter operably connected to a polynucleotide encoding any CAR described herein and a promoter operably connected to a polynucleotide encoding any PEBL described herein. In some embodiments, the promoter for CAR and the promoter for PEBL share less than 90% sequence identity, such as less than 90% identity, less than 80% identity, less than 75% sequence identity, less than 70% sequence identity, less than 65% sequence identity, less than 60% sequence identity, less than 55% sequence identity, etc. In some embodiments, the promoter for CAR and the promoter for PEBL share less than 80% sequence identity, such as 80% identity, 75% sequence identity, 70% sequence identity, 65% sequence identity, 60% sequence identity, 55% sequence identity, etc. In some embodiments, the promoter for CAR and the promoter for PEBL share at least 50% sequence identity, e.g., 50% sequence identity, 55% sequence identity, 60% sequence identity, 65% sequence identity, 70% sequence identity, 75% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity or more.

[0174] In some embodiments, the promoter used for CAR (referred to as the first promoter) is different from the promoter used for PEBL (referred to as the second promoter). The first promoter and the second promoter can have the same sequence. In other cases, the first promoter and the second promoter have different sequences.

[0175] In some embodiments, the first promoter and / or the second promoter have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity with the CMV promoter. In some embodiments, the first promoter and / or the second promoter comprises the CMV promoter. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO: 6.

[0176] In some embodiments, the first promoter and / or the second promoter have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity with the EF1α promoter. In some embodiments, the first promoter and / or the second promoter comprises the EF1α promoter. In some embodiments, the EF1α promoter comprises the sequence of SEQ ID NO: 7.

[0177] In some embodiments, the first promoter and / or the second promoter have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity with the EFS promoter. In some embodiments, the first promoter and / or the second promoter comprises the EFS promoter. In some embodiments, the EFS promoter comprises the sequence of SEQ ID NO: 8.

[0178] In some embodiments, the first promoter and / or the second promoter have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity with the murine stem cell virus (MSCV) promoter. In some embodiments, the first promoter and / or the second promoter comprises the MSCV promoter. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO: 9.

[0179] In some embodiments, the first promoter and / or the second promoter have at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or higher sequence identity with the phosphoglycerate kinase (PGK) promoter. In some embodiments, the first promoter and / or the second promoter comprises the PGK promoter. In some embodiments, the PGK promoter comprises the sequence of SEQ ID NO: 10.

[0180] In some embodiments, the retroviral construct from 5′ to 3′ comprises a first promoter operably linked to a polynucleotide encoding a CAR and a second promoter operably linked to a polynucleotide encoding a PEBL. In various embodiments, the retroviral construct from 5′ to 3′ comprises a second promoter operably linked to a polynucleotide encoding a PEBL and a first promoter operably linked to a polynucleotide encoding a CAR.

[0181] In some embodiments, the first promoter is located upstream of the second promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is an EF1α promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is an MSCV promoter. In some embodiments, the first promoter is a CMV promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an MSCV promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is an MSCV promoter and the second promoter is an EF1α promoter. In some embodiments, the first promoter is an MSCV promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an MSCV promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an MSCV promoter and the second promoter is an MSCV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is an EF1α promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is an MSCV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is a PGK promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EF1α promoter and the second promoter is an MSCV promoter. In some embodiments, the first promoter is an EF1α promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EF1α promoter and the second promoter is an EFS promoter. In some embodiments, the first promoter is an EF1α promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an EF1α promoter and the second promoter is an EF1α promoter. In some embodiments, the first promoter is the EFS promoter and the second promoter is the MSCV promoter. In some embodiments, the first promoter is the EFS promoter and the second promoter is the EF1α promoter. In some embodiments, the first promoter is the EFS promoter and the second promoter is the PGK promoter. In some embodiments, the first promoter is the EFS promoter and the second promoter is the CMV promoter. In some embodiments, the first promoter is the EFS promoter and the second promoter is the EFS promoter.

[0182] In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 17. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 18. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 19. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 20. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 21. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 22. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the retroviral constructs of the invention have at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to SEQ ID NO: 23. In some embodiments, the retroviral constructs of the invention comprise the nucleic acid sequence of SEQ ID NO: 23.

[0183] Antibodies that bind to CD7

[0184] In certain embodiments, the anti-CD7 scFv based on the TH69 antibody comprises a variable heavy chain (heavy chain variable region or VH) and a variable light chain (light chain variable region or VL) having an amino acid sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOs: 32 and 33, respectively. In some embodiments, the heavy chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH sequence set forth in SEQ ID NO: 32. In some embodiments, the light chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence set forth in SEQ ID NO: 33. In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 32. In some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 32. In some cases, the light chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 33. In some cases, the light chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 33. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0185] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 (SASQGISNYLN) of SEQ ID NO: 44, a VL CDR2 (YTSSLHS) of SEQ ID NO: 45, and a VL CDR3 (QQYSKLPYT) of SEQ ID NO: 46. In some embodiments, the anti-CD7 scFv comprises a VH CDR1 (SYAMS) of SEQ ID NO: 47, a VH CDR2 (SISSGGFTYYPDSVKG) of SEQ ID NO: 48, and a VH CDR3 (DEVRGYLDV) of SEQ ID NO: 49. In some embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO: 44, a VL CDR2 of SEQ ID NO: 45, a VL CDR3 of SEQ ID NO: 46, a VH CDR1 of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 48, and a VH CDR3 of SEQ ID NO: 49.

[0186] In some embodiments, the nucleic acid sequence encoding the VH has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 38. In some embodiments, the nucleic acid sequence encoding the VL has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 39.

[0187] In certain embodiments, an anti-CD7 scFv based on the 3A1F antibody comprises a variable heavy chain (heavy chain variable region or VH) and a variable light chain (light chain variable region or VL) having an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOs: 34 and 35, respectively. In some embodiments, the heavy chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH sequence set forth in SEQ ID NO: 34. In some embodiments, the light chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence set forth in SEQ ID NO: 35. In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 34. In some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 34. In some cases, the light chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 35. In some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 35. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0188] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 (RASQSISNNLH) of SEQ ID NO: 50, a VL CDR2 (SASQSIS) of SEQ ID NO: 51, and a VL CDR3 (QQSNSWPYT) of SEQ ID NO: 52. In some embodiments, the anti-CD7 scFv comprises a VH CDR1 (SYWMH) of SEQ ID NO: 53, a VH CDR2 (KINPSNGRTNYNEKFKS) of SEQ ID NO: 54, and a VH CDR3 (GGVYYDLYYYALDY) of SEQ ID NO: 55. In various embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO: 50, a VL CDR2 of SEQ ID NO: 51, a VLCDR3 of SEQ ID NO: 52, a VH CDR1 of SEQ ID NO: 53, a VH CDR2 of SEQ ID NO: 54, and a VH CDR3 of SEQ ID NO: 55.

[0189] In some embodiments, the nucleic acid sequence encoding the VH has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 40. In some embodiments, the nucleic acid sequence encoding the VL has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 41.

[0190] In certain embodiments, an anti-CD7 scFv based on the T3-3A1 antibody comprises a variable heavy chain (heavy chain variable region or VH) and a variable light chain (light chain variable region or VL) having an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOs: 36 and 37, respectively. In some embodiments, the heavy chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH sequence set forth in SEQ ID NO: 36. In some embodiments, the light chain variable region has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence set forth in SEQ ID NO: 37. In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 36. In some cases, the heavy chain variable region comprises 13 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acid substitutions in the sequence set forth in SEQ ID NO: 36. In some cases, the light chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 37. In some cases, the heavy chain variable region comprises 5 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the sequence set forth in SEQ ID NO: 37. Any of the amino acid substitutions described herein can be conservative or non-conservative substitutions.

[0191] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 (RASKSVSASGYSYMH) of SEQ ID NO: 56, a VL CDR2 (LASNLES) of SEQ ID NO: 57, and a VL CDR3 (QHSRELPYT) of SEQ ID NO: 58. In some embodiments, the anti-CD7 scFv comprises a VH CDR1 (SFGMH) of SEQ ID NO: 59, a VH CDR2 (YISSGSSTLHYADTVKG) of SEQ ID NO: 60, and a VH CDR3 (WGNYPHYAMDY) of SEQ ID NO: 61. In various embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO: 56, a VL CDR2 of SEQ ID NO: 57, a VL CDR3 of SEQ ID NO: 58, a VH CDR1 of SEQ ID NO: 59, a VH CDR2 of SEQ ID NO: 60, and a VH CDR3 of SEQ ID NO: 61.

[0192] In some embodiments, the nucleic acid sequence encoding the VH has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 42. In some embodiments, the nucleic acid sequence encoding the VL has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 43.

[0193] In some embodiments, the scFv of the invention comprises a variable heavy chain sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to the variable heavy chain sequence of an anti-CD7 antibody. In some embodiments, the scFv of the present invention comprises a variable light chain sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to the variable light chain sequence of an anti-CD7 antibody. For example, the anti-CD7 antibody can be any antibody recognized by those skilled in the art.

[0194] Table 3. Amino acid sequences of the VH and VL regions of anti-CD7 scFvs

[0195]

[0196] Table 4. Nucleic acid sequences of the VH and VL regions of anti-CD7 scFvs

[0197]

[0198]

[0199]

[0200] Downregulation of intracellular CD7 by CD7 PEBL

[0201] As described herein, when anti-CD7CAR is used in combination with downregulation of CD7 expression on effector T cells, the cytotoxicity of T cells is shown to be significantly increased. As described herein, downregulation of CD7 (e.g., elimination, reduction and / or repositioning) prevents the corresponding anti-CD7 CAR from exerting a killing effect, allowing greater T cell recovery after CAR expression compared to cells retaining the target antigen (e.g., CD7), and having more effective cytotoxicity against T leukemia / lymphoma cells. As will be understood by those skilled in the art, downregulation of CD7 expression on effector T cells can be achieved according to a variety of known methods, including, for example, protein expression blockers (PEBLs) for CD7 (as described in WO2016 / 126213), RNAi or gene editing methods for CD7, such as large-range nucleases, TALENs, CRISPR / Cas9 and zinc finger nucleases. The present invention describes PEBLs that bind to a target antigen and sequester the target antigen to the cytoplasm of the cell. The target antigen is synthesized and bound to the PEBL within the cell.

[0202] In certain embodiments, provided herein are polynucleotides comprising a nucleic acid sequence encoding a PEBL comprising a target binding molecule (e.g., a CD7 antigen binding domain) linked to a localization domain. In some cases, the PEBL comprises, from N-terminus to C-terminus, a CD7 antigen binding domain, an optional domain linker, and a cellular localization domain. In some embodiments, the PEBL further comprises a signal peptide fused to the N-terminus of the CD7 antigen binding domain. In some embodiments, the CD7 antigen binding domain comprises a VL domain, a domain linker, and a VH domain. Exemplary embodiments of PEBL are described in US 2018 / 0179280. Figure 3 E and Figure 17 , which is incorporated herein by reference.

[0203] As used herein, in the context of protein expression blockers, " connected " refers to the gene of the target binding molecule directly encoded in a frame (for example, without a joint) adjacent to one or more genes encoding one or more localization domains. Alternatively, the gene encoding the target binding molecule can be connected to one or more genes encoding one or more localization domains by a joint sequence as described herein, for example, as described in WO2016 / 126213. As will be appreciated by those skilled in the art, variants of such joint sequences and such joint sequences are known in the art. Design methods for the construct incorporated into joint sequences and methods for assessing functionality are readily available to those skilled in the art.

[0204] In some embodiments, the localization domain of a PEBL comprises an endoplasmic reticulum (ER) or Golgi retention sequence; or a proteosome localization sequence. In certain embodiments, the localization domain comprises an endoplasmic reticulum (ER) retention peptide from Table 5. In certain embodiments, the localization domain comprises a proteasome localization sequence listed in Table 5. Depending on the application, the localization domain can direct the PEBL to a specific cellular compartment, such as the Golgi or ER, the proteasome, or the cell membrane.

[0205] In some embodiments, proteasome localization is achieved by connecting the scFv sequence to a triple motif comprising 21 (TRIM21) targeting domain sequences and co-expressing a sequence encoding human TRIM21 E3 ubiquitin ligase protein. TRIM21 binds to the Fc domain of the antibody with high affinity and can recruit ubiquitin-proteosome complexes to degrade molecules (e.g., proteins and peptides) bound to the antibody. TRIM21 targeting domain sequence encodes an amino acid sequence selected from a group (e.g., IgG1, IgG2, or IgG4) of human immunoglobulin G (IgG) constant region (Fc) genes and is used to form a fusion protein comprising scFv and Fc domains. In this embodiment, exogenously expressed TRIM21 protein binds to a scFv-Fc fusion protein bound to a target protein (e.g., CD7) and guides the complex to the proteasome for degradation.

[0206] Details of the amino acid sequence of human TRIM21 E3 ligase protein can be found, for example, in NCBI Ref. Seq. No. NP_003132.2 of the NCBI protein database. Details of the nucleic acid sequence encoding human TRIM21 E3 ligase protein can be found, for example, in NCBI Ref. Seq. No. NM_003141.3 of the NCBI protein database.

[0207] In some cases, PEBL further comprises a hinge and transmembrane domain sequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD38, TCRα, CD32, CD64, VEGFR2, FAS or FGFR2B. In some embodiments, PEBL comprises a hinge and transmembrane domain selected from the hinge and transmembrane domain of CD8, the hinge and transmembrane domain of CD8β, the hinge and transmembrane domain of 4-1BB, the hinge and transmembrane domain of CD28, the hinge and transmembrane domain of CD34, the hinge and transmembrane domain of CD4, the hinge and transmembrane domain of FcεRIγ, the hinge and transmembrane domain of CD16, the hinge and transmembrane domain of OX40, the hinge and transmembrane domain of CD3ζ, the hinge and transmembrane domain of CD3β, the hinge and transmembrane domain of 4-1BB, the hinge and transmembrane domain of CD28, the hinge and transmembrane domain of CD34, the hinge and transmembrane domain of CD4, the hinge and transmembrane domain of FcεRIγ, the hinge and transmembrane domain of CD16, the hinge and transmembrane domain of OX40, the hinge and transmembrane domain of CD3ζ, the hinge and transmembrane domain of The hinge and transmembrane domain of the group consisting of the hinge and transmembrane domain of CD3ε, the hinge and transmembrane domain of CD3γ, the hinge and transmembrane domain of CD3δ, the hinge and transmembrane domain of TCRα, the hinge and transmembrane domain of CD32, the hinge and transmembrane domain of CD64, the hinge and transmembrane domain of VEGFR2, the hinge and transmembrane domain of FAS, and the hinge and transmembrane domain of FGFR2B.

[0208] In certain embodiments, the PEBL comprises one or more of the components listed in Table 5.

[0209] Table 5. Amino acid sequence information of selected components of CD7 PEBL

[0210]

[0211]

[0212] In some embodiments, the CD7 PEBL comprises a CD7 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33, and a VH-VL linker. The VH-VL linker can be (G4S) nlinker, wherein n can range from 1 to 6, for example, 1, 2, 3, 4, 5, or 6. In one embodiment, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33, and the amino acid sequence of SEQ ID NO: 79. In certain embodiments, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 32, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 33 and SEQ ID NO: 79. In other embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 32, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 33, and an amino acid sequence of SEQ ID NO: 79.

[0213] In some embodiments, the CD7 PEBL comprises a CD7 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 35, and a VH-VL linker. The VH-VL linker can be (G4S) n linker, wherein n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 35, and the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 34, an amino acid sequence of SEQ ID NO: 35, and an amino acid sequence of SEQ ID NO: 79. In certain embodiments, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 34, an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 35, and an amino acid sequence of SEQ ID NO: 79. In other embodiments, the CD7 PEBL comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 34, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 35, and an amino acid sequence of SEQ ID NO: 79.

[0214] In some embodiments, the CD7 PEBL comprises a CD7 antigen binding domain comprising the amino acid sequence of SEQ ID NO: 36, the amino acid sequence of SEQ ID NO: 37, and a VH-VL linker. The VH-VL linker may be (G4S) n linker, wherein n can range from 1 to 5, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 36, the amino acid sequence of SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CD7 PEBL comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 36, the amino acid sequence of SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 79. In certain embodiments, the CD7 PEBL comprises the amino acid sequence of SEQ ID NO: 36, the amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 79. In other embodiments, the CD7 PEBL comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:36, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:37, and an amino acid sequence of SEQ ID NO:79.

[0215] In some cases, the CD7 PEBL further comprises a localization domain selected from any of the sequences listed in SEQ ID NOs: 72-77. In some cases, the CD7 PEBL further comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide set forth in SEQ ID NO: 80. In other cases, the anti-CD7 protein expression blocking agent further comprises a CD8α hinge and transmembrane domain, such as, but not limited to, the CD8α hinge and transmembrane domain set forth in SEQ ID NO: 78.

[0216] In one embodiment, the CD7 PEBL encoded by the bicistronic vector described herein comprises the sequence of SEQ ID NO: 24 and a proline at the N-terminus. In some embodiments, the CD7 PEBL comprises the sequence of SEQ ID NO: 25. The N-terminal proline residue results from 2A cleavage. In some embodiments, the CD7 PEBL encoded by the bicistronic vector described herein comprises the sequence of SEQ ID NO: 26 and a proline at the N-terminus. In some embodiments, the CD7 PEBL comprises the sequence of SEQ ID NO: 27.

[0217] In some embodiments, the engineered immune cells of the present invention comprise CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises the sequence of SEQ ID NO: 24 and proline at the N-terminus or the sequence of SEQ ID NO: 25. In some embodiments, the engineered immune cells are CD4+ T cells comprising CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises the sequence of SEQ ID NO: 24 and proline at the N-terminus or the sequence of SEQ ID NO: 25. In some embodiments, the engineered immune cells are CD8+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises the sequence of SEQ ID NO: 24 and proline at the N-terminus or the sequence of SEQ ID NO: 25. In some embodiments, the engineered immune cells are CD3+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises the sequence of SEQ ID NO: 24 and proline at the N-terminus or the sequence of SEQ ID NO: 25.

[0218] In some embodiments, the engineered immune cells of the present invention comprise CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises the sequence of SEQ ID NO: 26 and proline at the N-terminus or the sequence of SEQ ID NO: 27. In some embodiments, the engineered immune cells are CD4+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises the sequence of SEQ ID NO: 26 and proline at the N-terminus or the sequence of SEQ ID NO: 27. In some embodiments, the engineered immune cells are CD8+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises the sequence of SEQ ID NO: 26 and proline at the N-terminus or the sequence of SEQ ID NO: 27. In some embodiments, the engineered immune cells are CD3+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises the sequence of SEQ ID NO: 26 and proline at the N-terminus or the sequence of SEQ ID NO: 27.

[0219] In some embodiments, the CD7 PEBL encoded by the dual-promoter vectors described herein comprises the sequence of SEQ ID NO: 24. In some embodiments, the CD7 PEBL encoded by the dual-promoter vectors described herein binds to CD7 and has at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the CD7 PEBL encoded by the dual-promoter vectors described herein comprises the sequence of SEQ ID NO: 26. In some embodiments, the CD7 PEBL encoded by the dual-promoter vectors described herein binds to CD7 and has at least 90% sequence identity to SEQ ID NO: 26.

[0220] In some embodiments, the nucleic acid sequence encoding the anti-CD7 PEBL comprises one or more nucleic acid sequences listed in Table 6.

[0221] In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 38, and the VL domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 39. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 38, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 39. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 38, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 39, or a codon-optimized variant thereof.

[0222] In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 40, and the VL domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 41. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 41. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 41, or a codon-optimized variant thereof.

[0223] In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 42, and the VL domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO: 43. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 42, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 43. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 42, and the VL domain of the anti-CD7 scFv of PEBL comprises a nucleotide sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 43, or a codon-optimized variant thereof.

[0224] Table 6. Nucleic acid sequence information of selected components of CD7 PEBL

[0225]

[0226]

[0227] In certain aspects of the invention, PEBL can bind to molecules expressed on the surface of cells, including but not limited to members of the CD1 family of glycoproteins, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

[0228] In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90% or more) sequence identity to SEQ ID NO: 2 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2.

[0229] In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90% or more) sequence identity to SEQ ID NO: 3 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3.

[0230] In some embodiments, the engineered immune cells of the present invention comprise a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 2. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 PEBL encoded by a bicistronic vector construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 2. Additionally, provided herein are populations comprising such cells.

[0231] In some embodiments, the engineered immune cells of the present invention comprise a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 PEBL encoded by a bicistronic vector construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 3. Additionally, provided herein are populations comprising such cells.

[0232] CD7-binding chimeric antigen receptor

[0233] In some embodiments, the CAR of the present invention comprises an intracellular signaling domain of 4-1BB and CD3ζ, and an antigen binding domain (e.g., a single-chain variable fragment or scFv) that specifically binds to CD7. The CD7 CAR of the present invention is sometimes referred to herein as "anti-CD7-41BB-CD3." In some embodiments, CAR further comprises a CD8α hinge domain and a transmembrane domain, such as, but not limited to, the amino acid sequence of SEQ ID NO: 84.

[0234] As will be appreciated by those skilled in the art, in certain embodiments, any of the amino acid sequences of the various components disclosed herein (e.g., scFv, intracellular signaling domain, linker, and combinations thereof) may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to a specific corresponding sequence disclosed herein. In some embodiments, the 4-1BB intracellular signaling domain may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 85, as long as it has the desired function. In certain embodiments, the intracellular signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 85.

[0235] In certain embodiments, the 4-1BB intracellular signaling domain can be replaced by another intracellular signaling domain from a costimulatory molecule, such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2. In some embodiments, the intracellular signaling domain of the CAR may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity to the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2.

[0236] As another example, in some cases, the 4-1BB intracellular signaling domain may also include another intracellular signaling domain (or a portion thereof) from a costimulatory molecule such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. In some embodiments, the additional intracellular signaling domain may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. In some embodiments, the additional intracellular signaling domain can have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2.

[0237] As another example, in certain embodiments, the CD3 zeta intracellular signaling domain can have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 86, so long as it has the desired function. In certain embodiments, the CD3 zeta intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 86.

[0238] In some cases, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or a portion thereof, as long as it has the desired function. The intracellular signaling domain of CAR may include at least 90% sequence identity with ITAM, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity. In certain embodiments, the intracellular signaling domain may have at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with FcεRIγ, CD4, CD7, CD8, CD28, OX40 or H2-Kb, as long as it has the desired function.

[0239] In certain embodiments, anti-CD7 CAR further includes a hinge domain and / or a transmembrane domain. The hinge and transmembrane sequences applicable to the present invention are known in the art and are provided in, for example, publication WO2016 / 126213, which is incorporated herein by reference in its entirety. In some embodiments, the hinge and transmembrane domain of anti-CD7 CAR may include signaling domains (such as hinges and transmembrane domains) from CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, FGFR2B or other transmembrane proteins.

[0240] In certain embodiments, the anti-CD7 CAR further comprises a CD8 peptide. A schematic diagram of an anti-CD7 CAR comprising the embodiments described herein is shown in US 2018 / 0179280. Figure 17 middle.

[0241] In some embodiments, the chimeric antigen receptor (CAR) can bind to a molecule expressed on the surface of a cell, including but not limited to a member of the CD1 family of glycoproteins, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

[0242] In certain embodiments, the isolated polynucleotide of the present invention comprises a nucleic acid sequence encoding a CAR according to Table 7. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a component of a CAR according to Table 7.

[0243] Table 7. Amino acid sequence information of selected components of CD7 CAR

[0244]

[0245]

[0246] In some embodiments, the CD7 CAR comprises a CD7 antigen binding domain, a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a CD8 hinge and transmembrane domain. In some embodiments, the CD7 antigen binding domain comprises a VH domain and a VL domain, and a VH-VL linker, such as, but not limited to, (G4S) n linker, wherein n can be 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In some embodiments, the CD7 CAR comprises, from N-terminus to C-terminus: a CD8 signal peptide, a CD7 antigen binding domain, a CD8 hinge and transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta intracellular signaling domain.

[0247] In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 31. Exemplary embodiments of the CD7 CAR of the present invention are described in Figures 42-45 Shown in.

[0248] In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 29. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 29. In some embodiments, the engineered immune cells are CD8 T cells comprising a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 29. In some embodiments, the engineered immune cell is a CD3+ T cell comprising a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 29. Additionally, populations comprising such cells are provided herein.

[0249] In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. In some embodiments, the engineered immune cell is a CD3+ cell comprising a CD7 CAR encoded by a bicistronic vector such that the CD7 CAR comprises the sequence of SEQ ID NO: 30 and additional amino acid residues at the N-terminus resulting from cleavage of the cleaved 2A peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. Additionally, populations of such cells are provided herein.

[0250] In some embodiments, the CD7 CAR encoded by the dual promoter vector described herein comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28. In some embodiments, the engineered immune cells are CD3+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 28. In some embodiments, the CD7 CAR encoded by the bicistronic vector described herein comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30. In some embodiments, the engineered immune cells are CD3+ T cells comprising a CD7 CAR encoded by a dual promoter vector, such that the CD7 CAR comprises the sequence of SEQ ID NO: 30. In addition, populations of such cells are provided herein.

[0251] In certain embodiments, the isolated polynucleotide of the CD7 CAR of the present invention comprises one or more nucleic acid sequences of Table 8. In some embodiments, the nucleic acid sequence comprises a sequence encoding one or more components of the CAR listed in Table 8.

[0252] Table 8. Nucleic acid sequence information of selected components of CD7 CAR

[0253]

[0254] In some embodiments, the polynucleotide encoding the CD7 CAR comprises a nucleic acid sequence of an antigen binding domain that binds to CD7, a nucleic acid sequence of a CD8α hinge and transmembrane domain, a nucleic acid sequence of a 4-1BB intracellular signaling domain, and an intracellular signaling domain of CD3ζ. In certain embodiments, the polynucleotide further comprises a nucleic acid sequence of a CD8 signal peptide.

[0255] In certain embodiments, the antigen binding domain is an anti-CD7 scFv. In some embodiments, the VH domain of the scFv comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 38, and the VL domain of the scFv comprises a nucleic acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 39.

[0256] In some embodiments, the VH domain of the scFv comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 40, and the VL domain of the scFv comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 41. In some embodiments, the VH domain of the scFv comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 42, and the VL domain of the scFv comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 43.

[0257] In some embodiments, the polynucleotide encoding the CD7 CAR comprises, from 5' to 3' end: a nucleic acid sequence that binds to the antigen binding domain of CD7, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98. In some embodiments, the polynucleotide encoding the CD7 CAR comprises, from 5' to 3' end: SEQ ID NO: 81, a nucleic acid sequence that binds to the antigen binding domain of CD7, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0258] In some embodiments, the CD7 CAR comprises a nucleic acid sequence that has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90% or higher) sequence identity to SEQ ID NO: 4 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity to SEQ ID NO: 3 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity to SEQ ID NO: 4.

[0259] In some embodiments, the CD7 CAR comprises a nucleic acid sequence that has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90% or more) sequence identity to SEQ ID NO: 5 and binds to CD7. In some embodiments, the CD7 CAR comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 3 and binds to CD7. In some embodiments, the CD7 PEBL comprises a nucleic acid sequence that has at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 5.

[0260] In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a bicistronic construct or a dual promoter construct comprising a nucleic acid sequence of a CD7 CAR having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity to SEQ ID NO: 4. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a bicistronic vector construct or a dual promoter construct comprising a nucleic acid sequence of a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the engineered immune cell is a CD3+ T cell comprising CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 4. Additionally, provided herein are populations comprising such cells.

[0261] In some embodiments, the engineered immune cells of the present invention comprise a CD7 CAR encoded by a bicistronic construct or a dual promoter construct, the dual promoter construct comprising a nucleic acid sequence of a CD7 CAR having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence identity to SEQ ID NO: 5. In some embodiments, the engineered immune cells are CD4+ T cells comprising a CD7 CAR encoded by a bicistronic vector construct comprising a nucleic acid sequence of CD7PEBL having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the engineered immune cells are CD8+ T cells comprising a CD7PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7PEBL having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the engineered immune cell is a CD3+ T cell comprising CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 5. Additionally, provided herein are populations comprising such cells.

[0262] Engineered immune cells expressing bicistronic vectors

[0263] In certain embodiments, an engineered immune cell is provided, comprising a bicistronic construct comprising: (i) a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and specifically binds to CD7; (ii) a polynucleotide encoding a target binding molecule connected to a localization domain, wherein the target binding molecule is an antigen binding domain that binds CD7, and the localization domain comprises an endoplasmic reticulum retention sequence; (iii) a nucleic acid sequence encoding a self-cleaving 2A peptide or an IRES sequence as exemplified herein.

[0264] In certain embodiments, in the case of a CAR and in the case of an antigen binding domain against CD7, the antigen binding domain that binds to CD7 comprises: the VH sequence set forth in SEQ ID NO: 32 and the VL sequence set forth in SEQ ID NO: 33; the VH sequence set forth in SEQ ID NO: 34 and the VL sequence set forth in SEQ ID NO: 35; the VH sequence set forth in SEQ ID NO: 36 or the VL sequence set forth in SEQ ID NO: 37. As described herein, in certain embodiments, the antigen binding domain comprises a VH and a VL whose sequences are at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the VH and VL sequences of SEQ ID NOs: 32 and 33, respectively, the VH and VL sequences of SEQ ID NOs: 34 and 35, respectively, and the VH and VL sequences of SEQ ID NOs: 36 and 37, respectively. In certain embodiments, as described herein, the antigen binding domain that binds to CD7 in the context of a CAR may be different from the antibody that binds to CD7 in the context of a target binding molecule (protein expression blocker or PEBL).

[0265] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5′ end to the 3′ end, a polynucleotide encoding a target binding molecule linked to a localization domain, wherein the target binding molecule binds CD7 (e.g., CD7 PEBL), an IRES sequence, and a polynucleotide encoding a chimeric antigen receptor for CD7 (e.g., CD7 CAR). In some cases, the engineered immune cell comprises a nucleic acid construct comprising SEQ ID NO: 11. In some embodiments, provided herein are engineered CD4+ cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 11. In other embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 11. In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 11.

[0266] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5′ end to the 3′ end: a polynucleotide encoding a chimeric antigen receptor for CD7, an IRES sequence, and a polynucleotide encoding a target binding molecule in combination with a localization domain, wherein the target binding molecule binds to CD7 (e.g., a PEBL for CD7). In some cases, the engineered immune cell comprises a nucleic acid construct comprising SEQ ID NO: 12 or Figure 26 In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 12 or Figure 26 In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 12 or Figure 26 In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 12 or Figure 26 sequence shown.

[0267] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5′ end to the 3′ end, a polynucleotide encoding a chimeric antigen receptor for CD7 (e.g., CD7 CAR), a polynucleotide encoding a self-cleaving 2A peptide, and a polynucleotide encoding a target binding molecule linked to a localization domain, wherein the target binding molecule binds CD7 (e.g., CD7 PEBL). In some cases, the engineered immune cell comprises a nucleic acid construct comprising SEQ ID NO: 13 or Figure 27 In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 13 or Figure 27 In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 13 or Figure 27 In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 13 or Figure 27 sequence shown.

[0268] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5′ end to the 3′ end: a polynucleotide encoding a target binding molecule linked to a localization domain, wherein the target binding molecule binds CD7, a nucleic acid sequence encoding a self-cleaving 2A peptide, and a polynucleotide encoding a chimeric antigen receptor for CD7.

[0269] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5' end to the 3' end: a promoter, a polynucleotide encoding a chimeric antigen receptor for CD7 (e.g., CD7 CAR), a nucleic acid sequence encoding a self-cleaving 2A peptide, and a polynucleotide encoding a target binding molecule connected to a localization domain, wherein the target binding molecule binds to CD7 (e.g., CD7 PEBL). In some cases, the engineered immune cell comprises a nucleic acid construct having at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOS: 14-16. In some cases, the engineered immune cell comprises a nucleic acid construct comprising any one of the nucleic acid sequences of SEQ ID NOS: 14-16. In some embodiments, the engineered immune cell comprises a nucleic acid construct having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 14. In some embodiments, the engineered immune cell comprises a nucleic acid construct having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 15. In some embodiments, the engineered immune cell comprises a nucleic acid construct having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 16.

[0270] In some embodiments, the engineered immune cell comprises a bicistronic construct comprising a nucleic acid construct comprising, from the 5′ end to the 3′ end: a promoter, a polynucleotide encoding a chimeric antigen receptor for CD7, a nucleic acid sequence encoding a self-cleaving 2A peptide, and a polynucleotide encoding a target binding molecule linked to a localization domain, wherein the target binding molecule binds CD7. In some cases, the promoter is selected from the group consisting of an MSCV promoter, a PGK promoter, an EF1α promoter, and an EFS promoter. In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 14 or Figures 28A-28B In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or Figure 28AIn some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or Figure 28A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or Figure 28A -The sequence shown in B.

[0271] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 15. In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or Figure 29A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or Figure 29A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or Figure 29A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or Figure 29A -The sequence shown in B.

[0272] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 16 or Figure 30A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or Figure 30A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or Figure 30A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or Figure 30A -The sequence shown in B.

[0273] In some embodiments, the engineered immune cells described herein or their populations comprise at least 10% CD7 CAR+ / CD7 negative T cells, at least 15% CD7 CAR+ / CD7 negative T cells, at least 20% CD7 CAR+ / CD7 negative T cells, at least 25% CD7 CAR+ / CD7 negative T cells, at least 30% CD7 CAR+ / CD7 negative T cells, at least 35% CD7 CAR+ / CD7 negative T cells, at least 40% CD7 CAR+ / CD7 negative T cells, at least 45% CD7 CAR+ / CD7 negative T cells, at least 50% CD7 CAR+ / CD7 negative T cells, at least 55% CD7 CAR+ / CD7 negative T cells, at least 60% CD7 CAR+ / CD7 negative T cells, at least 65% CD7 CAR+ / CD7 negative T cells, at least 70% CD7 CAR+ / CD7 negative T cells, at least 75% CD7 CAR+ / CD7 negative T cells, at least 80% CD7 CAR+ / CD7 negative T cells, at least 85% of CD7CAR+ / CD7 negative T cells, at least 90% of CD7CAR+ / CD7 negative T cells, at least 95% of CD7CAR+ / CD7 negative T cells at least 96% CD7CAR+ / CD7 negative T cells, at least 97% CD7CAR+ / CD7 negative T cells, at least 98% CD7CAR+ / CD7 negative T cells, at least 99% CD7CAR+ / CD7 negative T cells or 100% CD7CAR+ / CD7 negative T cells. In some embodiments, the engineered immune cells outlined herein include a substantially purified population of CD7CAR / CD7 negative T cells, wherein such cells express any one of the bicistronic constructs.

[0274] Engineered immune cells expressing dual-promoter vectors

[0275] In some embodiments, an engineered immune cell is provided, comprising a recombinant retroviral vector comprising (a) a first promoter operably linked to a first polynucleotide encoding any CAR described herein, and (b) a second promoter operably linked to a second polynucleotide encoding any PEBL described herein. In some embodiments, the engineered immune cell comprises any recombinant retroviral vector described herein, comprising a promoter driving CAR expression and another promoter driving PEBL expression.

[0276] In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 17. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 18. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 19. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 20. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 21. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 22. In some embodiments, the engineered immune cell comprises a recombinant retroviral vector comprising a nucleic acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO: 23. In some cases, the engineered immune cell is an engineered CD4+ T cell, or a population thereof, or a population comprising the same. In some cases, the engineered immune cell is an engineered CD8+ T cell, or a population thereof, or a population comprising the same.

[0277] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 17 or Figure 31A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or Figure 31A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or Figure 31A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or Figure 31A -The sequence shown in B.

[0278] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 18. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or Figure 32A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or Figure 32A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or Figure 32A -The sequence shown in B.

[0279] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 19 or Figure 33A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or Figure 33A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or Figure 33A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or Figure 33A -The sequence shown in B.

[0280] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 20 or Figure 34A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 20 or Figure 34A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 20 or Figure 34A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 20 or Figure 34A -The sequence shown in B.

[0281] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 21 or Figure 35A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 21 or Figure 35A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 21 or Figure 35A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 21 or Figure 35A -The sequence shown in B.

[0282] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 22 or Figure 36A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 22 or Figure 36A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 22 or Figure 36A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 22 or Figure 36A -The sequence shown in B.

[0283] In some cases, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 23 or Figure 37A In one embodiment, provided herein are engineered CD4+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 23 or Figure 37A In some embodiments, provided herein are engineered CD8+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 23 or Figure 37A In some embodiments, provided herein are engineered CD3+ T cells or populations thereof comprising a nucleic acid construct comprising SEQ ID NO: 23 or Figure 37A -The sequence shown in B.

[0284] In some embodiments, the engineered immune cells described herein or their populations comprise at least 10% CD7 CAR+ / CD7 negative T cells, at least 15% CD7 CAR+ / CD7 negative T cells, at least 20% CD7 CAR+ / CD7 negative T cells, at least 25% CD7 CAR+ / CD7 negative T cells, at least 30% CD7 CAR+ / CD7 negative T cells, at least 35% CD7 CAR+ / CD7 negative T cells, at least 40% CD7 CAR+ / CD7 negative T cells, at least 45% CD7 CAR+ / CD7 negative T cells, at least 50% CD7 CAR+ / CD7 negative T cells, at least 55% CD7 CAR+ / CD7 negative T cells, at least 60% CD7 CAR+ / CD7 negative T cells, at least 65% CD7 CAR+ / CD7 negative T cells, at least 70% CD7 CAR+ / CD7 negative T cells, at least 75% CD7 CAR+ / CD7 negative T cells, at least 80% CD7 CAR+ / CD7 negative T cells, at least 85% of CD7CAR+ / CD7 negative T cells, at least 90% of CD7CAR+ / CD7 negative T cells, at least 95% of CD7CAR+ / CD7 negative T cells at least 96% CD7CAR+ / CD7 negative T cells, at least 97% CD7CAR+ / CD7 negative T cells, at least 98% CD7CAR+ / CD7 negative T cells, at least 99% CD7CAR+ / CD7 negative T cells or 100% CD7CAR+ / CD7 negative T cells. In some embodiments, the engineered immune cells outlined herein include a substantially purified population of CD7CAR / CD7 negative T cells, wherein such cells express any one of the described dual promoter constructs.

[0285] CD7 CAR+ engineered immune cells with reduced endogenous CD7 expression

[0286] In some embodiments, the engineered immune cells described herein express CD7 CAR and have reduced endogenous CD7 expression or no endogenous CD7 expression compared to non-engineered immune cells. Such engineered immune cells express CD7 PEBL, which minimizes or eliminates the endogenous expression of CD7 on the surface of immune cells. In some embodiments, reduced CD7 expression refers to downregulation or partial downregulation of surface CD7 by cells. In some cases, reduced expression includes expression levels that are at least 5% lower (e.g., at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 24%, 25%, 28%, 40%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) compared to the expression levels of comparable wild-type or non-engineered cells. In some embodiments, the engineered immune cells outlined herein include a substantially purified population of CD7 CAR / CD7 negative T cells.

[0287] In some embodiments, the engineered immune cells described herein express a CD7 PEBL that has at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the engineered immune cells express a CD7 PEBL that has at least 90% sequence identity to SEQ ID NO: 25. In some embodiments, the engineered immune cells express a CD7 PEBL that has at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, the engineered immune cells express a CD7 PEBL that has at least 90% sequence identity to SEQ ID NO: 27.

[0288] In some embodiments, the engineered immune cells described herein express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the engineered immune cells express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cells express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the engineered immune cells express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0289] In some embodiments, the engineered immune cells described herein express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 24 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the engineered immune cells described herein express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 24 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30.

[0290] In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and a CD7 CARB having at least 90% sequence identity to SEQ ID NO: 31.

[0291] In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 26 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 26 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30.

[0292] In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cells express a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0293] In certain embodiments, the engineered immune cells are engineered T cells, engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages or engineered dendritic cells. In some embodiments, the engineered immune cells are engineered CD4+T cells. In some embodiments, the engineered immune cells are engineered CD8+T cells. In some embodiments, the engineered immune cells are engineered CD3+T cells. A colony of any of the engineered cells described herein is also provided.

[0294] In some embodiments, provided herein are populations of engineered immune cells (e.g., CD3+ T cells, CD4+ T cells, or CD8+ T cells) comprising at least about 50% (e.g., about 50%, 55%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 71%, 73%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) of CD7 CAR-positive, endogenous CD7-negative cells. In some embodiments, provided herein are populations of engineered immune cells comprising at least about 50% (e.g., about 50%, 55%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 71%, 73%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) of CD7 CAR-positive, endogenous CD7-negative CD4+ T cells. In some embodiments, provided herein is an engineered immune cell population comprising at least about 50% (about 50%, 55%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 71%, 73%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) CD7 CAR positive, endogenous CD7 negative CD8+ T cells. Such a cell population can be produced by peripheral blood mononuclear cells (PBMC), purified CD4+T cells, purified CD8+T cells, or a group comprising purified CD4+T cells and purified CD8+T cells.

[0295] In some embodiments, the engineered immune cells described herein are cultured to produce a high-purity CD7 CAR-T cell population having reduced endogenous CD7 expression. The purity level can be at least about 75 (e.g., about 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more)% of CD7 CAR-T cells without surface expression of CD7. Expression levels CD7 can be determined according to standard methods known to those skilled in the art, including but not limited to immunocytochemistry, flow cytometry, and FACS analysis.

[0296] In some embodiments, the engineered immune cells of the present invention include CD45RO+ cells. In some embodiments, the engineered immune cells include CCR7 negative cells. In some embodiments, the engineered immune cells include central memory T cells. In some embodiments, the engineered immune cells include effector memory T cells. In some embodiments, the engineered immune cells include effector T cells. In some embodiments, the engineered immune cells include naive T cells.

[0297] In some cases, the engineered immune cell population includes effector memory T cells, central memory T cells, effector T cells, and naive T cells. In some embodiments, the engineered immune cell population comprises a higher percentage of effector memory T cells and central memory T cells than effector T cells and naive T cells. In some embodiments, the engineered immune cell population comprises about 40% or more effector memory T cells.

[0298] In some cases, the engineered immune cell population comprises PD1 negative cells. In some cases, the engineered immune cell population comprises TIM-1 negative cells. In some embodiments, the population comprises about 60% or more PD1 negative, TIM-1 negative cells. In some embodiments, the population comprises about 4% to about 20% PD1 positive, TIM-1 positive cells.

[0299] In some embodiments, the engineered immune cells generate an immune response and secrete interferon-γ. The engineered immune cells induce T cell-mediated cytotoxicity in response to cancer cells, such as cancer cells expressing CD7.

[0300] In some embodiments, cells comprising a bicistronic expression vector as described herein can be used to generate a population of CD7CAR+ / CD7-negative cells. CD7CAR+ / CD7-negative T cells can be expanded and enriched over time. CD7CAR+ / CD7-negative T cells can be generated from cells including, but not limited to, large numbers of PBMCs, purified T cells comprising CD4 and CD8+ T cells, and purified CD3+ T cells. CD7CAR+ / CD7-negative T cells can be used to generate different subsets of T cells, including T EM cells, T CM cells, Teff cells and naive T cells.

[0301] On the other hand, there is also provided a method for producing engineered immune cells (e.g., engineered CD3+T cells, engineered CD4+T cells, and engineered CD8+T cells) having any embodiment described herein, the method comprising introducing any bicistronic construct or dual promoter construct of the present invention into immune cells. In some embodiments, engineered immune cells are derived from immune cells obtained from a subject, who will receive engineered immune cells as therapy. In some embodiments, engineered immune cells are derived from immune cells obtained from a donor, and the resulting engineered immune cells are administered to a subject as therapy.

[0302] In various aspects, a kit for producing engineered immune cells as described herein is also provided. This kit can be used to produce, for example, allogeneic or autologous T cells having anti-CD7 CAR-mediated cytotoxic activity. In some embodiments, the kit can be used to produce allogeneic effector T cells having anti-CD7 CAR-mediated cytotoxic activity. In certain embodiments, the kit can be used to produce autologous effector T cells having anti-CD7 CAR-mediated cytotoxic activity.

[0303] Thus, the kits provided herein comprise any of the bicistronic constructs or dual promoter constructs described herein.

[0304] In certain embodiments, the bicistronic construct further comprises sequences that allow, for example, cloning and / or expression (e.g., plasmid or vector sequences). For example, the nucleotide sequence can be provided as part of a plasmid to facilitate cloning into other plasmids and / or vectors (expression vectors or viral expression vectors), for example, for transfection, transduction, or electroporation into cells (e.g., immune cells).

[0305] Typically, the test kit is compartmentalized for ease of use and may include one or more containers containing reagents. In certain embodiments, all of the test kit components are packaged together. Alternatively, one or more individual components of the test kit may be provided in separate packaging from the other test kit components. The test kit may also include instructions for using the test kit components.

[0306] Drug delivery to engineered immune cells

[0307] In other aspects, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutic amount of an engineered immune cell having any embodiment described herein, thereby treating cancer in the subject in need thereof.

[0308] In certain embodiments, the method includes administering a therapeutic amount of engineered immune cells comprising a bicistronic viral construct comprising a polynucleotide comprising a polynucleotide comprising a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding a PEBL. In various embodiments, the method includes administering a therapeutic amount of any engineered immune cell described herein comprising a recombinant retroviral vector comprising: (a) a first promoter operably linked to a first polynucleotide encoding a CD7 chimeric antigen receptor (CD7CAR) as described herein; (b) a second promoter operably linked to a second polynucleotide encoding a CD7 protein expression blocker (CD7PEBL) as outlined herein.

[0309] In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 11 or Figure 25 In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 12 or Figure 26 In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 13 or Figure 27 In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or Figures 28A-28B In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or Figures 29A-29BIn some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or Figures 30A-30B In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or Figures 31A-31B In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or Figure 32A In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or Figure 33A In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 20 or Figure 34A In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 21 or Figure 35A In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 22 or Figure 36A In some embodiments, a therapeutic amount of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) or a population thereof comprising a nucleic acid construct comprising SEQ ID NO: 23 or Figure 37A -The sequence shown in B.

[0310] In some embodiments, a therapeutic amount of a population of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 25 is administered to a subject having cancer. In some embodiments, a therapeutic amount of a population of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 27 is administered to a subject having cancer.

[0311] In some embodiments, a therapeutic amount of a population of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 CAR of SEQ ID NO: 29 is administered to a subject having cancer. In some embodiments, a therapeutic amount of a population of engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 CAR of SEQ ID NO: 31 is administered to a subject having cancer.

[0312] In some embodiments, a therapeutic amount of an engineered immune cell population (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 25 and a CD7 CAR of SEQ ID NO: 29 is administered to a subject having cancer. In some embodiments, a therapeutic amount of an engineered immune cell population (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 27 and a CD7 CAR of SEQ ID NO: 29 is administered to a subject having cancer.

[0313] In some embodiments, a therapeutic amount of an engineered immune cell population (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 25 and a CD7 CAR of SEQ ID NO: 31 is administered to a subject having cancer. In some embodiments, a therapeutic amount of an engineered immune cell population (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a CD7 PEBL of SEQ ID NO: 27 and a CD7 CAR of SEQ ID NO: 31 is administered to a subject having cancer.

[0314] In some embodiments, a therapeutic amount of a population of engineered immune cells comprising SEQ ID NO: 95 (eg, engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) is administered to a subject having cancer.

[0315] In certain embodiments, the cancer is a T-cell malignancy, such as a T-cell leukemia or T-cell lymphoma, such as T-cell acute lymphoblastic leukemia, T-cell lymphocytic leukemia, T-cell large granular lymphocytic leukemia, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Cesari syndrome, primary cutaneous gamma-T-cell lymphoma, peripheral T-cell lymphoma-not otherwise specified, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma. In certain embodiments, the T-cell malignancy is early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0316] In some embodiments, the engineered immune cells are autologous to the subject in need of treatment, such as cancer treatment. In other embodiments, the engineered immune cells are allogeneic to the subject in need of treatment.

[0317] In certain embodiments, the engineered immune cells are administered to a subject by intravenous infusion, intra-arterial infusion, direct injection into the tumor and / or post-operative perfusion of the tumor bed, implantation in an artificial scaffold at the tumor site, intrathecal administration, and intraocular administration.

[0318] In certain embodiments, the engineered immune cells are administered to the subject by infusion. Methods for infusing immune cells (e.g., allogeneic immune cells or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient to improve the symptoms of the disease. Typically, 10 7 to 10 10 A dose of 10 cells, for example 9 The dose of cells is 10 9 A cell dose can be divided into several 10 9 The cells are administered in the form of a single cell dose. The frequency of infusion can be daily, once every 2 to 30 days, or even longer intervals (if necessary or indicated). The amount of infusion is typically at least 1 infusion per subject, and preferably, at least 3 infusions if tolerated or until the symptoms of the disease have improved. The cells can be injected intravenously at a rate of 50-250 ml / hr. Other suitable modes of administration include intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or post-operative perfusion of the tumor bed, implantation of an artificial stent at the tumor site, and intrathecal administration. Methods for adapting the present invention to this delivery mode are readily available to those skilled in the art.

[0319] In certain embodiments, the methods of treating cancer according to the present invention are combined with at least one other known cancer therapy, such as radiation therapy, chemotherapy, or other immunotherapy.

[0320] In other aspects, there is also provided a use of an engineered immune cell of any embodiment described herein for treating cancer, comprising administering a therapeutic amount of the engineered immune cell to a subject in need thereof. In certain embodiments, the cancer is a T cell malignancy. In certain embodiments, the T cell malignancy is early T cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0321] In certain embodiments, the engineered immune cells are administered to a subject by intravenous infusion, intraarterial infusion, intraperitoneal infusion, injection directly into the tumor after surgery and / or perfusion of the tumor bed, implantation of an artificial stent at the tumor site, or intrathecal injection.

[0322] Examples

[0323] Example 1: Blocking CD7 expression in chimeric antigen receptor T cells using twin expression vectors

[0324] This example demonstrates the blockade of CD7 expression in anti-CD7 CAR-T cells using a bicistronic expression construct.

[0325] method

[0326] Cell culture

[0327] 293T cells (ATCC CRL-3216) were maintained in DMEM (Gibco) containing 10% FBS (Hyclone), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco). Jurkat clone E6-1 cells (ATCC TIB-152) and NALM6 clone G5 cells (CRL-3273) were maintained in RPMI1640 (Gibco) containing 10% FBS (Hyclone), 100 U / mL penicillin, 100 μg / mL streptomycin (Gibco), and 1 GlutaMAX (Gibco).

[0328] Lentiviral production

[0329] 293T cells were co-transfected with the lentiviral transfer vector and the Virapower packaging plasmid mixture (Invitrogen) at a 1:3 ratio using Lipofectamine 2000 (Invitrogen). Six hours after transfection, the transfection medium was replaced with fresh DMEM (Gibco) and 10% FBS (Hyclone). After 48 hours, the viral supernatant was collected, passed through a 0.45 μM filter, and concentrated 100-fold using a Lenti-X concentrator (Clontech). The concentrated viral stock was stored at −150°C until use.

[0330] Retroviral production

[0331] 293T cells were co-transfected with the retroviral transfer vector and the pEQ and pRDF packaging plasmids using X-tremeGENE 9 DNA transfection reagent (Roche). Six hours after transfection, the transfection medium was replaced with fresh DMEM (Gibco) and 10% FBS (Hyclone). After 24 and 48 hours, viral supernatants were collected and filtered through a 0.45 μM filter. Retroviruses could be used fresh or stored at -150°C until use.

[0332] Lentiviral titration in 293T cells

[0333] 293T cells were transduced with different volumes of lentivirus in the presence of 5 μg / mL polypropylene glycol (Sigma). After 15 hours of overnight culture, the transduction medium was removed and the cells were treated with 10 U / mL DNase I (New England Biolabs) in fresh culture medium at 37°C for 15 minutes. The culture medium was then replaced with fresh DMEM containing 10% FBS for further culture. Transduced cells were collected for analysis ≥72 hours after transduction. Viral titers were determined using flow cytometry and RT-qPCR.

[0334] Transducing unit (TU) titers were calculated from flow cytometry data using the following formula: [TU / mL = (number of 293T cells in each sample x %CAR+ cells) ÷ virus volume in mL]. TU titers were calculated using samples within the linear range of %CAR+ cells and virus volume.

[0335] The integrated unit (IU) titer was calculated from the RT-qPCR data of genomic DNA using the following formula: [IU / ml = (number of 293T cells per sample × number of proviral gene copies per genome) ÷ viral volume in milliliters]. IU titers were calculated using samples within the linear range of proviral gene copy number and viral mass.

[0336] Lentiviral transduction of Jurkat cells

[0337] Lentivirus was added directly to Jurkat cells with or without 8 μg / mL trimethoprim (Sigma). A complete medium change was performed two days later to remove the lentivirus from the culture. Transduced cells were harvested for analysis ≥2 days after transduction.

[0338] Primary T cell culture

[0339] Frozen human primary peripheral blood mononuclear cells (PBMC) (ATCC Cat# PCS-800-011 and Stemcell Technologies Cat# 70025) were thawed, recovered overnight, and maintained at 1 million cells / mL (Hyclone) in RPMI1640 (Gibco) containing 10% FBS, 100 U / mL penicillin, 100 ug / mL streptomycin (Gibco), and 1x GlutaMAX (Gibco), TexMACS medium (Miltenyi Biotec) supplemented with 3% human AB serum (Sigma) or serum-free TexMACS medium. The medium was supplemented with 120 IU / mL interleukin 2 (Miltenyi Biotec) every 2 to 3 days.

[0340] PBMCs were cultured in bulk without further selection or used for T cell purification after overnight recovery. CD4+ and CD8+ T cells were isolated using CD4 microbeads (Miltenyi Biotec) and CD8 microbeads (Miltenyi Biotec). CD3+ T cells were isolated using CD3 microbeads (Miltenyi Biotec).

[0341] T cell expansion and activation were performed using T cell TransAct (Miltenyi Biotec) or immunomagnetic human T-activator CD3 / CD28 according to the manufacturer's recommendations. Immunomagnetic beads were added at a 1:1 bead-to-cell ratio. Bead removal was performed after 4 days.

[0342] Lentiviral transduction of primary T cells

[0343] Primary T cells were transduced 1 to 4 days after activation. Lentivirus was added directly to T cells for static transduction. A complete medium change was performed after two days to remove the lentivirus from the culture. Transduced cells were analyzed by flow cytometry ≥ 3 days after transduction.

[0344] Retroviral transduction of primary T cells

[0345] Retroviral transduction of primary T cells was performed using retroviral-based transduction. Untreated tissue culture plates were coated with 2.5 μg / cm2 of the retroviral-based transduction kit according to the manufacturer's recommendations. 2 RetroNectin recombinant human fibronectin fragment (Takara) was added to the retroviral supernatant on the retroviral plate and centrifuged at 1000 x g for 2 h at 32°C. The viral supernatant was then removed from the wells. Before adding T cells, the wells were rinsed with culture medium. Transduced cells were analyzed by flow cytometry ≥3 days after transduction.

[0346] flow cytometry

[0347] Antibody staining and washing were performed with staining buffer (1xPBS pH 7.4, 0.2% BSA, 0.02% sodium azide). Cells were incubated with antibodies on ice for 15 minutes and washed 3 times. The following antibodies were used for staining: anti-mouse F (ab') 2-biotin (Jackson Immunoresearch 115-066-072), streptavidin APC (Jackson Immunoresearch016-130-084), anti-human CD7-PE (BD 555361), anti-human CD3 eFluor780 (eBioscience 47-0037-42). 3 μg of mouse IgG1 isotype control antibody (BioXCell) was added, and cells stained with anti-mouse F (ab') 2-biotin were blocked for 5 minutes, and then the remaining antibodies were added. DAPI was used for live / dead identification at a concentration of 1 μg / mL. Stained cells were collected on an Invitrogen Attune NxT flow cytometer and analyzed using FlowJo v10 software.

[0348] Real-time quantitative PCR (RT-qPCR)

[0349] Genomic DNA was extracted from cells using the DNeasy Blood and Tissue Kit (Qiagen) and RNase A (Qiagen). Total RNA was extracted from cells using the MN NucleoSpinRNA Kit (Macherey-Nagel), and cDNA was synthesized using the MaximaFirst Strand cDNA Synthesis Kit (Thermo Scientific). All kits were used according to the manufacturer's recommendations. iTaq Universal SYBR Green Supermix (Bio-Rad) was used on a CFX96 Touch TM RT-qPCR was performed using a real-time PCR detection system (Bio-Rad).

[0350] The primers used were:

[0351] RPPH1-F: 5'-GAGGGAAGCTCATCAGTGGG-3' (SEQ ID NO: 87)

[0352] RPPH1-R: 5'-CATCTCCTGCCCAGTCTGAC-3' (SEQ ID NO: 88)

[0353] WPRE-F: 5'-CCTTTCCGGGACTTTCGCTTT-3' (SEQ ID NO: 89)

[0354] WPRE-R: 5'-GCAGAATCCAGGTGGCAACA-3' (SEQ ID NO: 90)

[0355] TH69CD7CAR-F: 5'-GCAGCCTTTCATGAGACCAG-3' (SEQ ID NO: 91)

[0356] TH69CD7CAR-R: 5'-TGCCCAGGTTCAGCTCATTA-3' (SEQ ID NO: 92)

[0357] TH69CD7PEBL-F: 5'-ACCTGCCGCATACAAGGATA-3' (SEQ ID NO: 93)

[0358] TH69CD7PEBL-R: 5'-CCACTGTGCAGACTAGAGGT-3' (SEQ ID NO: 94)

[0359] The primer efficiencies of all assayed primers ranged from 90% to 110%.

[0360] Fold changes of all genes were normalized to the housekeeping gene using the equation: [Fold change = 2̂-(Ct(target gene)-Ct(housekeeping gene))] The copy number of the target gene was normalized to the genomic copy number of RNaseP in 293T cells.

[0361] Western blotting

[0362] Cells were lysed with RIPA buffer (Pierce) and protease inhibitors (Pierce). Protein quantification was performed on cell lysates using a Bradford Coomassie protein assay kit (Thermo Scientific) according to the manufacturer's recommendations. Protein immunoblotting was performed using the Simple WesternWes automated protein immunoblotting system (ProteinSimple) with a 12-230 kDa Wes separation module. The following primary antibodies were used: anti-β-actin, clone 13E5 (Cell Signaling Technology); anti-Myc tag, clone 71D10 (Cell Signaling Technology); and anti-CD3ζ polyclonal antibody, catalog number ab226475 (Abcam). Secondary antibodies from the Wes anti-rabbit detection module were used. Data were analyzed using SimpleWestern software from Compass.

[0363] IFNγ secretion

[0364] Effector CAR-T cells were injected at 10 6 Cells were resuspended at a density of 100,000 cells / mL and 100,000 CAR-T cells were plated per well of a 96-well round-bottom plate. Target cells were co-cultured with effector CAR-T cells at various effector:target (E:T) ratios for 24 hours. After 24 hours, cells were centrifuged and the supernatant was collected and stored at -150°C. IFNγ secretion in the supernatant was assessed using the ELISA MAX Standard Set Human IFN-γ Kit (Biolegend) according to the manufacturer's recommendations.

[0365] Cytotoxicity assay

[0366] The target cells were 10 6 The cells were resuspended at a cell density of 100 μg / ml and loaded with 0.4 μg / ml calcein orange red AM (Invitrogen) for 10 minutes. The loaded cells were then washed three times to remove excess calcein. 100,000 target cells were seeded per well in a 96-well round-bottom plate. Target cells were co-cultured with effector CAR-T cells at various effector: target (E: T) ratios for 4 h. After 4 hours, DAPI was added to all wells and cells were collected on a flow cytometer. The number of remaining live target cells was counted in all wells. The percentage of cytotoxicity was calculated using the following equation:

[0367] Cytotoxicity percentage = [(SE) / S]*100%

[0368] S = The remaining live target cells in the target cell can only control the wells

[0369] E = viable target cells remaining in the experimental well after co-culture with effector T cells

[0370] result

[0371] Primary T cells were transduced with different retroviruses expressing (1) PEBL, (2) CAR, (3) PEBL and CARy in sequence, (4) PEBL-IRES-CAR, or (5) CAR-P2A-PEBL. Transduced cells were analyzed by flow cytometry for CD7 and CAR expression ( Figure 1A Cell lysates from primary T cells transduced with the indicated retroviruses were analyzed by Western blotting for β-actin, Myc-tagged PEBL, CAR, and endogenous CD3ζ expression ( Figure 1B ).

[0372] A dual-promoter lentiviral construct was prepared to express anti-CD7 CAR and anti-CD7 PEBL from a single vector. Figures 2A-2F As shown, the general format of the dual promoter construct from 5' to 3' end includes the first promoter anti-CD7 CAR - the second promoter anti-CD7 PEBL. The promoters tested include the MSCV promoter, EFS promoter, PGK promoter and EF1a promoter. The nucleic acid sequences of exemplary dual promoter constructs are provided as SEQ ID NOs: 19-23 and are shown in Figures 33A-33B 、 Figures 34A-34B 、 Figures 35A-35B 、 Figures 36A-36B and Figures 37A-37B Such constructs encode anti-CD7 CARs, including anti-CD7 CARs based on TH69 antibodies and anti-CD7 CARs based on 3A1F antibodies, and anti-CD7 PEBLs, including anti-CD7 PEBLs based on TH69 antibodies and anti-CD7 PEBLs based on 3A1F antibodies.

[0373] A dual-promoter lentiviral vector was transduced into cells to generate cells with partially downregulated surface CD7 expression and low expression of the anti-CD7 CAR. After prolonged culture, the percentage of cells expressing the CAR remained low. Figure 3 The expression of CAR (y-axis) and CD7 (x-axis) in cells 5 days after transduction and 14 days after transduction are shown. The figure shows the expression of cells (e.g., healthy donor cells, including healthy donor lymphocytes) transduced by exemplary dual promoter constructs, which are provided as SEQ ID NOs: 18-23 and are shown in FIG. Figures 32A-32B, 33A-33B, 34A-34B, 35A-35B, 36A-36B, 37A-37B. For example, cells transduced with a dual promoter lentiviral vector comprising an MSCV promoter-CD7 (TH69) CAR-EF1a promoter-CD7 (TH69) PEBL produced CD7 CAR-negative / CD7-negative cells (52.8%), CD7 CAR+ / CD7-negative cells (2.98%), CD7 CAR-negative / CD7+ cells (40.4%), and CD7 CAR+ / CD7+ cells (3.84%) on day 5 after transduction. On day 14 after transduction, cells transduced with MSCV promoter-CD7(TH69)CAR-EF1a promoter-CD7(TH69)PEBL contained a population of CD7CAR-negative / CD7-negative cells (42.6%), CD7CAR+ / CD7-negative cells (0.14%), CD7CAR-negative / CD7+ cells (54.9%), and CD7CAR+ / CD7+ cells (2.37%).

[0374] To optimize the expression of CD7 CAR and CD7 PEBL, a single-promoter bicistronic lentiviral construct was generated. Figure 4B 、 Figure 4B 、 4C An exemplary schematic diagram of such a configuration is provided in . Figure 4A A schematic diagram of an exemplary bicistronic construct comprising MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 14, is depicted. Figure 4B A schematic diagram of an exemplary bicistronic construct is depicted, comprising an EF1a promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 15. Figure 4c depicts a schematic diagram of an exemplary bicistronic construct, comprising an EFS promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL, such as one of SEQ ID NO: 16.

[0375] CD7 CAR+ / CD-negative T cells were generated by cells transduced with MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL lentivirus. Figure 5The figure shows the expansion and enrichment of CD7 CAR+ / CD7-negative T cells from day 0 to day 9 after transduction. For example, on day 0, 10.9% of the cells were CD7 CAR-negative / CD7-negative cells, 0.016% were CD7 CAR+ / CD7-negative cells, 87.9% were CD7 CAR-negative / CD7+ cells, and 1.21% were CD7 CAR+ / CD7+ cells. On day 3, 24.1% of the cells were CD7 CAR-negative / CD7-negative cells, 17.7% were CD7 CAR+ / CD7-negative cells, 53.8% were CD7 CAR-negative / CD7+ cells, and 4.33% were CD7 CAR+ / CD7+ cells. On day 6, 27.5% of cells were CD7 CAR-neg / CD7- cells, 63.7% were CD7 CAR+ / CD7-neg cells, 6.25% were CD7 CAR-neg / CD7+ cells, and 2.57% were CD7 CAR+ / CD7+ cells. On day 9, 16.1% of cells were CD7 CAR-neg / CD7-neg cells, 83.7% were CD7 CAR+ / CD7- cells, 0.012% were CD7 CAR-neg / CD7+ cells, and 0.095% were CD7 CAR+ / CD7+ cells.

[0376] CD7 CAR+ / CD7-negative cells generated by cells transduced with anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL bicistronic lentiviral vectors with different promoters were enriched over time. Figure 6The percentage of CD7 CAR+ / CD7-negative cells increased 5 days after transduction and 14 days after transduction. For example, cells transduced with the EFS promoter anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL lentiviral vector produced 67.3% CD7 CAR-negative / CD7- cells, 31.2% CD7 CAR+ / CD7-negative cells, 0.44% CD7 CAR-n negative / CD7+ cells, and 1.06% CD7 CAR+ / CD7+ cells on day 5 after transduction. On day 14 after transduction, 25.0% were CD7 CAR-negative / CD7- cells, 73.8% were CD7 CAR+ / CD7-negative cells, 0.80% were CD7 CAR-negative / CD7+ cells, and 0.46% were CD7 CAR+ / CD7+ cells. As a control, cells transduced with a lentiviral vector expressing an EF1a promoter upstream of the EF19 promoter on day 5 post-transduction showed that 34.7% of cells were CD19 CAR+ / CD7+, 60.1% were CD19 CAR-negative / CD7+, 4.58% were double negative, and 0.68% were CD19 CAR+ / CD7-negative. On day 14 post-transduction, 47.5% of cells were CD19 CAR+ / CD7+, 50.4% were CD19 CAR-negative / CD7+, 1.01% were double negative, and 1.06% were CD19 CAR+ / CD7-negative. Thus, little enrichment of CD19 CAR-expressing cells was detected over time.

[0377] To evaluate the consistency and reproducibility of the single promoter bicistronic vector, cells were transduced with two independent batches of MSCV promoter-anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL lentivirus. CD7 PEBL was myc-tagged and detected by western blot. CD7 CAR ( Figure 7B ). Figure 7A Flow cytometric analysis of CD7 CAR and CD7 expression in transduced cells is shown. The first batch produced a group of transduced cells including 53.8% CD7 CAR+ / CD7-negative cells and 46.1% CD7-negative / CD7-negative cells. The second batch produced transduced cells containing 65.5% CD7 CAR+ / CD7-negative cells and 34.5% CD7-negative / CD7-negative cells. Note that the untransduced cells included 98.6% CAR-negative / CD7+ cells.

[0378] It should be noted that the single promoter bicistronic vector described herein has been successfully used to generate CD7 PEBL-CAR-T cells from different starting cells, including bulk PBMCs, purified T cells containing CD4+ and CD8+ T cells, and purified CD3+ T cells. In addition, different T cell activation reagents were used, including Human T activator CD3 / CD28 (Gibco) and T Cell TransAct TM (Miltenyi Biotec). Figure 8 The results show that when cells transduced with anti-human CD7 (TH69) CAR-P2A-anti-human CD7 (TH69) PEBL were cultured over time, the percentage of CD7 CAR+ / CD7-negative T cells increased. Comparable expansion of CD7 CAR+ / CD7-negative T cells was detected between different starting cell types and between the two activators.

[0379] The cells described herein (e.g., CD7 CAR+ / CD7- negative T cells) were generated from CD4+ positively selected CD4+ and CD8+ positively selected T cells cultured in serum-free TexMACS medium or TexMACS medium supplemented with 3% human AB serum. T cells were transduced with CD7CAR-P2A-CD7PEBL lentivirus at an MOI of 10 to generate CD7-CAR+ T cells. On day 11 after cell activation, the total fold change of transduced cells was higher ( Figure 9A and Figure 9B ).

[0380] Purified CD4+ and CD8+ selected T cells transduced with CD7CAR-P2A-CD7PEBL lentivirus at different days after activation (day 1 to day 4) generated highly pure CAR+ T cells ( Figure 10A and Figure 10B During the manufacturing process, cells transduced on different days are expanded and proliferated. Figure 10B It was shown that when cells were transduced 1, 2, 3, or 4 days after activation, the transduced T cells exhibited an average of approximately 5- to 10-fold expansion.

[0381] The expression of CAR and endogenous CD7 in T cells transduced with different MOIs of CD7CAR-P2A-CD7PEBL lentivirus was measured by FACS. T cells transduced at lower MOIs had a lower percentage of CD7CAR+ / CD7-negative T cells early in the transduction process, but increased to match the percentage of CD7CAR+ / CD7-negative T cells obtained at higher MOIs (see, e.g., Figure 11For example, T cells from donor 1 transduced with lentivirus at an MOI of 3 were 9.76% CD7 CAR+ / CD7-negative T cells 3 days after transduction and 69.9% CD7 CAR+ / CD7-negative T cells 9 days after transduction. T cells from donor 1 transduced with lentivirus at an MOI of 10 were 17.7% CD7 CAR+ / CD7-negative 3 days after transduction and 83.7% CD7 CAR+ / CD7-negative 9 days after transduction.

[0382] Purified CD4+ and CD8+ T cells from three unique donors were transduced with CD7CAR-P2A-CD7PEBL lentivirus at the indicated MOI in two separate wells. The percentage of CAR+ cells was analyzed by flow cytometry. Cell pellets were collected and genomic DNA was extracted to determine vector copy number (VCN) by RT-qPCR analysis. Higher MOIs were associated with higher VCN ( Figure 12B ), but the percentages of CD7 CAR+T cells were similar at MOI 5 and 10 ( Figure 12A ).

[0383] The expression of various surface markers was measured in primary T cells transduced with the MSCV-CD7CAR-P2A-CD7PEBL lentivirus 11 days after activation. Figure 13A CD7 CAR and endogenous CD7 expression in transduced cells from three different donors is shown. Compared with CD14 / CD19 / CD56, CD3 expression was Figure 13B CD4 and CD8 expression in Figure 13C Shown in. Figure 13D The transduced cells gave rise to different T cell subsets, including T cells identified by CD45RO and CCR7 expression. EM cells, TCM cells, T eff cells and naive T cells. Figure 13D The expression of PD-1 and TIM-3 in transduced T cells is shown.

[0384] Through IFNγ secretion ( Figure 14A ) and cytotoxicity ( Figure 14B) The responses of transduced PEBL-CAR T cells to CD7+ Jurkat cells and CD7-negative Nalm6 cells were determined. IFN-g secretion was measured in the culture supernatant of PEBL-CAR T cells co-cultured with Jurkat or Nalm6 cells for 24 hours at the indicated E:T ratios (mean ± SD of technical replicates). PEBL-CAR-T cells demonstrated target-specific functional responses, as IFNγ was secreted by PEBL-CAR-T cells when cultured with CD7+ Jurkat cells (but not Nalm6 cells). In addition, in a cytotoxicity assay, PEBL-CAR-T cells killed CD7+ Jurkat cells but not CD7-negative Nalm6 cells.

[0385] This example demonstrates the generation and expansion of PEBL-CAR-T cells generated using a CD7 CAR-P2A-CD7 PEBL bicistronic lentiviral vector. These cells exhibited antigen-specific T cell functional responses (e.g., IFNγ secretion) and specific toxicity against CD7+ target cell lines. PEBL-CAR-T cells exhibited a high percentage of CD7-negative CAR+ T cell purity.

[0386] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0387] While the invention has been particularly shown and described with reference to example embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. Sequence Listing <110> MediSykes Medical Pte Ltd <120> Two types of gene vectors for generating CAR-T cells and their uses <130> 119419-5007-WO <140> Submit Here <141> 2019-11-13 <150> 62 / 767,069 <151> 2018-11-14 <160> 98 <170> PatentIn version 3.5 <210> 1 <211> 2708 <212> DNA <213> Artificial sequence <220> <223> CPPT-CMV-MCS-PGK-GFP-WPRE <400> 1 atcgatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aattagtagac 60 father cagacataca aactaaagaa ttacaaaaac aatacaaa aattcaaaat 120 ttcgggttt attaattaac agggacatgc agagatccag tttaagcttg ggagttccgc 180 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 240 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 300 tgggtggagt attacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 360 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 420 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 480 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 540 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 600 gactttccaa aatgcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 660 cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc 720 catccacgct gttttgacct ccatagaaga caccgactct actagaggat cgctagcgct 780 accggactca gatcgaattc ctcaagcttc ctcgagtgca gtcgacgcgg gcccgggatc 840 ccgcgactct agataattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc 900 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac 960 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc 1020 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag 1080 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga 1140 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt 1200 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg 1260 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac 1320 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgactggcc accggcttcc 1380 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 1440 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 1500 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 1560 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 1620 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 1680 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1740 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 1800 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 1860 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 1920 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 1980 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 2040 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 2100 taacgcgtct ggaacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2160 taacgcgtct ggaacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2160 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2220 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2220 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2280 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2280 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2340 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2340 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2400 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2400 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2460 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2460 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 2520 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 2520 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2580 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2580 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2640 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2640 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 2700 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 2700 ccgcctgg 2708 ccgcctgg 2708 <210> 2 <210> 2 <211> 855 <211> 855 <212> DNA <212> DNA <213> 人工序列 <213> Artificial sequence <220> <220> <223> 抗人CD7(TH69)PEBL的合成多核苷酸 <223> Synthetic polynucleotide against human CD7 (TH69) PEBL <400> 2 atggctctgc ctgtgaccgc actgctgctg ccctggctc tgctgctgca cgccgcaaga 60 cctgccgcat acaaggatat tcagatgact cagaccacaa gctccctgag cgcctccctg 120 ggagaccgag tgacaatctc ttgcagtgca tcacaggggaa ttagcaacta cctgaattgg 180 tatcagcaga agccagatgg cactgtgaaa ctgctgatct actatacctc tagtctgcac 240 agtggggtcc cctcacgatt cagcggatcc ggctctggga cagactacag cctgactatc 300 tccaacctgg agcccgaaga tattgccacc tactattgcc agcagtactc caagctgcct 360 tataccttg gcgggggaac aaagctggag attaaaaggg gaggaggagg aagcggagga 420 ggaggatccg gaggcggggg atctggagga ggaggaagtg aggtgcagct ggtcgaatct 480 ggaggaggac tggtgaagcc aggaggatct ctgaaactga gttgtgccgc ttcaggcctg 540 accttctcaa gctacgccat gagctgggtg cgacagacac ctgagaagcg gctggaatgg 600 gtcgctagca tctcctctgg cgggttcaca tactatccag actccgtgaa aggcagattt 660 actatctctc gggataacgc aagaaatatt ctgtacctgc agatgagttc actgaggagc 720 gaggacaccg caatgtacta ttgtgccagg gacgaagtgc gcggctatct ggatgtctgg 780 ggagctggca ctaccgtcac cgtctccagc gagcagaaac tgattagcga agaggacctg 840 aaagatgaac tgtaa 855 <210> 3 <211> 846 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide against human CD7 (3A1F) PEBL <400> 3 atggctctgc ccgtcaccgc tctgctgctg cctctggctc tgctgctgca cgccgcaaga 60 ccccaggtcc agctgcagga gtcaggggca gagctggtga aacccggagc cagtgtcaaa 120 ctgtcctgta aggccagcgg ctatactttc accagctact ggatgcactg ggtgaaacag 180 aggccaggac agggcctgga gtggatcggc aagattaacc ccagcaatgg gcgcaccaac 240 tacaacgaaa agtttaaatc caaggctaca ctgactgtgg acaagagctc ctctaccgca 300 tacatgcagc tgagttcact gacatctgaa gatagtgccg tgtactattg cgccagaggc 360 ggggtctact atgacctgta ctattacgca ctggattatt gggggcaggg aaccacagtg 420 actgtcagct ccggaggagg aggatccggc ggaggaggct ctgggggagg cgggagtgac 480 atcgagctga cacagtctcc agccactctg agcgtgaccc ctggcgattc tgtcagtctg 540 tcatgtagag ctagccagtc catctctaac aatctgcact ggtaccagca gaaatcacat 600 gaaagccctc ggctgctgat taagagtgct tcacagagca tctccgggat tccaagcaga 660 ttctctggca gtgggtcagg aaccgacttt acactgtcca ttaactctgt ggagaccgaa 720 gatttcggca tgtatttttg ccagcagagc aattcctggc cttacacatt cggaggcggg 780 actaaactgg agattaagag ggagcagaaa ctgattagcg aagaggacct gaaggacgaa 840 ctgtga 846 <210> 4 <211> 1482 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide of anti-human CD7 (TH69) CAR <400> 4 atggctctgc ctgtgactgc cctgctgctg cccctggccc tgctgctgca cgctgctaga 60 cccgccgcat acaaggacat tcagatgact cagactacat ccagcctgtc agcttccctg 120 ggcgatcgag tgactatcag ctgttcagca agccagggga tcagcaatta cctgaactgg 180 taccagcaga agcccgacgg aactgtcaag ctgctgatct actatacttc cagtctgcat 240 agcggggtcc ctagccgatt ctctgggagt ggaagcggca ccgactatag cctgaccatt 300 tccaacctgg aaccagagga cattgccacc tattactgtc agcagtattc aaagctgcccc 360 tataccttg gcggagggac taagctggag atcaagcgag gagggggagg atcaggggga 420 ggggggagcg gaggcggggg gagtggcggc ggagggtcag aagtccagct ggtcgagagt 480 gggggcggac tggtgaaacc tgggggctct ctgaaactga gctgtgcagc cagtggcctg 540 acattctcct catatgctat gtcttgggtg cggcagaccc cagaaaagag actggagtgg 600 gtggcttcaa tctcttccgg gggcttcact tactatcctg acagcgtgaa aggcagattc 660 actatctcca gggataacgc acggaatac ctgtatctgc agatgagctc actgagatcc 720 gaggacaccg caatgtacta ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg 780 ggagcaggca ctaccgtcac cgtgtctagc accactacac ctgcaccaag gcctcccaca 840 cccgctccca ctatcgcttc ccagccactg tccctgaggc ccgaggcctg caggccagca 900 gctggcggag ccgtgcatac tagggggctg gacttcgctt gcgacatcta catctgggcc 960 ccactggcag ggacatgcgg agtcctgctg ctgtccctgg tcatcacact gtactgcaag cggggggcgca aaaaactgct gtatatcttt aagcagcctt tcatgagacc agtgcagaca acccaggagg aagatgggtg ctcatgccgg tttcccgagg aggaggagg cggctgcgag 1140 ctgagggtga agttttcccg ctcagcagat gctcctgcct accagcaggg ccagaaccag ctgtataatg agctgaacct gggcagacgc gaagagtatg atgtgctgga caaaaggcgg ggaagacc ccgaaatggg aggaagcca aggcggaaaa accccccagga gggcctgtac aatgagctgc agaaggacaa aatggcagag gcttacagtg agattgggat gaaggagag 1380. agacggaggg gaaaagggca cgatggcctg taccaggggc tgagcacagc aaccaaagat acttatgacg cactgcacat gcaggcactg ccacccagat ga <210> 5 <211> 1476 <212> DNA <213> The snowstorm <220> <223> A CD7(3A1F) CAR cycle cycle <400> 5 atggctctgc ccgtcaccgc tctgctgctg cctctggctc tgctgctgca cgctgctcga 60 ccacaggtcc agctgcagga gtcaggagct gagctggtga agccaggggc aagcgtcaaa 120 ctgtcctgca aggcctctgg atatacattc actagctact ggatgcactg ggtgaaacag 180 agacccggac agggcctgga gtggatcgga aagattaacc ctagcaatgg caggaccaac 240 tacaacgaaa agtttaaatc caaggcaacc ctgacagtgg acaagagctc ctctacagcc 300 tacatgcagc tgagttcact gacttcagag gatagcgcag tgtactattg cgccagaggc 360 ggggtctact atgacctgta ctattacgcc ctggattatt gggggcaggg aaccacagtg 420 actgtcagct ccggaggagg aggaagcgga ggaggaggat ccggaggagg aggatctgac 480 atcgagctga cccagagtcc tgctacactg agcgtgactc caggcgattc tgtcagtctg 540 tcatgtcggg caagccagtc catctctaac aatctgcact ggtaccagca gaaatcccat 600 gaatctccac gactgctgat taagagtgcc tcacagagca tctccggcat tccctcccgg 660 ttctctggca gtgggtcagg aactgacttt accctgagta ttaactcagt ggagacagaa 720 gatttcggca tgtatttttg ccagcagagc aattcctggc cctacacttt cggaggcggg 780 accaaactgg agatcaagcg gactaccaca ccagctccaa gaccacctac ccctgcacca 840 acaattgcta gtcagccact gtcactgaga ccagaagcat gtaggcctgc agctggagga 900 gctgtgcaca ccagaggcct ggactttgcc tgcgatatct acatttgggc tcctctggca 960 ggaacctgtg gcgtgctgct gctgtctctg gtcatcacac tgtattgcaa acgagggcgg 1020 aagaaactgc tgtacatttt caagcagccc tttatgaggc ctgtgcagac tacccaggag 1080 gaagacggct gcagctgtcg cttccctgag gaagaggaag ggggatgtga gctgcgcgtc 1140 aaattttctc gaagtgcaga tgccccagct tatcagcagg gccagaacca gctgtacaac 1200 gagctgaatc tggggcggag agaggaatat gacgtgctgg ataagaggcg aggaagggac 1260 ccagaaatgg gagggaaacc acgacggaag aacccccagg agggcctgta taatgaactg 1320 cagaaagaca agatggcaga ggcctactct gaaatcggga tgaaaggaga gagaaggcgc 1380 ggcaaggggc atgatggcct gtaccagggg ctgagcacag caaccaaaga cacctatgac 1440 gcactgcaca tgcaggcact gccaccacgg taatga 1476 <210> 6 <211> 255 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides of CMV promoter <400> 6 aagcttggga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 60 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 120 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 180 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240 ggcattatgc ccagt 255 <210> 7 <211> 1179 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide of EF1a promoter <400> 7 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gagttaggc cagcttggca cttgatgtaa 1080 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140 gtggttcaaa gttttttct tccatttcag gtgtcgtga <210> 8 <211> 373 <212> DNA <213> The snowstorm <220> <223> EFS is a snowflake storage device <400> 8 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt fathergc gtgacgttc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg accggcgggtc tagaggatcc 240 ggtactcgag gaactgaaaa accagaaagt taactggtaa gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa ctgctcctca gtggatgttg 360 cctttacttc tag 373 <210> 9 <211> 384 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides of the MSCV promoter <400> 9 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag 60 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac 120 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat 240 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca 300 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca 360 acccctcact cggcgcgcca gtcc 384 <210> 10 <211> 509 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide of PGK promoter <400> 10 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 60 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 120 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 180 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 240 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 300 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 360 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 4​​​​​​​​​​​​​​​​​​​​​​​cctgccgcat acaaggatat tcagatgact cagaccacaa gctccctgag cgcctccctg 120 ggagaccgag tgacaatctc ttgcagtgca tcacaggggaa ttagcaacta cctgaattgg 180 tatcagcaga agccagatgg cactgtgaaa ctgctgatct actatacctc tagtctgcac 240 agtggggtcc cctcacgatt cagcggatcc ggctctggga cagactacag cctgactatc 300 tccaacctgg agcccgaaga tattgccacc tactattgcc agcagtactc caagctgcct 360 tataccttg gcgggggaac aaagctggag attaaaaggg gaggaggagg aagcggagga 420 ggaggatccg gaggcggggg atctggagga ggaggaagtg aggtgcagct ggtcgaatct 480 ggaggaggac tggtgaagcc aggaggatct ctgaaactga gttgtgccgc ttcaggcctg 540 accttctcaa gctacgccat gagctgggtg cgacagacac ctgagaagcg gctggaatgg 600 gtcgctagca tctcctctgg cgggttcaca tactatccag actccgtgaa aggcagattt 660 actatctctc gggataacgc aagaaatatt ctgtacctgc agatgagttc actgaggagc 720 gaggacaccg caatgtacta ttgtgccagg gacgaagtgc gcggctatct ggatgtctgg 780 ggagctggca ctaccgtcac cgtctccagc gagcagaaac tgattagcga agaggacctg 840 aaagatgaac tgtaacggga tcaattccgc ccccccccta acgttactgg ccgaagccgc 900 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt 960 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt 1020 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 1080 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 1140 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg 1200 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 1260 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct 1320 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 1380 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgataata ccatggctct 1440 gcctgtgact gccctgctgc tgcccctggc cctgctgctg cacgctgcta gacccgccgc 1500 atacaaggac attcagatga ctcagactac atccagcctg tcagcttccc tgggcgatcg 1560 agtgactatc agctgttcag caagccaggg gatcagcaat tacctgaact ggtaccagca gaagcccgac ggaactgtca agctgctgat ctactatact tccagtctgc atagcggggt 1680. ccctagccga ttctctggga gtggaagcgg caccgactat agcctgacca tttccaacct ggaccagag gacattgcca cctattactg tcagcagtat tcaaagctgc cctatacctt tggcggaggg actaagctgg agatcaagcg aggagggga ggatcagggg gaggggggag 1860. cggaggcggg gggagtggcg gcggagggtc agaagtccag ctggtcgaga gtgggggcgg 1920. actggtgaaa cctgggggct ctctgaaact gagctgtgca gccagtggcc tgacattctc ctcatatgct atgtcttggg tgcggcagac cccagaaaag agactggagt gggtggcttc aatctcttcc gggggcttca cttactatcc tgacagcgtg aaaggcagat tcactatctc cagggataac gcacggaata tcctgtatct gcagatgagc tcactgagat ccgaggacac cgcaatgtac tactgcgcaa gagacgaagt gcgggggtat ctggacgtgt ggggagcagg cactaccgtc accgtgcta gcaccactac acctgcacca aggcctccca cacccgctcc 2280 cactatcgct tcccagccac tgtccctgag gcccgaggcc tgcaggccag cagctggcgg 2340 agccgtgcat actagggggc tggacttcgc ttgcgacatc tacatctggg ccccactggc 2400 agggacatgc ggagtcctgc tgctgtccct ggtcatcaca ctgtactgca agcgggggcg 2460 caaaaaactg ctgtatatct ttaagcagcc tttcatgaga ccagtgcaga caacccagga 2520 ggaagatggg tgctcatgcc ggtttcccga ggaggaggaa ggcggctgcg agctgagggt 2580 gaagttttcc cgctcagcag atgctcctgc ctaccagcag ggccagaacc agctgtataa 2640 tgagctgaac ctgggcagac gcgaagagta tgatgtgctg gacaaaaggc ggggaagaga 2700 ccccgaaatg ggagggaagc caaggcggaa aaacccccag gagggcctgt acaatgagct 2760 gcagaaggac aaaatggcag aggcttacag tgagattggg atgaagggag agagacggag 2820 gggaaaaggg cacgatggcc tgtaccaggg gctgagcaca gcaaccaaag atacttatga 2880 cgcactgcac atgcaggcac tgccacccag atgacagcca ggggatttca ccactcaaag 2940 gccagacctg cagacgccca gattatgaga caca 2974 <210> 12 <211> 2974 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide against human CD7 (TH69) CAR-IRES-anti-human CD7 (TH69) PEBL <400> 12 atggctctgc ctgtgactgc cctgctgctg cccctggccc tgctgctgca cgctgctaga 60 cccgccgcat acaaggacat tcagatgact cagactacat ccagcctgtc agcttccctg 120 ggcgatcgag tgactatcag ctgttcagca agccagggga tcagcaatta cctgaactgg 180 taccagcaga agcccgacgg aactgtcaag ctgctgatct actatacttc cagtctgcat 240 agcggggtcc ctagccgatt ctctgggagt ggaagcggca ccgactatag cctgaccatt 3' tccaacctgg aaccagagga cattgccacc tattactgtc agcagtattc aaagctgccc 360 tatacctttg gcggagggac taagctggag atcaagcgag gagggggagg atcaggggga 420 ggggggagcg gaggcggggg gagtggcggc ggagggtcag aagtccagct ggtcgagagt 480 gggggcggac tggtgaaacc tgggggctct ctgaaactga gctgtgcagc cagtggcctg 540 Note: There seems to be an error in the original sequence where the 3' end marker "3'" in the translation of line 26 is not in the original. It should be corrected in the original if possible. Also, the last line break in the original seems to be a mistake as there is no content after it. This translation assumes the correct interpretation of the original text despite these potential issues.acattctcct catatgctat gtcttgggtg cggcagaccc cagaaaagag actggagtgg 600 gtggcttcaa tctcttccgg gggcttcact tactatcctg acagcgtgaa aggcagattc 660 actatctcca gggataacgc acggaatac ctgtatctgc agatgagctc actgagatcc 720 gaggacaccg caatgtacta ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg 780 ggagcaggca ctaccgtcac cgtgtctagc accactacac ctgcaccaag gcctcccaca 840 cccgctccca ctatcgcttc ccagccactg tccctgaggc ccgaggcctg caggccagca 900 gctggcggag ccgtgcatac taggggctg gactcgctt gcgacatcta catctgggcc 960 ccactggcag ggacatgcgg agtcctgctg ctgtccctgg tcatcacact gtactgcaag 1020 cgggggcgca aaaaactgct gtatatcttt aagcagcctt tcatgagacc agtgcagaca 1080 acccaggagg aagatgggtg ctcatgccgg tttcccgagg aggaggaagg cggctgcgag 1140 ctgagggtga agttttcccg ctcagcagat gctcctgcct accagcaggg ccagaaccag 1200 ctgtataatg agctgaacct gggcagacgc gaagagtatg atgtgctgga caaaaggcgg 1260 ggaagagacc ccgaaatggg agggaagcca aggcggaaaa accccacagga gggcctgtac 1320 1380 agacggaggg gaaagggca cgatggcctg taccaggggc tgagcacagc aaccaaagat 1440 acttatgacg cactgcacat gcaggcactg ccacccagat gacagccagg ggatttcacc 1500 actcaaaggc cagacctgca gacgcccaga ttatgagaca cacgggatca attccgcccc 1560 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg 1620 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc 1680 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg 1740 1800 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca 1860 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata 1920 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc 1980 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt 2040 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt 2100 tgaaaaacac gataatacca tggctctgcc tgtgaccgca ctgctgctgc ccctggctct 2160 gctgctgcac gccgcaagac ctgccgcata caaggatatt cagatgactc agaccacaag 2220 ctccctgagc gcctccctgg gagaccgagt gacaatctct tgcagtgcat cacagggaat 2280 tagcaactac ctgaattggt atcagcagaa gccagatggc actgtgaaac tgctgatcta 2340 ctatacctct agtctgcaca gtggggtccc ctcacgattc agcggatccg gctctgggac 2400 agactacagc ctgactatct ccaacctgga gcccgaagat attgccacct actattgcca 2460 gcagtactcc aagctgcctt atacctttgg cgggggaaca aagctggaga ttaaaagggg 2520 aggaggagga agcggaggag gaggatccgg aggcggggga tctggaggag gaggaagtga 2580 ggtgcagctg gtcgaatctg gaggaggact ggtgaagcca ggaggatctc tgaaactgag 2640 ttgtgccgct tcaggcctga ccttctcaag ctacgccatg agctgggtgc gacagacacc 2700 tgagaagcgg ctggaatggg tcgctagcat ctcctctggc gggttcacat actatccaga 2760 ctccgtgaaa ggcagattta ctatctctcg ggataacgca agaaatattc tgtacctgca 2820 gatgagttca ctgaggagcg aggacaccgc aatgtactat tgtgccaggg acgaagtgcg 2880 cggctatctg gatgtctggg gagctggcac taccgtcacc gtctccagcg agcagaaact 2940 gattagcgaa gaggacctga aagatgaact gtaa 2974 <210> 13 <211> 2400 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide against human CD7 (TH69) CAR-p2A-anti-human CD7 (TH69) PEBL <400> 13 atggctctgc ctgtgactgc cctgctgctg cccctggccc tgctgctgca cgctgctaga 60 cccgccgcat acaaggacat tcagatgact cagactacat ccagcctgtc agcttccctg 120 ggcgatcgag tgactatcag ctgttcagca agccagggga tcagcaatta cctgaactgg 180 taccagcaga agcccgacgg aactgtcaag ctgctgatct actatacttc cagtctgcat 240 agcggggtcc ctagccgatt ctctgggagt ggaagcggca ccgactatag cctgaccatt 300 tccaacctgg aaccagagga cattgccacc tattactgtc agcagtattc aaagctgcccc 360 tataccttg gcggagggac taagctggag atcaagcgag gagggggagg atcaggggga 420 ggggggagcg gaggcggggg gagtggcggc ggagggtcag aagtccagct ggtcgagagt 480 gggggcggac tggtgaaacc tgggggctct ctgaaactga gctgtgcagc cagtggcctg 540 acattctcct catatgctat gtcttgggtg cggcagaccc cagaaaagag actggagtgg 600 gtggcttcaa tctcttccgg gggcttcact tactatcctg acagcgtgaa aggcagattc 660 actatctcca gggataacgc acggaatac ctgtatctgc agatgagctc actgagatcc 720 gaggacaccg caatgtacta ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg 780 ggagcaggca ctaccgtcac cgtgtctagc accactacac ctgcaccaag gcctcccaca 840 cccgctccca ctatcgcttc ccagccactg tccctgaggc ccgaggcctg caggccagca 900 gctggcggag ccgtgcatac taggggctg gactcgctt gcgacatcta catctgggcc 960 ccactggcag ggacatgcgg agtcctgctg ctgtccctgg tcatcacact gtactgcaag 1020 cggggggcgca aaaaactgct gtatatcttt aagcagcctt tcatgagacc agtgcagaca acccaggagg aagatgggtg ctcatgccgg tttcccgagg aggaggagg cggctgcgag 1140 ctgagggtga agttttcccg ctcagcagat gctcctgcct accagcaggg ccagaaccag ctgtataatg agctgaacct gggcagacgc gaagagtatg atgtgctgga caaaaggcgg ggaagacc ccgaaatggg aggaagcca aggcggaaaa accccccagga gggcctgtac aatgagctgc agaaggacaa aatggcagag gcttacagtg agattgggat gaaggagag 1380. agacggaggg gaaaagggca cgatggcctg taccaggggc tgagcacagc aaccaaagat acttatgacg cactgcacat gcaggcactg ccacccagag gcagcggcgc cacaaacttc tctctgctaa agcaagcagg tgatgttgaa gaaaaccccg ggcctatggc tctgcctgtg accgcactgc tgctgcccct ggctctgctg ctgcacgccg caagacctgc cgcatacaag gatattcaga tgactcagac cacaagctcc ctgagcgcct ccctggggaga ccgagtgaca atctcttgca gtgcatcaca gggaattagc aactacctga atctcttgca gcagaagcca gatggcactg tgaaactgct gatctactat acctctagtc tgcacagtgg ggtcccctca 1800 cgattcagcg gatccggctc tgggacagac tacagcctga ctatctccaa cctggagccc 1860 gaagatattg ccacctacta ttgccagcag tactccaagc tgccttatac ctttggcggg 1920 ggaacaaagc tggagattaa aaggggagga ggaggaagcg gaggaggagg atccggaggc 1980 gggggatctg gaggaggagg aagtgaggtg cagctggtcg aatctggagg aggactggtg 2040 aagccaggag gatctctgaa actgagttgt gccgcttcag gcctgacctt ctcaagctac 2100 gccatgagct gggtgcgaca gacacctgag aagcggctgg aatgggtcgc tagcatctcc 2160 tctggcgggt tcacatacta tccagactcc gtgaaaggca gatttactat ctctcgggat 2220 aacgcaagaa atattctgta cctgcagatg agttcactga ggagcgagga caccgcaatg 2280 tactattgtg ccagggacga agtgcgcggc tatctggatg tctggggagc tggcactacc 2340 gtcaccgtct ccagcgagca gaaactgatt agcgaagagg acctgaaaga tgaactgtaa 2400 <210> 14 <211> 2803 <212> DNA <213> Synthetic Sequence <220> <223> Synthetic polynucleotide of MSCV promoter - anti - human CD7 (TH69) CAR - p2A - anti - human CD7 (TH69) PEBL <400> 14 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag 60 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac 120 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat 240 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca 300 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca 360 acccctcact cggcgcgcca gtccgaattc ggcttccacc atggctctgc ctgtgactgc 420 cctgctgctg cccctggccc tgctgctgca cgctgctaga cccgccgcat acaaggacat 480 tcagatgact cagactacat ccagcctgtc agcttccctg ggcgatcgag tgactatcag 540 ctgttcagca agccagggga tcagcaatta cctgaactgg taccagcaga agcccgacgg 600 aactgtcaag ctgctgatct actatacttc cagtctgcat agcggggtc ctagccgatt 660 ctctgggagt ggaagcggca ccgactatag cctgaccatt tccaacctgg aaccagagga 720 cattgccacc tattactgtc agcagtattc aaagctgccc tatacctttg gcggagggac 780 tagctggag atcaagcgag gagggggagg atcaggggga ggggggagcg gaggcggggg 840 gagtggcggc ggagggtcag aagtccagct ggtcgagagt gggggcggac tggtgaaacc 900 tgggggctct ctgaaactga gctgtgcagc cagtggcctg acattctcct catatgctat 960 gtcttgggtg cggcagaccc cagaaaagag actggagtgg gtggcttcaa tctcttccgg 1020 gggcttcact tactatcctg acagcgtgaa aggcagattc actatctcca gggataacgc 1080 acggaatatc ctgtatctgc agatgagctc actgagatcc gaggacaccg caatgtacta 1140 ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg ggagcaggca ctaccgtcac 1200 cgtgtctagc accactacac ctgcaccaag gcctcccaca cccgctccca ctatcgcttc 1260 ccagccactg tccctgaggc ccgaggcctg caggccagca gctggcggag ccgtgcatac 1320 tagggggctg gacttcgctt gcgacatcta catctgggcc ccactggcag ggacatgcgg 1380 agtcctgctg ctgtccctgg tcatcacact gtactgcaag cgggggcgca aaaaactgct 1440 gtatatcttt aagcagcctt tcatgagacc agtgcagaca acccaggagg aagatgggtg 1500 ctcatgccgg tttcccgagg aggaggaagg cggctgcgag ctgagggtga agttttcccg 1560 ctcagcagat gctcctgcct accagcaggg ccagaaccag ctgtataatg agctgaacct 1620 gggcagacgc gaagagtatg atgtgctgga caaaaggcgg ggaagagacc ccgaaatggg 1680 agggaagcca aggcggaaaa acccccagga gggcctgtac aatgagctgc agaaggacaa 1740 aatggcagag gcttacagtg agattgggat gaagggagag agacggaggg gaaaagggca 1800 cgatggcctg taccaggggc tgagcacagc aaccaaagat acttatgacg cactgcacat 1860 gcaggcactg ccacccagat gaggcagcgg cgccacaaac ttctctctgc taaagcaagc 1920 aggtgatgtt gaagaaaacc ccgggcctat ggctctgcct gtgaccgcac tgctgctgcc 1980 cctggctctg ctgctgcacg ccgcaagacc tgccgcatac aaggatattc agatgactca 2040 gaccacaagc tccctgagcg cctccctggg agaccgagtg acatctct gcagtgcatc 2100 acagggaatt agcaactacc tgaattgta tcagcagaag ccagatggca ctgtgaaact 2160 gctgatctac tatacctacta gtctgcacag tggggtcccc tcacgatca gcggatccgg 2220 ctctgggaca gactacagcc tgactctc caactggag cccgaagata ttgccaccta 2280 ctattgccag cagtactcca agctgcctta taccttttggc gggggaacaa agctggagat 2340 2400 aggaagtgag gtgcagctgg tcgaatctgg aggaggactg gtgaagccag gaggatctct 2460 gaaactgagt tgtgccgctt caggcctgac cttctcaagc tacgccatga gctgggtgcg 2520 acagacacct gagaagcggc tggaatgggt cgctagcatc tcctctggcg ggttcacata 2580 ctatccagac tccgtgaaag gcagatttac tatctctcgg gataacgcaa gaatattct 2640 gtacctgcag atgagttcac tgaggagcga ggacaccgca atgtactatt gtgccaggga 2700 cgaagtgcgc ggctatctgg atgtctgggg agctggcact accgtcaccg tctccagcga 2760 gcagaaactg attagcgaag aggacctgaa agatgaactg taa 2803 <210> 15 <211> 3598 <212> DNA <213> Artificial Sequence <220> <223> EF1a promoter - synthetic polynucleotide against human CD7 (TH69) CAR - p2A - anti - human CD7 (TH69) PEBL <400> 15 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 第360位 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540 注:原文中“第360位”是根据上下文推测添加的注释,因为原英文翻译中“360”前面没有合适的中文对应词,可能是原文表述有误。请根据实际情况进行调整。 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag aattcggctt ccaccatggc 1200 tctgcctgtg actgccctgc tgctgcccct ggccctgctg ctgcacgctg ctagacccgc 1260 cgcatacaag gatatcaga tgactcagac tacatccagc ctgtcagctt ccctggggcga 1320 tcgagtgact atcagctgtt cagcaagcca ggggatcagc aattacctga actggtacca 1380 gcagaagcccc gacggaacctg tcaagctgct gattactat acttccagtc tgcatagcgg 1440 ggtccctagc cgattctctg ggagtggaag cggcaccgac tatagctga ccattccaa 1500 cctggaacca gaggacattg ccacctatta ctgtcagcag tattcaagc tgcctatac 1560 ctttggcgga gggactaagc tggagatca gcgaggagggg ggaggatcag gggagggggg 1620 gagcggaggc gcggcggagg gtcagaagtc cagctggtcg agagtgggggg 1680 cggactgtg aaacctgggg gctctgaa actgagctgt gcagccagtg gcctgacatt 1740 ctcctcatat gctatgtctt gggtgcggca gaccccagaa agagactgg agtgggtggc 1800 1860 ctccagggat acgcacgga atatcctgta tctgcagatg acctcactga gatccgagga 1920 caccgcaatg tactactgcg cagagacga agtgcgggg tattctggacg tgtgggagc 1980 agcactacc gtcaccgtgt ctagcaccac tacacctgca ccaggccctc ccacaccgc 2040 tcccactatc gcttcccagc cactgtccct gaggcccgag gcctgcaggc cagcagctgg 2100 cggagccgtg catactaggg ggctggactt cgcttgcgac atctacatct gggccccact 2160 ggcagggaca tgcggagtcc tgctgctgtc cctgtcatc acacgtact gcaagcgggg 2220 gcgcaaaaaa ctgctgtata tctttaagca gcctttcatg agaccagtgc agacaaccca 2280 ggaggagat gggtgctcat gccggtttcc cgaggaggag gaggcggct gcgagctgag 2340 ggtgaagttt tccccctcag cagatgctcc tgcctaccag cagggccaga accagctgta 2400 taatgagctg aacctgggca gacgcgaga gtatgatgtg ctggacaaaa ggcggggaag 2460 agaccccgaa atgggaggga agccaaggcg gaaaacccc caggaggggcc tgtacaatga 2520 gctgcagaag gandaaatgg cagaggctta cagtgagatt gggatgaagg gagagagacg 2580 gaggggaaaa gggcacgatg gcctgtacca ggggctgagc acagcaacca aagatactta 2640 tgacgcactg cacatgcagg cactgccacc cagatgaggc agcggcgcca caaacttctc 2700 tctgctaaag caagcaggtg atgttgaaga aaaccccggg cctatggctc tgcctgtgac 2760 cgcactgctg ctgcccctg ctctgctgct gcacgccgca agacctgccg catacaagga 2820 tattcagatg actcagacca caagctccct gagcgcctcc ctgggagacc gagtgacaat 2880 ctcttgcagt gcatcacagg gaattagcaa ctacctgaat tggtatcagc agaagccaga 2940 tggcactgtg aaactgctga tctactatac ctctagtctg cacagtgggg tccctcacg 3000 attcagcgga tccggctctg ggacagacta cagcctgact atctccaacc tggagcccga 3060 agatattgcc acctactatt gccagcagta ctccaagctg ccttatacct ttggcggggg 3120 aacaaagctg gagattaaaa ggggaggagg aggaagcgga ggaggaggat ccggaggcgg 3180 gggatctgga ggaggagaa gtgaggtgca gctggtcgaa tctggaggag gactggtgaa 3240 gccaggagga tctctgaaac tgagttgtgc cgcttcaggc ctgaccttct caagctacgc 3300 catgagctgg gtgcgacaga cacctgagaa gcggctggaa tgggcgcta gcatctcctc 3360 tggcgggttc acatactatc cagactccgt gaaaggcaga tttactatct ctcggggataa 3420 cgcaagaaat attctgtacc tgcagatgag ttcactgagg agcgaggaca ccgcaatgta ctattgtgcc agggacgaag tgcgcggcta tctggatgtc tggggagctg gcactaccgt 3540 caccgtctcc agcgagcaga aactgattag cgaagaggac ctgaagatg aactgtaa <210> 16 <211> 2792 <212> DNA <213> The snowstorm <220> <223> EF1a is also known as the mammalian CD7(TH69) gene CAR-p2A-expressing CD7(TH69)PEBL <400> 16 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gccttttttcc cgagggtggg ggagaaccgt fathergc gtgacgttc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg accggcgggtc tagaggatcc 240 ggtactcgag gaactgaaaa accagaaagt taactggtaa gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa ctgctcctca gtggatgttg 360 cctttacttc taggaattcg gcttccacca tggctctgcc tgtgactgcc ctgctgctgc 420 ccctggccct gctgctgcac gctgctagac ccgccgcata caaggacatt cagatgactc 480 agactacatc cagcctgtca gcttccctgg gcgatcgagt gactatcagc tgttcagcaa 540 gccaggggat cagcaattac ctgaactggt accagcagaa gcccgacgga actgtcaagc 600 tgctgatcta ctatacttcc agtctgcata gcggggtccc tagccgattc tctgggagtg 660 gaagcggcac cgactatagc ctgaccattt ccaacctgga accagaggac attgccacct 720 attactgtca gcagtattca aagctgccct atacctttgg cggagggact aagctggagaga 780 tcaagcgagg aggggagga tcaaggggag gggggagcgg aggcgggggg agtggcggcg 840 gagggtcaga agtccagctg gtcgagagtg ggggcggact ggtgaaacct gggggctctc 900 tgaaactgag ctgtgcagcc agtggcctga cattctcctc atatgctatg tcttgggtgc 960 ggcagacccc agaaaagaga ctggagtggg tggcttcaat ctcttccggg ggcttcactt 1020 1080 tgtatctgca gatgagctca ctgagatccg aggacaccgc aatgtactac tgcgcaagag 1140 acgaagtgcg ggggtatctg gacgtgtggg gagcaggcac taccgtcacc gtgtctagca 1200 ccactacacc tgcaccaagg cctcccacac ccgctcccac tatcgcttcc cagccactgt 1260 ccctgaggcc cgaggcctgc aggccagcag ctggcggagc cgtgcatact agggggctgg 1320 acttcgcttg cgacatctac atctgggccc cactggcagg gacatgcgga gtcctgctgc 1380 tgtccctggt catcacactg tactgcaagc gggggcgcaa aaaactgctg tatatcttta 1440 agcagccttt catgagacca gtgcagacaa cccaggagga agatgggtgc tcatgccggt 1500 ttcccgagga ggaggaaggc ggctgcgagc tgagggtgaa gttttcccgc tcagcagatg 1560 ctcctgccta ccagcagggc cagaaccagc tgtataatga gctgaacctg ggcagacgcg 1620 aagagtatga tgtgctggac aaaaggcggg gaagagaccc cgaaatggga gggaagccaa 1680 ggcggaaaaa cccccaggag ggcctgtaca atgagctgca gaaggacaaa atggcagagg 1740 cttacagtga gattgggatg aagggagaga gacggagggg aaaagggcac gatggcctgt 1800 accaggggct gagcacagca accaaagata cttatgacgc actgcacatg caggcactgc 1860 cacccagatg aggcagcggc gccacaaact tctctctgct aaagcaagca ggtgatgttg 1920 aagaaaaccc cgggcctatg gctctgcctg tgaccgcact gctgctgccc ctggctctgc 1980 tgctgcacgc cgcaagacct gccgcataca aggatattca gatgactcag accacaagct 2040 ccctgagcgc ctccctggga gaccgagtga caatctcttg cagtgcatca cagggaatta 2100 gcaactacct gaattggtat cagcagaagc cagatggcac tgtgaaactg ctgatctact 2160 atacctctag tctgcacagt ggggtcccct cacgattcag cggatccggc tctgggacag 2220 actacagcct gactatctcc aacctggagc ccgaagatat tgccacctac tattgccagc 2280 agtactccaa gctgccttat acctttggcg ggggaacaaa gctggagatt aaaaggggag 2340 gaggaggaag cggaggagga ggatccggag gcgggggatc tggaggagga ggaagtgagg 2400 tgcagctggt cgaatctgga ggaggactgg tgaagccagg aggatctctg aaactgagtt 2460 gtgccgcttc aggcctgacc ttctcaagct acgccatgag ctgggtgcga cagacacctg 2520 agaagcggct ggaatgggtc gctagcatct cctctggcgg gttcacatac tatccagact 2580 agaagcggct ggaatgggtc gctagcatct cctctggcgg gttcacatac tatccagact 2580 ccgtgaaagg cagatttact atctctcggg ataacgcaag aaatattctg tacctgcaga 2640 ccgtgaaagg cagatttact atctctcggg ataacgcaag aaatattctg tacctgcaga 2640 tgagttcact gaggagcgag gacaccgcaa tgtactattg tgccagggac gaagtgcgcg 2700 tgagttcact gaggagcgag gacaccgcaa tgtactattg tgccagggac gaagtgcgcg 2700 gctatctgga tgtctgggga gctggcacta ccgtcaccgt ctccagcgag cagaaactga 2760 gctatctgga tgtctgggga gctggcacta ccgtcaccgt ctccagcgag cagaaactga 2760 ttagcgaaga ggacctgaaa gatgaactgt aa 2792 ttagcgaaga ggacctgaaa gatgaactgt aa 2792 <210> 17<210> 17 <211> 3346<211> 3346 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220><220> <223> MSCV promoter - synthetic polynucleotide against human CD7 (TH69) <223> MSCV promoter - synthetic polynucleotide against human CD7 (TH69) CAR - PGK promoter - anti - human CD7 (TH69) PEBL CAR - PGK promoter - anti - human CD7 (TH69) PEBL <400> 17 <400> 17 ttaattaatt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt 60 ttaattaatt tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt 60 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag 120 ggcaagctag cttaagtaac gccattttgc aaggcatgga aaatacataa ctgagaatag 120 <000168 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg ccagtccgaa 420 ttcggcttcc accatggctc tgcctgtgac tgccctgctg ctgcccctgg ccctgctgct 480 gcacgctgct agacccgccg catacaagga cattcagatg actcagacta catccagcct 540 gtcagcttcc ctgggcgatc gagtgactat cagctgttca gcaagccagg ggatcagcaa 600 ttacctgaac tggtaccagc agaagcccga cggaactgtc aagctgctga tctactatac 660 ttccagtctg catagcgggg tccctagccg attctctggg agtggaagcg gcaccgacta 720 tagcctgacc atttccaacc tggaaccaga ggacattgcc acctattact gtcagcagta 780 ttcaaagctg ccctatacct ttggcggagg gactaagctg gagatcaagc gaggaggggg 840 aggatcaggg ggagggggga gcggaggcgg ggggagtggc ggcggagggt cagaagtcca 900 gctggtcgag agtgggggcg gactggtgaa acctgggggc tctctgaaac tgagctgtgc 960 agccagtggc ctgacattct cctcatatgc tatgtcttgg gtgcggcaga ccccagaaaa 1020 gagactggag tgggtggctt caatctcttc cggggggcttc acttactatc ctgacagcgt 1080 gaaaggcaga ttcactatct ccagggataa cgcacggaat atcctgtatc tgcagatgag ctcactgaga tccgaggaca ccgcaatgta ctactgcgca agagacgaag tgcgggggta tctggacgtg tggggagcag gcactaccgt caccgtgtct agcaccacta cacctgcacc 1260 aaggcctccc acacccgctc ccactatcgc ttcccagcca ctgtccctga ggcccgaggc 1320 ctgcaggcca gcagctggcg gagccgtgca tactaggggg ctggacttcg cttgcgacat ctacatctgg gccccactgg cgggacatg cggagtcctg ctgctgtccc tggtcatcac 1440 actgtactgc aagcgggggc gcaaaaaact gctgtatatc tttaagcagc ctttcatgag accagtgcag acaacccagg aggaagatgg gtgctcatgc cggtttcccg aggagaga 1560 aggcggctgc gagctgaggg tgaagttttc ccgctcagca gatgctcctg cctaccagca 1620 gggccagaac cagctgtata atgagctgaa cctgggcaga cgcgaagagt atgatgtgct ggacaaaagg cggggagag accccgaat gggagggag ccaaggcgga aaaaccccca ggagggcctg tacaatgagc tgcagaagga caaaatggca gaggcttaca gtgagattgg 1800 gatgaaggga gagagacgga ggggaaaagg gcacgatggc ctgtaccagg ggctgagcac 1860 agcaaccaaa gatacttatg acgcactgca catgcaggca ctgccaccca gatgactcga 1920 gtgcagtcga cgcgggcccg ggatcccgcg actctagata attctaccgg gtaggggagg 1980 cgcttttccc aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct 2040 acacaagtgg cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc 2100 cgttctttgg tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc 2160 ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc 2220 tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc 2280 aatagcagct ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg 2340 gcgggctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg 2400 cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg 2460 gcctttcgac tggccaggct tccaccatgg ctctgcctgt gaccgcactg ctgctgcccc 2520 tggctctgct gctgcacgcc gcaagacctg ccgcatacaa ggatattcag atgactcaga 2580 ccacaagctc cctgagcgcc tccctgggag accgagtgac aatctcttgc agtgcatcac 2640 agggaattag caactacctg aattggtatc agcagaagcc agatggcact gtgaaactgc 2700 tgatctacta tacctctagt ctgcacagtg gggtcccctc acgattcagc ggatccggct 2760 ctgggacaga ctacagcctg actatctcca acctggagcc cgaagatatt gccacctact 2820 attgccagca gtactccaag ctgccttata cctttggcgg gggaacaaag ctggagatta 2880 aaaggggagg aggaggaagc ggaggaggag gatccggagg cgggggatct ggaggaggag 2940 gaagtgaggt gcagctggtc gaatctggag gaggactggt gaagccagga ggatctctga 3000 aactgagttg tgccgcttca ggcctgacct tctcaagcta cgccatgagc tgggtgcgac 3060 agacacctga gaagcggctg gaatgggtcg ctagcatctc ctctggcggg ttcacatact 3120 atccagactc cgtgaaaggc agatttacta tctctcggga taacgcaaga aatattctgt 3180 acctgcagat gagttcactg aggagcgagg acaccgcaat gtactattgt gccagggacg 3240 aagtgcgcgg ctatctggat gtctggggag ctggcactac cgtcaccgtc tccagcgagc 3300 agaaactgat tagcgaagag gacctgaaag atgaactgta acgcgt 3346 <210> 18 <211> 3938 <212> DNA <213> Artificial Sequence <220> <223> MSCV Promoter - Synthetic Polynucleotide of Anti - human CD7 CAR (TH69) - EF1a Promoter - Anti - human CD7 <400> 18 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag 60 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac 120 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat 240 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca 300 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca 360 acccctcact cggcgcgcca gtccgaattc ggcttccacc atggctctgc ctgtgactgc 420 cctgctgctg ccctggccc tgctgctgca cgctgctaga cccgccgcat acaaggacat 480 tcagatgact cagactacat ccagcctgtc agcttccctg ggcgatcgag tgactatcag 540 ctgttcagca agccagggga tcagcaatta cctgaactgg taccagcaga agcccgacgg 600 aactgtcaag ctgctgatct actatacttc cagtctgcat agcggggtc ctagccgatt 660 ctctgggagt ggaagcggca ccgactatag cctgaccatt tccaacctgg aaccagagga 720 cattgccacc tattactgtc agcagtattc aaagctgccc tatacctttg gcggagggac 780 tagctggag atcaagcgag gagggggagg atcaggggga ggggggagcg gaggcggggg 840 gagtggcggc ggagggtcag aagtccagct ggtcgagagt gggggcggac tggtgaaacc 900 tgggggctct ctgaaactga gctgtgcagc cagtggcctg acattctcct catatgctat 960 gtcttgggtg cggcagaccc cagaaaagag actggagtgg gtggcttcaa tctcttccgg 1020 gggcttcact tactatcctg acagcgtgaa aggcagattc actatctcca gggataacgc 1080 acggaatatc ctgtatctgc agatgagctc actgagatcc gaggacaccg caatgtacta 1140 ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg ggagcaggca ctaccgtcac cgtgtctagc accactacac ctgcaccag gcctcccaca cccgctccca ctatcgcttc ccagccactg tccctgaggc ccgaggcctg caggccagca gctggcggag ccgtgcatac 1320 tagggggctg gacttcgctt gcgacatcta catctgggcc ccactggcag ggacatgcgg agtcctgctg ctgtccctgg tcatcacact gtactgcaag cggggcgca aaaaactgct 1440 gtatatcttt aagcagcctt tcatgagacc agtgcagaca acccaggagg aagatgggtg ctcatgccgg tttcccgagg aggaggagg cggctgcgag ctgagggtga agttttcccg 1560 ctcagcagat gctcctgcct accagcaggg ccagaaccag ctgtataatg agctgaacct gggcagacgc gaagagtatg atgtgctgga caaaaggcgg ggagagacc ccgaatggg agggaagcca aggcggaaaa accccccagga gggcctgtac aatgagctgc agaaggacaa aatggcagag gcttacagtg agattgggat gaagggagag agacggaggg gaaaagggca cgatggcctg taccaggggc tgagcacagc aaccaaagat acttatgacg cactgcacat gcaggcactg ccacccagat gagtcgacgg ctccggtgcc cgtcagtggg cagagcgcac 1920 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag 1980 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 2040 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 2100 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac 2160 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga 2220 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc 2280 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg 2340 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg 2400 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct 2460 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg 2520 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc 2580 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg 2640 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg 2700 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc 2760 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga 2820 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt 2880 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga 2940 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg 3000 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt 3060 gtcgtgaccc gggggcttcc accatggctc tgcctgtgac cgcactgctg ctgcccctgg 3120 ctctgctgct gcacgccgca agacctgccg catacaagga tattcagatg actcagacca 3180 caagctccct gagcgcctcc ctgggagacc gagtgacaat ctcttgcagt gcatcacagg 3240 gaattagcaa ctacctgaat tggtatcagc agaagccaga tggcactgtg aaactgctga 3300 tctactatac ctctagtctg cacagtgggg tcccctcacg attcagcgga tccggctctg 3360 ggacagacta cagcctgact atctccaacc tggagcccga agatattgcc acctactatt 3420 gccagcagta ctccaagctg ccttatacct ttggcggggg aacaaagctg gagattaaaa 3480 ggggaggagg aggaagcgga ggaggaggat ccggaggcgg gggatctgga ggaggaggaa 3540 gtgaggtgca gctggtcgaa tctggaggag gactggtgaa gccaggagga tctctgaaac 3600 tgagttgtgc cgcttcaggc ctgaccttct caagctacgc catgagctgg gtgcgacaga 3660 cacctgagaa gcggctggaa tgggtcgcta gcatctcctc tggcgggttc acatactatc 3720 cagactccgt gaaaggcaga tttactatct ctcgggataa cgcaagaaat attctgtacc 3780 tgcagatgag ttcactgagg agcgaggaca ccgcaatgta ctattgtgcc agggacgaag 3840 tgcgcggcta tctggatgtc tggggagctg gcactaccgt caccgtctcc agcgagcaga 3900 aactgattag cgaagaggac ctgaaagatg aactgtaa 3938 <210> 19 <211> 3132 <212> DNA <213> Artificial Sequence <220> <223> MSCV translocates to CD7 CAR (TH69)-EFS and CD7 (TH69) PEBL <400> 19 ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc atttgcaag 60 gcatggaaaa tacataactg agaatagaga agttcagatc aaggttagga acagagagac 120 agcagaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc 180 aagaacagat ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat 240 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca 300 gttcgcttct cgcttctgtt cgcgcttc tgctccccga gctcaataaa agagcccaca 360 acccctcact cggcgcgcca gtccgaattc ggcttccacc atggctctgc ctgtgactgc 420 cctgctgctg ccctggccc tgctgctgca cgctgctaga cccgccgcat acaaggacat 480 tcagatgact cagactacat ccagcctgtc agcttccctg ggcgatcgag tgactatcag 540 ctgttcagca agccagggga tcagcaatta cctgaactgg taccagcaga agcccgacgg 600 aactgtcaag ctgctgatct actatacttc cagtctgcat agcggggtc ctagccgatt 660 ctctgggagt ggaagcggca ccgactatag cctgaccatt tccaacctgg aaccagagga 720 cattgccacc tattactgtc agcagtattc aaagctgccc tatacctttg gcggagggac 780 tagctggag atcaagcgag gagggggagg atcaggggga ggggggagcg gaggcggggg 840 gagtggcggc ggagggtcag aagtccagct ggtcgagagt gggggcggac tggtgaaacc 900 tgggggctct ctgaaactga gctgtgcagc cagtggcctg acattctcct catatgctat 960 gtcttgggtg cggcagaccc cagaaaagag actggagtgg gtggcttcaa tctcttccgg 1020 gggcttcact tactatcctg acagcgtgaa aggcagattc actatctcca gggataacgc 1080 acggaatatc ctgtatctgc agatgagctc actgagatcc gaggacaccg caatgtacta 1140 ctgcgcaaga gacgaagtgc gggggtatct ggacgtgtgg ggagcaggca ctaccgtcac 1200 cgtgtctagc accactacac ctgcaccaag gcctcccaca cccgctccca ctatcgcttc 1260 ccagccactg tccctgaggc ccgaggcctg caggccagca gctggcggag ccgtgcatac 1320 tagggggctg gacttcgctt gcgacatcta catctgggcc ccactggcag ggacatgcgg 1380 agtcctgctg ctgtccctgg tcatcacact gtactgcaag cggggcgca aaaaactgct 1440 gtatatcttt aagcagcctt tcatgagacc agtgcagaca acccaggagg aagatgggtg ctcatgccgg tttcccgagg aggaggagg cggctgcgag ctgagggtga agttttcccg 1560 ctcagcagat gctcctgcct accagcaggg ccagaaccag ctgtataatg agctgaacct gggcagacgc gaagagtatg atgtgctgga caaaaggcgg ggagagacc ccgaatggg agggaagcca aggcggaaaa accccccagga gggcctgtac aatgagctgc agaaggacaa aatggcagag gcttacagtg agattgggat gaagggagag agacggaggg gaaaagggca cgatggcctg taccaggggc tgagcacagc aaccaaagat acttatgacg cactgcacat gcaggcactg ccacccagat gagtcgacgg gcagagcgca catcgcccac agtccccgag 1920 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcg tagtcgccgt gaacgttctt tttcgcaacg ggttgccgc cagaacacag gtgtcgtgac cgcgggtcta gaggatccgg tactcgagga actgaaaaac cagaaagtta 2160 actggtaagt ttagtctttt tgtcttttat ttcaggtccc ggatccggtg gtggtgcaaa 2220 tcaaagaact gctcctcagt ggatgttgcc tttacttcta gcccggggc ttccaccatg 2280 gctctgcctg tgaccgcact gctgctgccc ctggctctgc tgctgcacgc cgcaagacct 2340 gccgcataca aggatattca gatgactcag accacaagct ccctgagcgc ctccctggga 2400 gaccgagtga caatctcttg cagtgcatca cagggaatta gcaactacct gaattggtat 2460 cagcagaagc cagatggcac tgtgaaactg ctgatctact atacctctag tctgcacagt 2520 ggggtcccct cacgattcag cggatccggc tctgggacag actacagcct gactatctcc 2580 aacctggagc ccgaagatat tgccacctac tattgccagc agtactccaa gctgccttat 2640 acctttggcg ggggaacaaa gctggagatt aaaaggggag gagggagaag cggagggaga 2700 ggatccggag gcgggggatc tggaggagga ggaagtgagg tgcagctggt cgaatctgga 2760 ggaggactgg tgaagccagg aggatctctg aaactgagtt gtgccgcttc aggcctgacc 2820 ttctcaagct acgccatgag ctgggtgcga cagacacctg agaagcggct ggaatgggtc 2880 gctagcatct cctctggcgg gttcacatac tatccagact ccgtgaaagg cagatttact 2940 atctctcggg ataacgcaag aaatattctg tacctgcaga tgagttcact gaggagcgag 3000 gacaccgcaa tgtactattg tgccagggac gaagtgcgcg gctatctgga tgtctgggga 3060 gctggcacta ccgtcaccgt ctccagcgag cagaaactga ttagcgaaga ggacctgaaa 3120 gatgaactgt aa 3132 <210> 20 <211> 4057 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide of PGK promoter - anti - human CD7 CAR (3A1F) - EF1a promoter - anti - human CD7 (TH69) PEBL <400> 20 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 60 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 120 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 180 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 240 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 300 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 360 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 420 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 480 ctcctcttcc tcatctccgg gcctttcgag aattcggctt ccaccatggc tctgcccgtc 540 accgctctgc tgctgcctct ggctctgctg ctgcacgctg ctcgaccaca ggtccagctg 600 caggagtcag gagctgagct ggtgaagcca ggggcaagcg tcaaactgtc ctgcaaggcc 660 tctggatata cattcactag ctactggatg cactgggtga aacagagacc cggacagggc 720 ctggagtgga tcggaaagat taaccctagc aatggcagga ccaactacaa cgaaaagttt 780 aaatccaagg caaccctgac agtggacaag agctcctcta cagcctacat gcagctgagt 840 tcactgactt cagaggatag cgcagtgtac tattgcgcca gaggcggggt ctactatgac 900 ctgtactatt acgccctgga ttattggggg cagggaacca cagtgactgt cagctccgga 960 ggaggaggaa gcggaggagg aggatccgga ggaggaggat ctgacatcga gctgacccag 1020 agtcctgcta cactgagcgt gactccaggc gattctgtca gtctgtcatg tcgggcaagc 1080 cagtccatct ctaacaatct gcactggtac cagcagaaat cccatgaatc tccacgactg 1140 ctgattaaga gtgcctcaca gagcatctcc ggcattccct cccggttctc tggcagtggg 1200 tcaggaactg actttaccct gagtattaac tcagtggaga cagaagattt cggcatgtat 1260 ttttgccagc agagcaattc ctggccctac actttcggag gcgggaccaa actggagatc 1320 aagcggacta ccacaccagc tccaagacca cctacccctg caccaacaat tgctagtcag 1380 ccactgtcac tgagaccaga agcatgtagg cctgcagctg gaggagctgt gcacaccaga 1440 ggcctggact ttgcctgcga tatctacatt tgggctcctc tggcaggaac ctgtggcgtg 1500 ctgctgctgt ctctggtcat cacactgtat tgcaaacgag ggcggaagaa actgctgtac 1560 attttcaagc agccctttat gaggcctgtg cagactaccc aggaggaaga cggctgcagc 1620 tgtcgcttcc ctgaggaaga ggaaggggga tgtgagctgc gcgtcaaatt ttctcgaagt 1680 gcagatgccc cagcttatca gcagggccag aaccagctgt acaacgagct gaatctgggg 1740 cggagagagg aatatgacgt gctggataag aggcgaggaa gggacccaga aatgggaggg 1800 aaaccacgac ggaagaaccc ccaggagggc ctgtataatg aactgcagaa agacaagatg 1860 gcagaggcct actctgaaat cgggatgaaa ggagagagaa ggcgcggcaa ggggcatgat 1920 ggcctgtacc aggggctgag cacagcaacc aaagacacct atgacgcact gcacatgcag 1980 gcactgccac cacggtaatg agtcgacggc tccggtgccc gtcagtgggc agagcgcaca 2040 tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga 2100 aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 2160 ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 2220 tttgccgcca gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg 2280 ggttatggcc cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat 2340 cccgagcttc gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct 2400 tcgcctcgtg cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg 2460 tggcaccttc gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga 2520 tgacctgctg cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg 2580 cacactggta tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc 2640 acatgttcgg cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct 2700 caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg 2760 gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc 2820 cctgctgcag ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca 2880 cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag 2940 taccgggcgc cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta 3000 ggttgggggg aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa 3060 gttaggccag cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga 3120 tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg 3180 tcgtgacccg ggggcttcca ccatggctct gcctgtgacc gcactgctgc tgcccctggc 3240 tctgctgctg cacgccgcaa gacctgccgc atacaaggat attcagatga ctcagaccac 3300 aagctccctg agcgcctccc tgggagaccg agtgacaatc tcttgcagtg catcacaggg 3360 aattagcaac tacctgaatt ggtatcagca gaagccagat ggcactgtga aactgctgat 3420 ctactatacc tctagtctgc acagtggggt cccctcacga ttcagcggat ccggctctgg 3480 gacagactac agcctgacta tctccaacct ggagcccgaa gatattgcca cctactattg 3540 ccagcagtac tccaagctgc cttatacctt tggcggggga acaaagctgg agattaaaag 3600 gggaggagga ggaagcggag gaggaggatc cggaggcggg ggatctggag gaggaggaag 3660 tgaggtgcag ctggtcgaat ctggaggagg actggtgaag ccaggaggat ctctgaaact 3720 gagttgtgcc gcttcaggcc tgaccttctc aagctacgcc atgagctggg tgcgacagac 3780 acctgagaag cggctggaat gggtcgctag catctcctct ggcgggttca catactatcc 3840 agactccgtg aaaggcagat ttactatctc tcgggataac gcaagaaata ttctgtacct 3900 gcagatgagt tcactgagga gcgaggacac cgcaatgtac tattgtgcca gggacgaagt 3960 gcgcggctat ctggatgtct ggggagctgg cactaccgtc accgtctcca gcgagcagaa 4020 actgattagc gaagaggacc tgaaagatga actgtaa 4057 <210> 21 <211> 4054 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide of PGK promoter - anti - human CD7 CAR (TH69) - EF1a promoter - anti - human CD7 (3A1F) PEBL <400> 21 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 60 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 120 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 180 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 240 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 300 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 360 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 420 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 480 ctcctcttcc tcatctccgg gcctttcgag aattcggctt ccaccatggc tctgcctgtg 540 actgccctgc tgctgcccct ggccctgctg ctgcacgctg ctagacccgc cgcatacaag 600 gacattcaga tgactcagac tacatccagc ctgtcagctt ccctgggcga tcgagtgact 660 atcagctgtt cagcaagcca ggggatcagc aattacctga actggtacca gcagaagccc 720 gacggaactg tcaagctgct gatctactat acttccagtc tgcatagcgg ggtccctagc 780 cgattctctg ggagtggaag cggcaccgac tatagcctga ccatttccaa cctggaacca 840 gaggacattg ccacctatta ctgtcagcag tattcaaagc tgccctatac ctttggcgga 900 gggactaagc tggagatcaa gcgaggaggg ggaggatcag ggggaggggg gagcggaggc 960 ggggggagtg gcggcggagg gtcagaagtc cagctggtcg agagtggggg cggactggtg 1020 aaacctgggg gctctctgaa actgagctgt gcagccagtg gcctgacatt ctcctcatat 1080 gctatgtctt gggtgcggca gaccccagaa aagagactgg agtgggtggc ttcaatctct 1140 tccgggggct tcacttacta tcctgacagc gtgaaaggca gattcactat ctccagggat 1200 aacgcacgga atatcctgta tctgcagatg agctcactga gatccgagga caccgcaatg 1260 tactactgcg caagagacga agtgcggggg tatctggacg tgtggggagc aggcactacc 1320 gtcaccgtgt ctagcaccac tacacctgca ccaaggcctc ccacacccgc tcccactatc 1380 gcttcccagc cactgtccct gaggcccgag gcctgcaggc cagcagctgg cggagccgtg 1440 catactaggg ggctggactt cgcttgcgac atctacatct gggccccact ggcagggaca 1500 tgcggagtcc tgctgctgtc cctggtcatc acactgtact gcaagcgggg gcgcaaaaaa 1560 ctgctgtata tctttaagca gcctttcatg agaccagtgc agacaaccca ggaggaagat 1620 gggtgctcat gccggtttcc cgaggaggag gaaggcggct gcgagctgag ggtgaagtttt 1680 tcccgctcag cagatgctcc tgcctaccag cagggccaga accagctgta taatgagctg 1740 aacctgggca gacgcgaaga gtatgatgtg ctggacaaaa ggcggggaag agaccccgaa 1800 atgggaggga agccaaggcg gaaaaacccc caggagggcc tgtacaatga gctgcagaag 1860 gacaaaatgg cagaggctta cagtgagatt gggatgaagg gagagagacg gaggggaaaa 1920 gggcacgatg gcctgtacca ggggctgagc acagcaacca aagatactta tgacgcactg 1980 cacatgcagg cactgccacc cagatgagtc gacggctccg gtgcccgtca gtgggcagag 2040 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc 2100 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt 2160 cccgaggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc 2220 aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc 2280 tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt 2340 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga 2400 gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga 2460 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat 2520 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 2580 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc 2640 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg 2700 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg 2760 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt 2820 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 2880 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc 2940 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg 3000 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag 3060 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag 3120 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 3180 caggtgtcgt gacccggggg cttccaccat ggctctgccc gtcaccgctc tgctgctgcc 3240 tctggctctg ctgctgcacg ccgcaagacc ccaggtccag ctgcaggagt caggggcaga 3300 gctggtgaaa cccggagcca gtgtcaaact gtcctgtaag gccagcggct atactttcac 3360 cagctactgg atgcactggg tgaaacagag gccaggacag ggcctggagt ggatcggcaa 3420 gattaacccc agcaatgggc gcaccaacta caacgaaaag tttaaatcca aggctacact 3480 gactgtggac aagagctcct ctaccgcata catgcagctg agttcactga catctgaaga 3540 tagtgccgtg tactattgcg ccagaggcgg ggtctactat gacctgtact attacgcact 3600 ggattattgg gggcagggaa ccacagtgac tgtcagctcc ggaggaggag gatccggcgg 3660 aggaggctct gggggaggcg ggagtgacat cgagctgaca cagtctccag ccactctgag 3720 cgtgacccct ggcgattctg tcagtctgtc atgtagagct agccagtcca tctctaacaa 3780 tctgcactgg taccagcaga aatcacatga aagccctcgg ctgctgatta agagtgcttc 3840 acagagcatc tccgggattc caagcagatt ctctggcagt gggtcaggaa ccgactttac 3900 actgtccatt aactctgtgg agaccgaaga ttcggcatg tatttttgcc agcagagcaa 3960 ttcctggcct tacacattcg gaggcgggac taaactggag attaagaggg agcagaaact 4020 gattagcgaa gaggacctga aggacgaact gtga 4054 <210> 22 <211> 4063 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide of PGK promoter - anti - human CD7 CAR (TH69) - EF1a promoter - anti - human CD7 (TH69) PEBL <400> 22 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 60 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 120 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 180 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 240 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 300 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 360 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 420 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 480 ctcctcttcc tcatctccgg gcctttcgag aattcggctt ccaccatggc tctgcctgtg 540 actgccctgc tgctgcccct ggccctgctg ctgcacgctg ctagacccgc cgcatacaag 600 gacattcaga tgactcagac tacatccagc ctgtcagctt ccctgggcga tcgagtgact 660 atcagctgtt cagcaagcca ggggatcagc aattacctga actggtacca gcagaagccc 720 gacggaactg tcaagctgct gatctactat acttccagtc tgcatagcgg ggtccctagc 780 cgattctctg ggagtggaag cggcaccgac tatagcctga ccatttccaa cctggaacca 840 gaggacattg ccacctatta ctgtcagcag tattcaaagc tgccctatac ctttggcgga 900 gggactaagc tggagatcaa gcgaggaggg ggaggatcag ggggaggggg gagcggaggc 960 ggggggagtg gcggcggagg gtcagaagtc cagctggtcg agagtggggg cggactggtg 1020 aaacctgggg gctctctgaa actgagctgt gcagccagtg gcctgacatt ctcctcatat 1080 gctatgtctt gggtgcggca gaccccagaa aagagactgg agtgggtggc ttcaatctct 1140 tccgggggct tcacttacta tcctgacagc gtgaaaggca gattcactat ctccagggat 1200 aacgcacgga atatcctgta tctgcagatg agctcactga gatccgagga caccgcaatg 1260 tactactgcg aggagacga aggcgggggg tactactgacg tgtgggaggc agcactacc 1320 gtcaccgtgt ctagcaccac tacacctgca ccaggccctc ccaccccgc tcccactc 1380 gctcccagc cactccct gaggcccgag gcctgcaggc cagcagctgg cggagccgtg 1440 catactaggg gggctggactt cgcttgcgac atctacatct gggccccact ggcagggac 1500 tgcggagtcc tgctgctgtc cctgtcatc acacctgtact gcaagcgggg gcgcaaaaaa 1560 ctgctgtata tctttaagca gccttcatg agaccagtgc agacaaccca ggaggaagat 1620 gggtgctcat gccggtttcc cgaggag gaaggcggct gcgagctgag ggtgaagtttt 1680 tcccgctcag cagatgctcc tgcctaccag cagggccaga accagctgta taatgagctg 1740 aacctgggca gacgcgaga gtatgatgtg ctggacaaaa ggcggggaag agaccccgaa 1800 atgggaggga agccaaggcg gaaaacccc caggaggggcc tgtacaatga gctgcagaag 1860 gawaaatgg cagaggctta cagtgagatt gggatgaagg gagagacg gagggaaaa 1920s gggcacgatg gcctgtacca ggggctgagc acagcaacca aagatactta tgacgcactg 1980 cacatgcagg cactgccacc cagatgagtc gacggctccg gtgcccgtca gtgggcagag 2040 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc 2100 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt 2160 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc 2220 aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc 2280 tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt 2340 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga 2400 gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga 2460 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat 2520 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa 2580 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc 2640 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg 2700 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg 2760 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt 2820 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt 2880 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc 2940 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg 3000 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag 3060 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag 3120 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt 3180 caggtgtcgt gacccggggg cttccaccat ggctctgcct gtgaccgcac tgctgctgcc 3240 cctggctctg ctgctgcacg ccgcaagacc tgccgcatac aaggatattc agatgactca 3300 gaccacaagc tccctgagcg cctccctggg agaccgagtg acaatctctt gcagtgcatc 3360 acagggaatt agcaactacc tgaattgta tcagcagaag ccagatggca ctgtgaaact 3420 gctgatctac tataccctta gtctgcacag tggggtcccc tcacgatca gcggatccgg 3480 ctctgggaca gactacagcc tgactctc caactggag cccgaagata ttgccaccta 3540 ctattgccag cagtactcca agctgcctta taccttttggc gggggaacaa agctggagat 3600 3660 aggaagtgag gtgcagctgg tcgaatctgg aggaggactg gtgaagccag gaggatctct 3720 gaaactgagt tgtgccgctt caggcctgac cttctcaagc tacgccatga gctgggtgcg 3780 acagacacct gagaagcggc tggaatgggt cgctagcatc tcctctggcg ggttcacata 3840 ctatccagac tccgtgaaag gcagatttac tatctcgg gataacgcaa gaatattct 3900 gtacctgcag atgagttcac tgaggagcga ggacaccgca atgtactatt gtgccaggga 3960 cgaagtgcgc ggctatctgg atgtctgggg agctggcact accgtcaccg tctccagcga 4020 gcagaaactg attagcgaag aggacctgaa agatgaactg taa 4063 <210> 23 <211> 3257 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide of PGK promoter - anti - human CD7 CAR (TH69) - EFS promoter - anti - human CD7 (TH69) PEBL <400> 23 attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 60 cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 120 cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 180 cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 240 agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 300 aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 360 gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 420 aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 480 ctcctcttcc tcatctccgg gcctttcgag aattcggctt ccaccatggc tctgcctgtg 540 actgccctgc tgctgcccct ggccctgctg ctgcacgctg ctagacccgc cgcatacaag 600 gatattcaga tgactcagac tacatccagc ctgtcagctt ccctggggcga tcgagtgact 660 atcagctgtt cagcaagcca ggggatcagc aattacctga actggtacca gcagaagcccc 720 gacggaactg tcaagctgct gatctactat acttccagtc tgcatagcgg ggtccctagc 780 cgattctctg ggagtggaag cggcaccgac tatagctga ccatttccaa cctggaacca 840 gaggacattg ccacctatta ctgtcagcag tattcaagc tgcctatac ctttggcgga 900 gggactaagc tggagatcaa gcgaggagggg ggaggatcag ggggaggggg gagcggaggc 960 ggggggtg gcggcggagg gtcagaagtc cagctggtcg agagtggggg cggactggtg 1020 aaacctgggg gctctctgaa actgagctgt gcagccagtg gcctgacatt ctcctcatat 1080 gctatgtctt gggtgcggca gaccccagaa agagactgg agtggtggc ttcaatctct 1140 tccggggct tcacttacta tctgacagc gtgaaaggca gattcactat ctcaggt 1200 aacgcacgga atatcctgta tctgcagatg agctcactga gatccgagga caccgcaatg 1260 tactactgcg aggagacga aggcgggggg tactactgacg tgtgggaggc agcactacc 1320 gtcaccgtgt ctagcaccac tacacctgca ccaggccctc ccaccccgc tcccactc 1380 gctcccagc cactccct gaggcccgag gcctgcaggc cagcagctgg cggagccgtg 1440 catactaggg gggctggactt cgcttgcgac atctacatct gggccccact ggcagggac 1500 tgcggagtcc tgctgctgtc cctgtcatc acacctgtact gcaagcgggg gcgcaaaaaa 1560 ctgctgtata tctttaagca gccttcatg agaccagtgc agacaaccca ggaggaagat 1620 gggtgctcat gccggtttcc cgaggag gaaggcggct gcgagctgag ggtgaagtttt 1680 tcccgctcag cagatgctcc tgcctaccag cagggccaga accagctgta taatgagctg 1740 aacctgggca gacgcgaga gtatgatgtg ctggacaaaa ggcggggaag agaccccgaa 1800 atgggaggga agccaaggcg gaaaacccc caggaggggcc tgtacaatga gctgcagaag 1860 gawaaatgg cagaggctta cagtgagatt gggatgaagg gagagacg gagggaaaa 1920s gggcacgatg gcctgtacca ggggctgagc acagcaacca aagatactta tgacgcactg 1980 cacatgcagg cactgccacc cagatgagtc gacgggcaga gcgcacatcg cccacagtcc 2040 ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg 2100 taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac 2160 cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa 2220 cacaggtgtc gtgaccgcgg gtctagagga tccggtactc gaggaactga aaaaccagaa 2280 agttaactgg taagtttagt ctttttgtct tttatttcag gtcccggatc cggtggtggt 2340 gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctagcccg ggggcttcca 2400 ccatggctct gcctgtgacc gcactgctgc tgcccctggc tctgctgctg cacgccgcaa 2460 gacctgccgc atacaaggat attcagatga ctcagaccac aagctccctg agcgcctccc 2520 tgggagaccg agtgacaatc tcttgcagtg catcacaggg aattagcaac tacctgaatt 2580 ggtatcagca gaagccagat ggcactgtga aactgctgat ctactatacc tctagtctgc 2640 acagtggggt cccctcacga ttcagcggat ccggctctgg gacagactac agcctgacta 2700 tctccaacct ggagcccgaa gatattgcca cctactattg ccagcagtac tccaagctgc 2760 cttatacctt tggcggggga acaaagctgg agattaaaag gggaggagga ggaagcggag 2820 gaggaggatc cggaggcggg ggatctggag gaggaggaag tgaggtgcag ctggtcgaat 2880 ctggaggagg actggtgaag ccaggaggat ctctgaaact gagttgtgcc gcttcaggcc 2940 tgaccttctc aagctacgcc atgagctggg tgcgacagac acctgagaag cggctggaat 3000 gggtcgctag catctcctct ggcgggttca catactatcc agactccgtg aaaggcagat 3060 ttactatctc tcgggataac gcaagaaata ttctgtacct gcagatgagt tcactgagga 3120 gcgaggacac cgcaatgtac tattgtgcca gggacgaagt gcgcggctat ctggatgtct 3180 ggggagctgg cactaccgtc accgtctcca gcgagcagaa actgattagc gaagaggacc 3240 tgaaagatga actgtaa 3257 <210> 24 <211> 284 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide of Anti-CD7 (TH69) PEBL <400> 24 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 Ala Ala Tyr Lys Asp Ile Gln Met Thr Gln Thr 20 25 30 Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys 35 40 45 Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Ser Leu His 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 85 90 95 Ser Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Tyr Ser Lys Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 145 150 155 160 Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala 165 170 175 Ala Ser Gly Leu Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln 180 185 190 Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Ser Gly Gly 195 200 205 Phe Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 210 215 220 Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser 225 230 235 240 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Glu Val Arg Gly Tyr 245 250 255 Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Glu Gln 260 265 270 Lys Leu Ile Ser Glu Glu Asp Leu Lys Asp Glu Leu 275 280 <210> 25 <211> 285[[ID=~31]] <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide against CD7 (TH69) PEBL variant <400> 25 Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu 1 5 10 15 Leu His Ala Ala Arg Pro Ala Ala Tyr Lys Asp Ile Gln Met Thr Gln 20 25 30 Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 35 40 45 Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 50 55 60 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Ser Leu 65 70 75 80 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr 100 105 110 Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr Thr Phe Gly Gly Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 145 150 155 160 Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys 165 170 175 Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg 180 185 190 Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Ser Gly 195 200 205 Gly Phe Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 210 215 220 Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Ser Leu Arg 225 230 235 240 Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Glu Val Arg Gly 245 250 255 Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Glu 260 265 270 Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys Asp Glu Leu 275 280 285 <210> 26 <211> 281 <212> PRT <213> artificial sequence <220> <223> Anti-human CD7(3A1F)PEBL's synthetic peptide <400> 26 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 Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Lys Ile Asn Pro Ser Asn Gly Arg Thr Asn 65 70 75 80 Tyr Asn Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Gly Val Tyr Tyr Asp Leu Tyr Tyr 115 120 125 Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp 165 170 175 Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu 180 185 190 His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys 195 200 205 Ser Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser 210 215 220 Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu 225 230 235 240 Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Tyr Thr 245 250 255 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Glu Gln Lys Leu Ile 260 265 270 Ser Glu Glu Asp Leu Lys Asp Glu Leu 275 280 <210> 27 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide against human CD7 (3A1F) PEBL variant <400> 27 Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu 1 5 10 15[[ID=​ 20 25 30 Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly 50 55 60 Gln Gly Leu Glu Trp Ile Gly Lys Ile Asn Pro Ser Asn Gly Arg Thr 65 70 75 80 Asn Tyr Asn Glu Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Val Tyr Tyr Asp Leu Tyr 115 120 125 Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Ile Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 165 170 175 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 180 185 190 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 195 200 205 Lys Ser Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 225 230 235 240 Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Tyr 245 250 255 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Glu Gln Lys Leu 260 265 270 Ile Ser Glu Glu Asp Leu Lys Asp Glu Leu 275 280 <210> 28 <211> 493 <212> PRT <213> artificial sequence <220> <223> Anti-human CD7(TH69)CAR's synthetic peptide <400> 28 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 Ala Ala Tyr Lys Asp Ile Gln Met Thr Gln Thr 20 25 30 Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys 35 40 45 Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Ser Leu His 65 70 75 80 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 85 90 95 Ser Leu Thr Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr 100 105 110 Cys Gln Gln Tyr Ser Lys Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 145 150 155 160 Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala 165 170 175 Ala Ser Gly Leu Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln 180 185 190 Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Ser Gly Gly 195 200 205 Phe Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 210 215 220 Asp Asn Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser 225 230 235 240 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Asp Glu Val Arg Gly Tyr 245 250 255 Leu Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu [[ID=…]]<220>​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <223> Synthetic polypeptide of anti-human CD7 (TH69) CAR variant <400> 29 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 Ala Ala Tyr Lys Asp Ile Gln Met Thr Gln Thr 20 25 30 Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys 35 40 45 Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 50 55 60 Pro Asp Gly Thr Val Lys Leu Leu ...

Claims

1. A bicistronic retroviral vector comprising: (a) a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR) having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, wherein the anti-CD7 CAR comprises an anti-CD7 antibody or an antigen-binding fragment thereof having (i) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (CDR1) of SEQ ID NO: 47, a VH CDR2 of SEQ ID NO: 48, and a VH CDR3 of SEQ ID NO: 49; and (ii) a light chain variable region (VL) comprising a VL CDR1 of SEQ ID NO: 44, a VL CDR2 of SEQ ID NO: 45, and a VL CDR3 of SEQ ID NO: 46; (b) a second polynucleotide encoding a 2A self-cleaving peptide, wherein the 2A self-cleaving peptide is selected from the group consisting of a F2A peptide (foot-and-mouth disease virus 2A peptide), an E2A peptide (equine rhinitis virus 2A peptide), a P2A peptide (porcine tetanus virus-1 2A peptide) and a T2A peptide (TaV 2A); (c) a third polynucleotide encoding an anti-CD7 protein expression blocker (PEBL), the anti-CD7 PEBL having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 24, wherein the anti-CD7 PEBL comprises the same anti-CD7 antibody or antigen-binding fragment thereof as the anti-CD7 CAR; and (d) MSCV promoter or EFS promoter; and The bicistronic retroviral vector comprises the MSCV promoter or EFS promoter operably linked to the first polynucleotide from the 5′ to the 3′ end, the first polynucleotide is operably linked to the second polynucleotide, and the second polynucleotide is operably linked to the third polynucleotide.

2. The bicistronic retroviral vector according to claim 1, wherein The bicistronic retroviral vector comprises the MSCV promoter.

3. The bicistronic retroviral vector according to claim 1 or 2, wherein The MSCV promoter is encoded by the nucleic acid sequence of SEQ ID NO:

9.

4. The bicistronic retroviral vector according to claim 1, wherein The bicistronic retroviral vector comprises the EFS promoter.

5. The bicistronic retroviral vector according to claim 1 or 4, wherein The EFS promoter is encoded by the nucleic acid sequence of SEQ ID NO:

8.

6. The bicistronic retroviral vector according to claim 1 or 2, having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

14.

7. The bicistronic retroviral vector according to claim 6, comprising the nucleic acid sequence of SEQ ID NO:

14.

8. The bicistronic retroviral vector according to claim 1 or 4, having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

16. 9 . The bicistronic retroviral vector according to claim 8 , comprising the nucleic acid sequence of SEQ ID NO:

16.

10. The bicistronic retroviral vector according to claim 1 or 2, wherein: The retroviral vector is a lentiviral vector.

11. An engineered immune cell comprising the bicistronic retroviral vector of any one of claims 1 to 10.

12. The engineered immune cell according to claim 11, wherein The engineered immune cells are allogeneic T cells.

13. The engineered immune cell according to claim 11, wherein The engineered immune cells are autologous T cells.

14. The engineered immune cell according to claim 12 or 13, wherein: The engineered immune cells have reduced CD7 surface expression compared to otherwise identical immune cells that (i) do not express the anti-CD7 PEBL and (ii) express the anti-CD7 CAR.

15. A pharmaceutical composition comprising the engineered immune cell according to claim 11 and a pharmaceutically effective carrier.

16. Use of the engineered immune cell according to any one of claims 11 to 13 or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating cancer in a subject.

17. A method for producing engineered immune cells, comprising transducing immune cells with the bicistronic retroviral vector of any one of claims 1 to 10, and recovering the engineered immune cells.

18. The method according to claim 17, wherein: The immune cells are selected from the group consisting of peripheral blood mononuclear cells, isolated CD4+T cells, isolated CD8+T cells and isolated CD3+T cells.

19. The method according to claim 17 or 18, wherein: The engineered immune cells have reduced CD7 surface expression compared to otherwise identical immune cells that (i) do not express the anti-CD7 PEBL and (ii) express the anti-CD7 CAR.

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