Two-gene vectors for generating car-t cells and uses thereof

Abstract

To provide two-gene retroviral vector compositions for use in cancer therapy comprising polynucleotides encoding anti-CD7 chimeric activation receptor (CAR) and anti-CD7 protein expression blocker.SOLUTION: The invention provides a bicistronic retroviral vector comprising: (a) a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR) having at least 90% sequence identity to a specific amino acid sequence; (b) a second polynucleotide encoding an internal ribosomal entry site (IRES) or a ribosomal codon skipping site; (c) a third polynucleotide encoding an anti-CD7 protein expression blocker (PEBL) having at least 90% sequence identity to a specific amino acid sequence, where the first polynucleotide is operably linked to the second polynucleotide that is operably linked to the third polynucleotide.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 767,069, filed November 14, 2018, the disclosure of which is incorporated herein by reference in its entirety. Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The Sequence Listing was created on November 14, 2019, is entitled "119419-5007-WO-Sequence-Listing_ST25.txt" and is 139,264 bytes in size. [Background technology]

[0002] Chimeric antigen receptors (CARs) can redirect immune cells to specifically recognize and kill tumor cells. CARs are artificial multimolecular proteins composed of a single-chain variable region (scFv) of an antibody linked to a signaling molecule via a transmembrane domain. When the scFv ligates its cognate antigen, signaling is triggered, leading to tumor cell killing 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 using CAR-expressing autologous T lymphocytes have shown positive responses in patients with B-cell refractory leukemia and lymphoma (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).

[0003] The development of CAR technologies targeting T-cell malignancies has lagged significantly behind the progress of their B-cell counterparts. New therapies for T-cell malignancies are needed, but progress has been slow so far. In particular, effective immunotherapeutic options are lacking, and treatment of T-cell acute lymphoblastic leukemia (T-ALL) relies on intensive chemotherapy and hematopoietic stem cell transplantation. Despite aggressive treatment regimens associated with significant morbidity, results with these approaches are far from satisfactory.

[0004] CAR-T cells have recently been developed in which the target antigen of CAR-T itself is expressed in the CAR-T cell (Png et al., Blood, 2017, 1(25):2348-2360, WO2018 / 098306). To avoid suicide (e.g., fratricide), CAR-T cells also express PEBL, which serves to reduce the expression of the target antigen on the cell surface of the CAR-T. To produce viable CAR-T cells, a protein expression blocker (PEBL) protein was first expressed to bind and sequester the target protein before the subsequent expression of CAR. Co-expression of CAR and PEBL resulted in fratricide, since the target antigen was already present on the cell surface of the resulting engineered T cells. Notably, the existing cell surface target antigen is not subject to sequestration by the newly expressed PEBL protein and is recognized and targeted by the newly expressed CAR protein.

[0005] Instead of simultaneous expression, sequential expression can be performed, except for PEBL in T cells followed by The sequential expression of the CAR in each step poses several challenges for the clinical implementation of PEBL CAR-T cells. First, in the sequential engineering of T cells, separate viral vectors, one for PEBL and one for CAR, need to be separately manufactured and administered. This not only increases the cost and time but also complicates the experimental manipulation to produce engineered CAR-T cells. Furthermore, the sequential engineering of T cells results in a complex mix of engineered cells in the final clinical product, creating challenges regarding product characterization, uniformity, and efficacy. Because only a small fraction of T cells integrate the genes introduced at each engineering step, the final product (engineered T cells) contains some cells that received only the PEBL gene, some cells that received only the CAR gene, and some cells that received both genes.

[0006] In summary, there is a significant unmet need for new therapeutic options for patients with T-cell malignancies. Furthermore, methods are needed to produce engineered CAR-T cells and to eliminate CAR-mediated T-cell suicide or fratricide. Summary of the Invention

[0007] In some embodiments, the present invention provides a bicistronic retroviral vector comprising: (a) a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR) comprising 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; and (c) a third polynucleotide encoding an anti-CD7 protein expression blocker (PEBL) comprising 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, which is operably linked to the third polynucleotide.

[0008] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of any one of SEQ ID NOs: 28-31.

[0009] In some embodiments, the anti-CD7 PEBL comprises the amino acid sequence of any one of SEQ ID NOs:24-27.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the IRES is derived from encephalomyocarditis virus (EMCV) or an enterovirus.

[0013] In some embodiments, the ribosomal codon skipping site comprises a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A peptide (foot and mouth disease virus 2A peptide), E2A peptide (equine rhinitis A virus 2A peptide), P2A peptide (porcine teschovirus-12A peptide), and T2A peptide (torsea asigna virus 2A).

[0014] In some embodiments, the bicistronic retroviral vector comprises at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0015] In some embodiments, the bicistronic retroviral vector comprises the sequence of SEQ ID NO: In some embodiments, the bicistronic retroviral vector comprises the nucleic acid sequence of SEQ ID NO:13.

[0016] In some embodiments, the bicistronic retroviral vector further comprises a promoter element.

[0017] 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.

[0018] In some embodiments, the promoter element comprises at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6-10.

[0019] In some embodiments, the promoter element comprises any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs:6-10.

[0020] In some embodiments, the bicistronic retroviral vector comprises at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs:14-16.

[0021] In some embodiments, the bicistronic retroviral vector comprises any one nucleic acid sequence selected from the group consisting of SEQ ID NOs:14-16.

[0022] In some embodiments, the retroviral vector is a lentiviral vector.

[0023] In some embodiments, provided herein is an engineered immune cell comprising any one of the bicistronic retroviral vectors outlined herein.

[0024] In some embodiments, the engineered immune cells are allogeneic T cells. In some embodiments, the engineered immune cells are autologous T cells.

[0025] In some embodiments, the engineered immune cells have reduced CD7 surface expression compared to the corresponding immune cells and express an anti-CD7 CAR.

[0026] In some embodiments, provided herein is a pharmaceutical composition comprising any of the engineered immune cells described herein and a pharma- ceutical effective carrier.

[0027] In some embodiments, provided herein is a method of treating cancer in a subject comprising administering a therapeutically effective amount of any of the engineered immune cells described herein or a pharmaceutical composition thereof.

[0028] In some embodiments, provided herein is a method of producing an engineered immune cell, comprising transducing an immune cell with any one of the bicistronic retroviral vectors described herein and harvesting the engineered immune cell.

[0029] In some embodiments, 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.

[0030] In some embodiments, the engineered immune cells have reduced CD7 surface expression compared to the corresponding immune cells and express an anti-CD7 CAR.

[0031] In some embodiments, 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) comprising 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) comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26.

[0032] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30.

[0033] In some embodiments, the anti-CD7 PEBL comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26.

[0034] 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.

[0035] In some embodiments, the first promoter element and / or the second promoter element comprises at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6-10.

[0036] In some embodiments, the first promoter element and / or the second promoter element comprises any one nucleic acid sequence selected from the group consisting of SEQ ID NOs:6-10.

[0037] In some embodiments, the first promoter and the second promoter share less than 95% sequence identity.

[0038] 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.

[0039] 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.

[0040] In some embodiments, the recombinant retroviral vector comprises at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 18-23.

[0041] In some embodiments, the retroviral vector is a lentiviral vector.

[0042] Also provided are engineered immune cells comprising any one of the recombinant retroviral vectors described herein.

[0043] In some embodiments, the engineered immune cells are allogeneic T cells. In some embodiments, the engineered immune cells are autologous T cells.

[0044] In some embodiments, the engineered immune cells have reduced CD7 surface expression compared to the corresponding immune cells and express an anti-CD7 CAR.

[0045] In some embodiments, provided herein is a pharmaceutical composition comprising any of the engineered immune cells described herein and a pharma- ceutical effective carrier.

[0046] In some embodiments, provided herein is a method of treating cancer in a subject comprising administering a therapeutically effective amount of any of the engineered immune cells described herein or a pharmaceutical composition thereof.

[0047] In some embodiments, provided herein is a method of producing an engineered immune cell, comprising transducing an immune cell with any one of the recombinant retroviral vectors described herein and harvesting the engineered immune cell.

[0048] In some embodiments, 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.

[0049] In some embodiments, the engineered immune cells have reduced CD7 surface expression compared to the corresponding immune cells and express an anti-CD7 CAR. [Brief description of the drawings]

[0050] [Figure 1A-1B] Expression of CAR and PEBL by transduced primary T cells by flow cytometry (Figure 1A) and Western blot (Figure 1B). Primary T cells were transduced with the indicated retroviruses (e.g., PEBL, CAR, PEBL and CAR sequentially, PEBL-IRES-CAR, and CAR-P2A-PEBL) and analyzed by flow cytometry for CD7 and CAR expression. Cell lysates from primary T cells transduced with the indicated retroviruses were analyzed by Western blot for β-actin, Myc-tagged PEBL, CAR and endogenous CD3ζ expression. [Fig. 2A-2F]Provided are exemplary schematics of bicistronic promoter 1-CAR-promoter 2-PEBL lentiviral constructs. Figure 2A shows 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 that of SEQ ID NO: 19. Figure 2B shows 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 that of SEQ ID NO: 18. Figure 2C shows 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 that of SEQ ID NO: 23. Figure 2D shows 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 that of SEQ ID NO: 22. Figure 2E shows a schematic diagram of an exemplary dual promoter construct comprising a PGK promoter-anti-human CD7(3A1F)CAR-EF1a promoter-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 20. Figure 2F shows a schematic diagram of an exemplary dual promoter construct comprising a PGK promoter-anti-human CD7(TH69)CAR-EF1a promoter-anti-human CD7(3A1F)PEBL, such as that of SEQ ID NO: 21. [Diagram 3] Figure 1 shows expression of CAR and CD7 by transduced primary T cells by flow cytometry. Primary T cells were transduced with the indicated dual promoter lentiviruses and analyzed by flow cytometry 5 and 14 days after transduction. [Figure 4A-4C]Provided are exemplary schematic diagrams of bicistronic CAR-P2A-PEBL lentiviral constructs. Figure 4A shows a schematic diagram of an exemplary bicistronic construct comprising an MSCV promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 14. Figure 4B shows a schematic diagram of an exemplary bicistronic construct comprising an EF1a promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 15. Figure 4C shows 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 that of SEQ ID NO: 16. [Diagram 5] Shows CAR and CD7 expression by primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed by flow cytometry 3, 6, and 9 days after transduction. [Figure 6] Figure 1 shows expression of CAR and CD7 by transduced primary T cells by flow cytometry. 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. [Figure 7A-7B] Expression of CAR and PEBL by transduced primary T cells by flow cytometry (Figure 7A) and Western blot (Figure 7B). Primary T cells were transduced with two independently produced lots of MSCV-CD7CAR-P2A-CD7PEBL lentivirus and analyzed for CD7 and CAR expression by flow cytometry. Cell lysates from transduced cells were analyzed by Western blot for β-actin, Myc-tagged PEBL, CAR and endogenous CD3ζ expression. [Figure 8]Expression of CAR and CD7 by transduced primary T cells by flow cytometry. Bulk PBMC, CD4+ and CD8+ positively-selected T cells, and CD3+ positively-selected T cells were activated with either Dynabeads or TransAct and transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus. Cells were analyzed by flow cytometry 4, 7, and 10 days after transduction. [Figure 9A] Shows expression of CAR and CD7 by 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. [Figure 9B] The total fold expansion of transduced cells 11 days after activation is shown (mean ± SEM of biological replicates). [Figure 10A-10B] The percentage of CAR+ T cells (Figure 10A) and total fold expansion (Figure 10B) of transduced cells at 11 days post activation (mean ± SEM of biological replicates) are shown. Primary T cells were cultured in serum-free TexMACS medium and transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus 1, 2, 3, or 4 days post activation. [Figure 11] Shown is the expression of CAR and CD7 by transduced primary T cells by flow cytometry. CD4+ and CD8+ positive selected T cells were activated by TransAct and 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. [Figure 12A-12B]The percentage of CAR+ T cells (Figure 12A) and transgene vector copy number (VCN) (Figure 12B) of transduced cells at 11 days post-activation (average of biological replicates) are shown. Primary T cells were cultured in serum-free TexMACS medium and transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus at MOI 3, 5, or 10. T cells were analyzed for CAR expression by flow cytometry. Genomic DNA was extracted from transduced cells and transgene VCN was determined by RT-qPCR analysis. [Figures 13A-13E] Figure 13 shows the expression of various surface markers on primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus 11 days after activation. Transduced cells were analyzed by flow cytometry for CAR and CD7 (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). Triplicate analysis is for primary T cells from three unique donors transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus at MOI 10. [Figure 14A-14B] Functional response of PEBL-CAR-T cells generated with MSCV-CD7CAR-P2A-CD7PEBL lentivirus to CD7+ Jurkat and CD7- Nalm6 cells by IFNγ secretion (FIG. 14A) and cytotoxicity (FIG. 14B). IFNγ secretion was measured in culture supernatants of PEBL-CAR-T cells co-cultured with Jurkat or Nalm6 cells for 24 h at the indicated E:T ratios (means of technical replicates ± SD). Cytolytic activity of PEBL-CAR T cells was measured after 4 h of co-culture with Jurkat or Nalm6 cells at the indicated E:T ratios (means of technical replicates ± SD). [Figure 15] 1 shows the nucleic acid sequence of CPPT-CMV-MCS-PGK-GFP-WPRE (SEQ ID NO: 1), where CPPT is in bold, the CMV promoter is single underlined, the PGK promoter is double underlined, GFP is bold / single underlined, and the WPRE element is bold / double underlined. [Figure 16] 1 shows the nucleic acid sequence of an exemplary anti-human CD7 PEBL based on the antibody TH69 (SEQ ID NO:2). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 ends the sequence. [Figure 17] 1 shows the nucleic acid sequence of an exemplary anti-human CD7 PEBL based on the antibody 3A1F (SEQ ID NO: 3). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 ends the sequence. [Figure 18] 1 shows the nucleic acid sequence of an exemplary anti-human CD7 CAR based on the antibody TH69 (SEQ ID NO: 4). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, the CD3ζ signaling domain is bold / double underlined, and the stop codon ends the sequence. [Figure 19] 1 shows the nucleic acid sequence of an exemplary anti-human CD7 CAR based on the antibody 3A1F (SEQ ID NO:5). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain, the CD3ζ signaling domain is bold / double underlined, and the stop codon ends the sequence. [Figure 20]1 shows the nucleic acid sequence of an exemplary CMV promoter (SEQ ID NO:6). [Figure 21] 1 shows the nucleic acid sequence of an exemplary EF1α promoter (SEQ ID NO:7). [Figure 22] 1 shows the nucleic acid sequence of an exemplary EFS promoter (SEQ ID NO:8). [Diagram 23] 1 shows the nucleic acid sequence of an exemplary MSCV promoter (SEQ ID NO:9). [Figure 24] 1 shows the nucleic acid sequence of an exemplary PGK promoter (SEQ ID NO:10). [Diagram 25] 1 shows 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 normal font, IRES is in bold, and anti-human CD7(TH69)CAR is double underlined. [Figure 26] 1 shows 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 in normal font, IRES is in bold, and anti-human CD7(TH69)PEBL is single underlined. [Figure 27] 1 shows 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 normal font, P2A is in bold, and anti-human CD7(TH69)PEBL is single underlined. [Fig. 28A-28B] 1 shows the nucleic acid sequence of an exemplary bicistronic construct comprising MSCV promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL (SEQ ID NO: 14). The MSCV promoter is double underlined, the restriction enzyme sites and Kozak sequence are between the MSCV promoter and 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. [Figure 29A-29B]1 shows the nucleic acid sequence of an exemplary bicistronic construct comprising EF1a promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL (SEQ ID NO: 15). The EF1α promoter is double underlined, the restriction enzyme site and Kozak sequence are between the EF1α 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. [Fig. 30A-30B] 1 shows the nucleic acid sequence of an exemplary bicistronic construct comprising the 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 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. [Fig. 31A-31B] 1 shows the nucleic acid sequence of an 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 (italicized) and Kozak sequence are between the MSCV promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and PGK promoter, the PGK promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the PGK promoter and PEBL, the anti-human CD7(TH69)PEBL is bold / single underlined, and the restriction enzyme site (italicized) ends the sequence. [Fig. 32A-32B]1 shows the nucleic acid sequence of an 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 (italicized) and Kozak sequence are between the MSCV promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EF1a promoter, the EF1a promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EF1a promoter and PEBL, and the anti-human CD7(TH69)PEBL is bold / single underlined. [Fig. 33A-33B] 1 shows the nucleic acid sequence of an 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 (italicized) and Kozak sequence are between the MSCV promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EFS promoter, the EFS promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EFS promoter and PEBL, and the anti-human CD7(TH69)PEBL is bold / single underlined. [Fig. 34A-34B] 1 shows 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 (italicized) and Kozak sequence are between the PGK promoter and CAR, the anti-human CD7(3A1F)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EF1a promoter, the EF1a promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EF1a promoter and PEBL, and the anti-human CD7(TH69)PEBL is bold / single underlined. [Fig. 35A-35B] 1 shows 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 (SEQ ID NO: 21). The PGK promoter is double underlined, the restriction enzyme site (italicized) and Kozak sequence are between the PGK promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EF1a promoter, the EF1a promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EF1a promoter and PEBL, and the anti-human CD7(3A1F)PEBL is bold / single underlined. [Fig. 36A-36C] 1 shows 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 (italicized) and Kozak sequence are between the PGK promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EF1a promoter, the EF1a promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EF1a promoter and PEBL, and the anti-human CD7(TH69)PEBL is bold / single underlined. [Fig. 37A-37B] 2 shows the nucleic acid sequence of an exemplary dual promoter construct comprising PGK promoter-anti-human CD7(TH69)CAR-EFS promoter-anti-human CD7(TH69)PEBL (SEQ ID NO: 23). The PGK promoter is double underlined, the restriction enzyme site (italicized) and Kozak sequence are between the PGK promoter and CAR, the anti-human CD7(TH69)CAR is bold, the restriction enzyme site (italicized) is between the CAR and EFS promoter, the EFS promoter is single underlined, the restriction enzyme site (italicized) and Kozak sequence are between the EFS promoter and PEBL, and the anti-human CD7(TH69)PEBL is bold / single underlined. [Figure 38] 1 shows the amino acid sequence of an exemplary anti-human CD7 PEBL based on antibody TH69 (SEQ ID NO: 24). The CD8 signal peptide starts at position 1 and is of regular type, the anti-CD7 VL domain is in 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. [Figure 39] 2 shows the amino acid sequence of an exemplary anti-human CD7 PEBL mutant based on the antibody TH69 (SEQ ID NO:25). The N-terminal proline is in italics, the CD8 signal peptide starts at position 2 and is of regular type, the anti-CD7 VL domain is in 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. [Diagram 40] 1 shows the amino acid sequence of an exemplary anti-human CD7 PEBL based on antibody 3A1F (SEQ ID NO:26). The CD8 signal peptide starts at position 1 and is of regular type, the anti-CD7 VL domain is in 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. [Diagram 41] 2 shows the amino acid sequence of an exemplary anti-human CD7 PEBL mutant based on antibody 3A1F (SEQ ID NO:27). The N-terminal proline is in italics, the CD8 signal peptide starts at position 2 and is of regular type, the anti-CD7 VL domain is in 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. [Diagram 42]2 shows the amino acid sequence of an exemplary anti-human CD7 CAR based on the antibody TH69 (SEQ ID NO: 28). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain and is of normal type, and the CD3ζ signaling domain is bold / double underlined. [Diagram 43] 2 shows the amino acid sequence of an exemplary anti-human CD7 CAR variant based on the antibody TH69 (SEQ ID NO: 29). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is in bold / single underlined, the CD8a hinge and transmembrane domain is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain and is of normal type, the CD3ζ signaling domain is in bold / double underlined, and the amino acids C-terminal to the CD3ζ signaling domain occur via ribosomal skipping at the P2A site. [Diagram 44] 1 shows the amino acid sequence of an exemplary anti-human CD7 CAR based on the antibody 3A1F (SEQ ID NO: 30). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in 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 is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain and is of normal type, and the CD3ζ signaling domain is bold / double underlined. [Diagram 45]1 shows the amino acid sequence of an exemplary anti-human CD7 CAR variant based on antibody 3A1F (SEQ ID NO:31). The CD8 signal peptide starts at position 1, the anti-CD7 VL domain is in bold, the linker between the VL and VH domains is single underlined, the anti-CD7 VH domain is in bold / single underlined, the CD8a hinge and transmembrane domain is double underlined, the 4-1BB signaling domain is between the CD8a hinge and transmembrane domain and the CD3ζ signaling domain and is of normal type, the CD3ζ signaling domain is in bold / double underlined, and the amino acids C-terminal to the CD3ζ signaling domain occur via ribosomal skipping at the P2A site. [Figure 46] 9 shows the amino acid sequence of an exemplary anti-human CD7 CAR based on the antibody TH69, TH69-P2A-anti-human CD7 PEBL (SEQ ID NO: 95). The CD7 CAR is in normal font, P2A is double underlined, and CD7 PEBL is bold / single underlined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] introduction The present invention provides methods for the co-expression of a fratricide-inducing chimeric antigen receptor (e.g., CAR) and a fratricide-preventive protein (e.g., PEBL) in T cells, resulting in viable CAR-expressing cytotoxic T lymphocytes (CAR-T) that target T cell antigens.

[0052] Viral vectors have been created that can express two or more genes from a single construct. Typically, these vectors use either bicistronic elements or two-promoter configurations. In the case of bicistronic vectors, a sequence element is introduced between the two genes that allows translation of two proteins from a single messenger RNA. Examples include internal ribosome entry site sequences (IRES) and virus-derived "codon skipping" peptide sequences (P2A, T2A, F2A, E2A, etc.). In the case of vectors designed with two promoters, separate promoter elements are configured upstream of each gene so that each promoter transcribes the mRNA of its proximally linked gene. In some embodiments, the expression vector (e.g., construct) comprises a first promoter operably linked to CAR and a second promoter operably linked to PEBL.

[0053] Methods for making and testing various bicistronic and two-promoter design vectors for the expression of two different genes (e.g., a gene encoding a CAR and a gene encoding a PEBL) are described herein. Unexpectedly, it was discovered that a particular two-gene vector can direct the expression of both PEBL and CAR proteins in T cells so that the resulting engineered T cells can survive, proliferate, and kill target cells. The relative timing and level of expression of each gene in the identified two-gene vector allowed for downregulation of the target antigen before the CAR could cause excessive fratricide in the engineered T cells.

[0054] Described herein are fratricide-resistant CAR-T cells expressing a CAR directed against CD7, such CAR-T cells having reduced or no surface expression of CD7. The invention is based in part on the co-expression of a chimeric antigen receptor (CAR) directed against CD7 and a protein expression blocker (PEBL) directed against CD7 in immune cells (e.g., T cells) using a bicistronic construct, such as a bicistronic viral vector. In one aspect, the invention relates to engineered immune cells (e.g., engineered T cells) comprising a bicistronic construct comprising a polynucleotide sequence encoding an anti-CD7 CAR and a polynucleotide sequence encoding an anti-CD7 PEBL. In some embodiments, the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody (e.g., a single-chain variable fragment or scFv) that specifically binds to CD7. The CD7 CAR of the invention may be referred to herein as "anti-CD7-41BB-CD3ζ". In some embodiments, the CAR also comprises a CD8α hinge and a transmembrane domain. In some embodiments, anti-CD7 PEBL comprises an antibody (e.g., scFv) that specifically binds to CD7 and an intracellular localization sequence. In certain embodiments, anti-CD7 PEBL comprises an antibody (e.g., scFv) that specifically binds to CD7, CD8α hinge and transmembrane domains, and an intracellular localization sequence.

[0055] CD7 is a 40 kDa type I transmembrane glycoprotein that is a major marker for T-cell malignancies and is highly expressed in all cases of T-cell ALL, including early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Anti-CD7 CARs induce T cells to It has been shown that anti-CD7 CARs can exert specific cytotoxicity against malignant tumors of T cells. Furthermore, it has been shown that the cytotoxicity of T cells is significantly increased when anti-CD7 CARs are used in combination with downregulation of CD7 expression on effector T cells. Downregulation (e.g., elimination, reduction, and / or relocalization) of CD7 on T cells via expression of anti-CD7 PEBL prevented the fratricidal effect exerted by the corresponding anti-CD7 CAR. This promoted the recovery of T cells after CAR expression and made their cytotoxicity against T leukemia / lymphoma cells more effective compared to cells that retained the target antigen (e.g., CD7). definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0056] 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.

[0057] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0058] The term "about" in connection with a reference numerical value, and its grammatical equivalents as used herein, can include a range of values ​​from that value plus or minus 10%. For example, the amount "about 10" includes the amount 9 to 11. The term "about" in connection with a reference numerical value can also include a range of values ​​from that value plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0059] As used herein, the term "nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide 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. Furthermore, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, "nucleic acid" can refer to one or both strands of the molecule. As used herein, nucleic acid and polynucleotide are interchangeable.

[0060] The term "nucleotide sequence" with respect to nucleic acids refers to a contiguous series of nucleotides linked by covalent bonds, such as phosphorus linkages (e.g., phosphodiester, alkyl and aryl phosphonate, phosphorothioate, phosphotriester linkages), and / or non-phosphorus linkages (e.g., peptide and / or sulfamate linkages). In certain embodiments, the nucleotide sequence encoding the target binding molecule linked to, for example, a localization domain, is a heterologous sequence (e.g., a gene originating from a different species or cell type).

[0061] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Thus, nucleotides include, for example, naturally occurring bases such as adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine. ) and nucleotides containing modified bases known in the art.

[0062] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting 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 placed 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, where necessary to join two protein coding regions, are in the same reading frame.

[0063] The term "sequence identity" means that two nucleotide sequences or two amino acid sequences share at least, 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 when optimally aligned, such as by the program GAP or BESTFIT using default gap weights. In sequence comparison, typically, one sequence serves as a reference sequence (e.g., parent sequence) and is compared to a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0064] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm 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 search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), using the algorithms GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software. The sequence identity and sequence similarity can be determined by computerized implementation of the BLAST algorithm (see, generally, Ausubel et al., Current Protocols in Molecular Biology). One example of an algorithm suitable for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (publicly available from the National Institutes of Health NCBI Internet server). Typically, default program parameters can be used to perform sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0065] As will be appreciated by those of skill in the art, in some embodiments, the nucleic acid further comprises a plasmid sequence, which may include, for example, one or more of a promoter sequence, a selection marker sequence, or a target gene recombination sequence.

[0066] The term "promoter" or "promoter element" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate specific transcription of a polynucleotide sequence.

[0067] The term "retroviral vector" may refer to a gamma retroviral 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, for example, a polynucleotide encoding a CAR and a polynucleotide encoding PEBL. A retroviral vector may lack viral structural genes such as gag, pol, env, etc. Exemplary retroviral (e.g., gamma retroviral) vectors include mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), murine leukemia virus (MLV), spleen focus forming virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Maetzig et al., Viruses, 2011;3(6):677-713.

[0068] The term "bicistronic expression" is typically achieved by operably linking the polynucleotides described herein to a promoter and incorporating the bicistronic construct into an expression vector. The vector may be suitable for replication and integration eukaryotes. A typical cloning vector includes transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Nucleic acids can be cloned into many 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. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0069] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known 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) that incorporate a recombinant polynucleotide.

[0070] Furthermore, the expression vector can be provided to the cell in the form of a viral vector.Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include a replication origin functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0071] Additional promoter elements, e.g., enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but many promoter elements are It has been shown that promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Exemplary promoters include the immediate early cytomegalovirus (CMV), EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters. Strong constitutive promoter sequences can be used that are capable of driving high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may 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 leukemia 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 leukemia 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. In some embodiments, the promoter is an inducible promoter, providing a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0072] As used herein, "antibody" refers to an intact antibody or an antigen-binding fragment of an antibody, including modified or engineered intact antibodies or antigen-binding fragments, 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), minibodies, and diabodies.

[0073] The terms "specifically (or selectively) bind" or "specifically (or selectively) immunoreactive" when referring to a protein or peptide often refer to a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a specified antibody will bind to a particular protein at least twice background, and more typically more than 10-100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies may be selected to obtain only those polyclonal antibodies that specifically immunoreact with a selected antigen and not with other proteins. This selection may be accomplished by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0074] In certain embodiments, the antibody that binds to CD7 is a single chain variable fragment antibody ("scFv antibody"). scFv refers to an antibody fragment that contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, 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, a variety of suitable linkers are provided in the art and can be designed and tested for optimal function as disclosed herein.

[0075] As used herein, an "engineered" immune cell includes an immune cell that is genetically modified compared to a naturally occurring immune cell. For example, an engineered T cell produced according to the methods of the invention carries a nucleic acid that includes a nucleotide sequence that does not naturally occur in the T cell from which it is derived, such as the nucleic acids exemplified herein.

[0076] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to a cell that has been separated from the cells with which it is naturally associated in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0077] 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 surface expression of endogenous CD7. In some embodiments, low or no surface expression of endogenous CD7 is due to expression of PEBL for human CD7, which prevents or impedes the translocation of endogenous CD7 protein to the surface of the T cell. In some examples, surface expression of CD7 can be determined using standard methods known to those skilled in the art, such as, but not limited to, immunocytochemistry, flow cytometry, or FACS.

[0078] The term "autologous" as used herein and its grammatical equivalents may refer to originating from the same entity. For example, a sample (e.g., cells) may be removed, processed, and later returned to the same subject (e.g., patient). Autologous process is distinct from allogeneic process, in which donor and recipient are different subjects.

[0079] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0080] As used herein, the terms "treat," "treating," or "treatment" refer to combating 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 criteria.

[0081] As used herein, the term "subject" refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In certain embodiments, the subject is a human. A "subject in need of treatment" refers to a mammalian animal or mammalian subject that is a candidate for treatment by inducing T cells to exert specific cytotoxicity against malignant tumor T cells. "Treatment" refers to a subject (e.g., a patient) having or at risk of developing a disease or condition that can be treated (e.g., ameliorated, ameliorated, prevented).

[0082] As defined herein, a "therapeutic amount" refers to an amount that, when administered to a subject, is sufficient to achieve a desired therapeutic effect in the subject (treat a condition associated with a T cell malignancy) under the conditions of administration. The effective amount of an agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art, and will depend on several factors, including, for example, the particular agent selected, the subject's age, sensitivity, tolerance to the drug, and overall health. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As described in detail below, anti-CD7 CARs (also referred to as "CD7 CARs") may comprise an antigen binding domain that targets CD7 based on the TH69 antibody. In some embodiments, the antigen binding domain of the CD7 CAR is based on the 3A1F antibody. In some embodiments, the antigen binding domain of the CD7 CAR is based on the T3-3A1 antibody. In some embodiments, the CD7 CAR of the 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 one 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 engineered immune cells of the 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 of SEQ ID NO:29. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 30. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31. In some cases, the engineered immune cells comprise a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0083] In some embodiments, the CD7 PEBL of the present 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 present invention comprises an amino acid sequence having at least 90% sequence identity to that 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 examples, the engineered immune cells of the present invention comprise CD7 PEBL of SEQ ID NO:24. In some examples, the engineered immune cells of the present invention comprise CD7 PEBL having at least 90% sequence identity to SEQ ID NO:25. In some examples, the engineered immune cells comprise CD7 PEBL of SEQ ID NO:24. In some examples, the engineered immune cells comprise CD7 PEBL having at least 90% sequence identity to SEQ ID NO:25. In some examples, the engineered immune cells comprise CD7 PEBL of SEQ ID NO:26. In some examples, the engineered immune cells comprise CD7 PEBL having at least 90% sequence identity to SEQ ID NO:26. In some examples, the engineered immune cells comprise a CD7 PEBL of SEQ ID NO: 27. In some examples, the engineered immune cells comprise a CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27.

[0084] In some embodiments, the engineered immune cells or populations of engineered immune cells of the 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 of SEQ ID NO: 24 and a CD7 CAR of SEQ ID NO: 28. , 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 cell or population of engineered immune cells comprises a CD7 PEBL of SEQ ID NO:24 and a CD7 CAR of 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:24 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: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 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 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 with at least 90% sequence identity SEQ ID NO:26 and a CD7 CAR with at least 90% sequence identity 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 with at least 90% sequence identity SEQ ID NO:25 and a CD7 CAR with at least 90% sequence identity 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 with at least 90% sequence identity SEQ ID NO:25 and a CD7 CAR with at least 90% sequence identity 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 with at least 90% sequence identity SEQ ID NO:27 and a CD7 CAR with at least 90% sequence identity 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 with at least 90% sequence identity SEQ ID NO:27 and a CD7 CAR with at least 90% sequence identity SEQ ID NO:31.

[0085] 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 harboring the bicistronic or dual promoter construct are generated from PBMCs. In some embodiments, the engineered immune cells harboring the bicistronic or dual promoter construct are generated from purified CD4+ T cells. In some embodiments, the engineered immune cells harboring the bicistronic or dual promoter construct are generated from purified CD8+ T cells. In some embodiments, the engineered immune cells harboring the bicistronic or dual promoter construct are generated from a population of cells comprising purified CD4+ T cells and purified CD8+ T cells. In some embodiments, the engineered immune cells harboring the bicistronic or dual promoter construct are generated from a population of cells comprising purified CD3+ T cells. Bicistronic expression constructs Provided herein is a recombinant bicistronic viral construct or vector comprising a polynucleotide encoding a CAR and a polynucleotide encoding a PEBL, as described herein. In some embodiments, the recombinant bicistronic viral construct comprises an internal ribosome entry site (IRES) sequence between the nucleic acid sequence of the CAR and the nucleic acid sequence of the PEBL. In some embodiments, the recombinant bicistronic viral construct comprises a ribosome codon skipping site sequence (also referred to as a sequence encoding a 2A self-cleaving peptide) between the nucleic acid sequence of the CAR and the nucleic acid sequence of the PEBL. In some embodiments of the bicistronic construct, the polynucleotide encoding the CAR is located upstream (5' end) of the IRES sequence, and the polynucleotide encoding the PEBL is located downstream (3' end) of the IRES. In some cases, the nucleic acid sequence encoding the CAR is operably linked to the IRES sequence, and the IRES sequence is operably linked to the nucleic acid sequence encoding the PEBL. In some cases, the nucleic acid sequence encoding the PEBL is operably linked to the IRES sequence, and the IRES sequence is operably linked to the nucleic acid sequence encoding the CAR.

[0086] In some embodiments of the bicistronic construct, the polynucleotide encoding the CAR is located upstream (5' end) of the polynucleotide encoding the 2A self-cleaving peptide, and the polynucleotide encoding PEBL is located downstream (3' end) of the polynucleotide encoding the 2A self-cleaving peptide. In some cases, the nucleic acid sequence encoding the CAR is operably linked to the nucleic acid sequence encoding the 2A self-cleaving peptide, which is operably linked to the nucleic acid sequence encoding PEBL. In some cases, the nucleic acid sequence encoding the PEBL is operably linked to the nucleic acid sequence encoding the 2A self-cleaving peptide, which is operably linked to the nucleic acid sequence encoding the CAR.

[0087] The mechanism of ribosomal codon skipping via 2A peptide sequences is useful for generating two proteins from one transcript, where the normal peptide bond is disrupted at the 2A sequence, resulting in two discontinuous protein fragments from one translation event. Self-cleaving 2A peptides (e.g., 2A cleavage sites) are described in Kim et al., PLoS One, 2011,6(4):e18556.

[0088] In some embodiments, the IRES is derived from an encephalomyocarditis virus. In some embodiments, the IRES is derived 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).

[0089] In some embodiments, the ribosomal codon skipping site is based on a 2A self-cleaving peptide (see, e.g., Table 2). In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of P2A, E2A, F2A, and T2A. In some examples, 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 examples, 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 examples, 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 examples, 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.

[0090] In some embodiments, the viral construct (e.g., retroviral construct) comprises a 2A self-cleaving peptide selected from the group consisting of P2A, E2A, F2A, and T2A. (e.g., a 2A peptide cleavage site), and the polynucleotide encoding the 2A self-cleaving peptide links the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding PEBL. In other words, the polynucleotide encoding the 2A self-cleaving peptide is between the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding PEBL. As described above, in some embodiments, the construct comprises or consists of, from the 5' to the 3' end, a nucleic acid sequence encoding a CAR, a nucleic acid sequence encoding a P2A self-cleaving peptide, and a nucleic acid sequence encoding PEBL. In some embodiments, the construct comprises or consists of, from the 5' to the 3' end, a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a P2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of, from the 5' to the 3' end, a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding an E2A self-cleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of, from the 5' to the 3' end, a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding an F2A autocleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein. In some embodiments, the construct comprises or consists of, from the 5' to the 3' end, a nucleic acid sequence encoding any CD7 CAR described herein, a nucleic acid sequence encoding a T2A autocleaving peptide, and a nucleic acid sequence encoding any CD7 PEBL described herein.

[0091] In some embodiments, the construct comprises or consists of, from the 5' to 3' end, a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding the P2A autocleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of, from the 5' to 3' end, a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding the E2A autocleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of, from the 5' to 3' end, a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding the F2A autocleaving peptide, and a nucleic acid sequence encoding a CAR. In some embodiments, the construct comprises or consists of, from the 5' to 3' end, a nucleic acid sequence encoding PEBL, a nucleic acid sequence encoding the T2A autocleaving peptide, and a nucleic acid sequence encoding a CAR.

[0092] 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 the 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 the 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 the 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 the nucleic acid of SEQ ID NO:66.

[0093] In some embodiments, the nucleic acid sequence encoding PEBL is positioned (e.g., located) 5' to the nucleic acid sequence encoding CAR. In some embodiments, the nucleic acid sequence encoding CAR is positioned 5' to the nucleic acid sequence encoding PEBL. In some embodiments, the bicistronic construct comprises, from the 5' end to the 3' end: (a1) SEQ ID NO:4, SEQ ID NO:63, and SEQ ID NO:2; (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; 4) 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) (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: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.

[0094] In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 16, SEQ ID NO: 63, and the sequence of FIG. 18. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 16, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 19. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 17, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 18. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 17, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 19.

[0095] In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 18, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 16. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 19, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 16. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 18, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 17. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to 3' end, the sequence of FIG. 19, any one of SEQ ID NOs: 63-66, and the sequence of FIG. 17.

[0096] In some embodiments, the bicistronic construct comprises or consists of, from the 5' to the 3' end, a polynucleotide encoding CD7(TH67)CAR of SEQ ID NO:28, a polynucleotide encoding a P2A peptide of SEQ ID NO:67, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO:24. In some embodiments, the bicistronic construct comprises, from the 5' to the 3' end, a polynucleotide encoding CD7(TH67)CAR of SEQ ID NO:28, a polynucleotide encoding a P2A peptide of SEQ ID NO:67, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO:24. 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding CD7(TH67)PEBL of FIG. 38. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to the 3' end, 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 CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, the bicistronic construct comprises or consists of, from the 5' to the 3' end, a polynucleotide encoding the CD7(TH67)CAR of FIG. 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding CD7(3A1F)PEBL of FIG. 40. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO:30, a polynucleotide encoding a P2A peptide of SEQ ID NO:67, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO:24. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of Figure 44, a polynucleotide encoding a P2A peptide of SEQ ID NO:67, and a polynucleotide encoding CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO:30, a polynucleotide encoding a P2A peptide of SEQ ID NO:67, and a polynucleotide encoding CD7(3A1F)PEBL of SEQ ID NO:26.In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO: 67, and a polynucleotide encoding the CD7(3A1F)PEBL of Figure 40. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, 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 or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(TH67)CAR of Figure 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises or consists, from the 5' to 3' end, of a polynucleotide encoding CD7(TH67)CAR of SEQ ID NO:28, a polynucleotide encoding a P2A peptide of SEQ ID NO:68, and a polynucleotide encoding CD7(3A1F)PEBL of SEQ ID NO:26. In some embodiments, the bicistronic construct comprises or consists, from the 5' to 3' end, of a polynucleotide encoding CD7(TH67)CAR of Figure 42, a polynucleotide encoding a P2A peptide of SEQ ID NO:68, and a polynucleotide encoding CD7(3A1F)PEBL of Figure 40. In some embodiments, the bicistronic construct comprises or consists, from the 5' to 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO:30, a polynucleotide encoding a P2A peptide of SEQ ID NO:68, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO:24.In some embodiments, the bicistronic construct comprises or consists of, from the 5' to the 3' end, a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO:68, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises, from the 5' to the 3' end, a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO:68, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38. A bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO: 30, a polynucleotide encoding a P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding CD7(3A1F)PEBL of SEQ ID NO: 26. In some embodiments, a bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of Figure 44, a polynucleotide encoding a P2A peptide of SEQ ID NO: 68, and a polynucleotide encoding CD7(3A1F)PEBL of Figure 40. In some embodiments, a bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(TH67)CAR of SEQ ID NO: 28, a polynucleotide encoding a P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO: 24. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(TH67)CAR of Figure 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, 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 or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(TH67)CAR of Figure 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of Figure 40.In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO:30, a polynucleotide encoding a P2A peptide of SEQ ID NO:69, and a polynucleotide encoding CD7(TH67)PEBL of SEQ ID NO:24. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of Figure 44, a polynucleotide encoding a P2A peptide of SEQ ID NO:69, and a polynucleotide encoding CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding CD7(3A1F)CAR of SEQ ID NO:30, a polynucleotide encoding a P2A peptide of SEQ ID NO:69, and a polynucleotide encoding CD7(3A1F)PEBL of SEQ ID NO:26. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO: 69, and a polynucleotide encoding the CD7(3A1F)PEBL of Figure 40. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, 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 or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(TH67)CAR of Figure 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38.In some embodiments, the bicistronic construct comprises or consists of, from the 5' to the 3' end, a polynucleotide encoding CD7(TH67)CAR of SEQ ID NO:28, a polynucleotide encoding a P2A peptide of SEQ ID NO:70, and a polynucleotide encoding CD7(3A1F)PEBL of SEQ ID NO:26. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(TH67)CAR of Figure 42, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of Figure 40. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, 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 or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(TH67)PEBL of Figure 38. In some embodiments, the bicistronic construct comprises or consists, from the 5' to the 3' end, 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 or consists, from the 5' to the 3' end, of a polynucleotide encoding the CD7(3A1F)CAR of Figure 44, a polynucleotide encoding the P2A peptide of SEQ ID NO: 70, and a polynucleotide encoding the CD7(3A1F)PEBL of Figure 40.

[0097] In some embodiments, the polynucleotide sequence encoding PEBL is positioned 5' (upstream) of the IRES site, which is positioned 5' of the polynucleotide sequence encoding CAR. In some embodiments, the polynucleotide sequence encoding CAR is positioned 5' of the IRES site, which is positioned 5' of the polynucleotide sequence encoding PEBL.

[0098] In some embodiments, the polynucleotide sequence encoding PEBL is positioned 5' (upstream) of a ribosomal codon skipping site, which is positioned 5' of the polynucleotide sequence encoding CAR. In some embodiments, the polynucleotide sequence encoding CAR is positioned 5' of a ribosomal codon skipping site, which is positioned 5' of the polynucleotide sequence encoding PEBL.

[0099] In some embodiments, provided herein is a recombinant bicistronic construct comprising at least 90% sequence identity to one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In some embodiments, the recombinant bicistronic construct comprises at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:63. In some embodiments, the recombinant bicistronic construct comprises at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:64. In some embodiments, the recombinant bicistronic construct comprises at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:65. In some embodiments, the recombinant bicistronic construct comprises 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 the group consisting of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0100] [Table 1]

[0101] [Table 2]

[0102] The present invention provides vectors, such as expression vectors, into which any of the polynucleotides described herein are inserted. In some embodiments, the vector is derived from a retrovirus, such as a lentivirus. Such vectors are suitable tools for achieving long-term gene transfer, since they allow long-term and stable integration of exogenous polynucleotides (e.g., transgenes) and their propagation in daughter cells. Unlike vectors derived from tumor retroviruses, such as murine leukemia viruses, lentiviral vectors can transduce non-proliferating cells. Lentiviral vectors are also less immunogenic. In other embodiments, the vector is an adenoviral vector. In certain embodiments, the vector is a plasmid.

[0103] In some embodiments, the promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the CMV promoter. In some embodiments, the promoter comprises a CMV promoter. 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 CMV promoter of SEQ ID NO:6.

[0104] In some embodiments, the promoter comprises at least 90%, e.g., 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 an 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.

[0105] In some embodiments, the promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the EFS promoter. In some embodiments, the promoter comprises an 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.

[0106] In some embodiments, the promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the murine stem cell virus (MSCV) promoter. In some embodiments, the promoter comprises an MSCV promoter. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO:9. In some embodiments, any of the constructs described herein comprises or consists of the MSCV promoter of SEQ ID NO:9.

[0107] In some embodiments, the promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to phosphoglycerate kinase (PGK). In some embodiments, the promoter comprises a 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.

[0108] 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 shown in FIG. 25. In some embodiments, the bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the sequence of SEQ ID NO: 11. The bicistronic vector comprises, from the 5' end to the 3' end, a nucleic acid sequence comprising a nucleic acid sequence encoding CD7 PEBL, an IRES sequence, and a nucleic acid sequence encoding CD7 CAR, and optionally at the 5' end, a promoter selected from the group consisting of a CMV promoter (e.g., SEQ ID NO: 6 or FIG. 20), an EF1a promoter (e.g., SEQ ID NO: 7 or FIG. 21), an EFS promoter (e.g., SEQ ID NO: 8 or FIG. 22), an MSCV promoter (e.g., SEQ ID NO: 9 or FIG. 23), and a PGK promoter (e.g., SEQ ID NO: 10 or FIG. 24).

[0109] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 12. The bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the sequence. An exemplary embodiment of such a sequence is shown in Figure 26. The bicistronic vector comprises, from the 5' end to the 3' end, a nucleic acid sequence comprising a nucleic acid sequence encoding a CD7 CAR, an IRES sequence, and a nucleic acid sequence encoding a CD7 PEBL, optionally at the 5' end, a promoter selected from the group consisting of a CMV promoter, an EF1a promoter, an EFS promoter, an MSCV promoter, and a PGK promoter.

[0110] In some embodiments, the bicistronic vector comprises a nucleic acid sequence of SEQ ID NO: 13. An exemplary embodiment of such a sequence is shown in Figure 27. In some embodiments, the bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the sequence of SEQ ID NO: 13. The bicistronic vector comprises, from the 5' end to the 3' end, a nucleic acid sequence comprising 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 the group consisting of a CMV promoter, an EF1a promoter, an EFS promoter, an MSCV promoter, and a PGK promoter.

[0111] In some embodiments, the bicistronic vector comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the sequence of SEQ ID NO: 14. The bicistronic vector comprises, from the 5' to the 3' end, a promoter, a CD7 The bicistronic vector comprises a nucleic acid sequence comprising a nucleic acid sequence encoding a CAR, a P2A sequence, and a nucleic acid sequence encoding a CD7 PEBL. In some examples, 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 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 shown in Figures 28A-B.

[0112] In some embodiments, the bicistronic vector comprises a nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the sequence of SEQ ID NO: 15. In some examples, 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 shown in Figures 29A-B.

[0113] In some embodiments, the bicistronic vector comprises a nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the bicistronic vector comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, sequence identity to the sequence of SEQ ID NO: 16. In some examples, 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 shown in Figures 30A-B. Dual promoter retroviral constructs Provided herein is a recombinant retroviral construct (or vector) for simultaneously expressing CAR and PEBL in a cell, such as a T cell. In some embodiments, the retroviral construct comprises a promoter operably linked to a polynucleotide encoding any of the CARs described herein, and a promoter operably linked to a polynucleotide encoding any of the PEBLs described herein. In some embodiments, the promoter of the CAR and the promoter of the PEBL share less than 90% sequence identity, e.g., 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 of the CAR and the promoter of the PEBL share 80% or less sequence identity, e.g., 80% identity, 75% sequence identity, 70% sequence identity, 65% sequence identity, 60% sequence identity, 55% sequence identity, etc. In some embodiments, the CAR promoter and the PEBL promoter 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.

[0114] In some embodiments, the promoter of CAR (referred to as the first promoter) is different from the promoter of PEBL (referred to as the second promoter). The first promoter and the second promoter can have the same sequence. In other examples, the first promoter and the second promoter have different sequences.

[0115] In some embodiments, the first promoter and / or the second promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the CMV promoter. In some embodiments, the first promoter and / or the second promoter comprises a CMV promoter. In some embodiments, the CMV promoter comprises the sequence of SEQ ID NO:6.

[0116] In some embodiments, the first promoter and / or the second promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to 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.

[0117] In some embodiments, the first promoter and / or the second promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to the EFS promoter. In some embodiments, the first promoter and / or the second promoter comprises an EFS promoter. In some embodiments, the EFS promoter comprises the sequence of SEQ ID NO:8.

[0118] In some embodiments, the first promoter and / or the second promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to a murine stem cell virus (MSCV) promoter. In some embodiments, the first promoter and / or the second promoter comprises an MSCV promoter. In some embodiments, the MSCV promoter comprises the sequence of SEQ ID NO:9.

[0119] In some embodiments, the first promoter and / or the second promoter comprises at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more sequence identity to a phosphoglycerate kinase (PGK) promoter. In some embodiments, the first promoter and / or the second promoter comprises a PGK promoter. In some embodiments, the PGK promoter comprises the sequence of SEQ ID NO: 10.

[0120] In some embodiments, the 5' to 3' retroviral construct 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 5' to 3' retroviral construct comprises a second promoter operably linked to a polynucleotide encoding a PEBL and a first promoter operably linked to a polynucleotide encoding a CAR.

[0121] 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 an EFS promoter and the second promoter is an MSCV promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is an EF1α promoter. In some embodiments, the first. In some embodiments, the first promoter is an EFS promoter and the second promoter is a PGK promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is a CMV promoter. In some embodiments, the first promoter is an EFS promoter and the second promoter is an EFS promoter.

[0122] In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 17. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 18. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 18. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 19. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 20. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 20. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 21. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence of SEQ ID NO: 21. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 22. In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence of SEQ ID NO: 22.In some embodiments, a retroviral construct of the invention comprises a nucleic acid sequence having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or more, identity to SEQ ID NO: 23. In some embodiments, a retroviral construct of the invention comprises the nucleic acid sequence of SEQ ID NO:23. Antibodies that bind to CD7 In certain embodiments, an 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 amino acid sequences each 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% sequence 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. The heavy chain variable region may comprise 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 of SEQ ID NO: 32. The light chain variable region may comprise at least 90% sequence identity to the VL sequence of SEQ ID NO: 33. The heavy chain variable region may comprise 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 some examples, 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 certain examples, the heavy chain variable region comprises 10 or fewer amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the sequence set forth in SEQ ID NO: 32. In some examples, 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 particular examples, the light chain variable region comprises 10 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions in the sequence set forth in SEQ ID NO: 33. Any of the amino acid substitutions described herein may be conservative or non-conservative substitutions.

[0123] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO: 44 (SASQGISNYLN), a VL CDR2 of SEQ ID NO: 45 (YTSSLHS), and a VL CDR3 of SEQ ID NO: 46 (QQYSKLPYT). In some embodiments, the anti-CD7 scFv comprises a VH CDR1 of SEQ ID NO: 47 (SYAMS), a VH CDR2 of SEQ ID NO: 48 (SISSGGFTYYPDSVKG), and a VH CDR3 of SEQ ID NO: 49 (DEVRGYLDV). The 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.

[0124] In some embodiments, the nucleic acid sequence encoding the VH comprises 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 other embodiments, the nucleic acid sequence encoding the VL comprises 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.

[0125] 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) each having a 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% sequence 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. The heavy chain variable region may comprise 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 of SEQ ID NO: 34. The light chain variable region may comprise at least 90% sequence identity, at least The heavy chain variable region may comprise 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 some examples, 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 certain examples, the heavy chain variable region comprises 10 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 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 certain cases, the heavy chain variable region comprises 10 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions in the sequence set forth in SEQ ID NO: 35. Any of the amino acid substitutions described herein may be conservative or non-conservative substitutions.

[0126] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO:50 (RASQSISNNLH), a VL CDR2 of SEQ ID NO:51 (SASQSIS), and a VL CDR3 of SEQ ID NO:52 (QQSNSWPYT). In some embodiments, the anti-CD7 scFv comprises a VH CDR1 of SEQ ID NO:53 (SYWMH), a VH CDR2 of SEQ ID NO:54 (KINPSNGRTNYNEKFKS), and a VH CDR3 of SEQ ID NO:55 (GGVYYDLYYYALDY). 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 VL CDR3 of SEQ ID NO:52, a VH CDR1 of SEQ ID NO:53, a VH CDR3 of SEQ ID NO:54. CDR2, and a VH CDR3 of SEQ ID NO:55.

[0127] In some embodiments, the nucleic acid sequence encoding the VH comprises 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 other embodiments, the nucleic acid sequence encoding the VL comprises 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.

[0128] 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) each having a 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% sequence 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. The heavy chain variable region may comprise 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 of SEQ ID NO: 36. The light chain variable region may comprise at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 37. The heavy chain variable region may comprise at least one amino acid substitution (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the sequence set forth in SEQ ID NO: 36. In some examples, the heavy chain variable region comprises at least one amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the sequence set forth in SEQ ID NO: 36. In some cases, the light chain variable region comprises at least one amino acid substitution (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the sequence set forth in SEQ ID NO: 37. In certain cases, the heavy chain variable region comprises five or fewer amino acid substitutions (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) in the sequence set forth in SEQ ID NO: 37. Any of the amino acid substitutions described herein may be conservative or non-conservative substitutions.

[0129] In some embodiments, the anti-CD7 scFv comprises a VL CDR1 of SEQ ID NO: 56 (RASKSVSASGYSYMH), a VL CDR2 of SEQ ID NO: 57 (LASNLES), and a VL CDR3 of SEQ ID NO: 58 (QHSRELPYT). In some embodiments, the anti-CD7 scFv comprises a VH CDR1 of SEQ ID NO: 59 (SFGMH), a VH CDR2 of SEQ ID NO: 60 (YISSGSSTLHYADTVKG), and a VH CDR3 of SEQ ID NO: 61 (WGNYPHYAMDY). 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.

[0130] In some embodiments, the nucleic acid sequence encoding the VH comprises 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 nucleic acid sequence set forth in SEQ ID NO:42. In other embodiments, the nucleic acid sequence encoding the VL comprises 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.

[0131] In some embodiments, an scFv of the invention comprises 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 invention comprises 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 a CD7 antibody. For example, an anti-CD7 antibody can be any one recognized by one of skill in the art.

[0132] [Table 3]

[0133] [Table 4]

[0134] CD7 PEBL-mediated downregulation of intracellular CD7 As described herein, it has been shown that the cytotoxicity of T cells is significantly increased when anti-CD7 CARs are used in combination with downregulation of CD7 expression on effector T cells. As shown herein, downregulation of CD7 (e.g., elimination, reduction, and / or relocalization) prevents the fratricide effect exerted by the corresponding anti-CD7 CAR, allowing greater T cell recovery after CAR expression and more effective cytotoxicity against T leukemia / lymphoma cells compared to cells that retain the target antigen (e.g., CD7). As will be understood by those skilled in the art, downregulation of CD7 expression on effector T cells can be achieved according to various known methods, including, for example, protein expression blocker (PEBL) against CD7 (described in WO2016 / 126213), RNAi against CD7, or gene editing methods, such as, for example, meganucleases, TALEN, CRISPR / Cas9, zinc finger nucleases, etc. In the present invention, PEBL is described that binds to a target antigen and sequesters the target antigen in the cytoplasm of the cell. The target antigen is synthesized and binds to PEBL intracellularly.

[0135] In certain embodiments, provided herein is a polynucleotide comprising a nucleic acid sequence encoding PEBL comprising a target binding molecule (e.g., a CD7 antigen binding domain) linked to a localization domain. In some examples, 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, 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 shown in Figure 3E and Figure 17 of US2018 / 0179280, which is incorporated herein by reference.

[0136] As used herein, "linked" in the context of protein expression blockers refers to a gene encoding a target binding molecule directly (e.g., without a linker) in frame 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 via a linker sequence, for example, as described in WO2016 / 126213. As will be understood by those skilled in the art, such linker sequences and variants of such linker sequences are known in the art. Methods for designing constructs incorporating linker sequences and methods for evaluating functionality are readily available to those skilled in the art.

[0137] In some embodiments, the localization domain of PEBL comprises an endoplasmic reticulum (ER) or Golgi retention sequence, or a proteasome localization sequence. In certain embodiments, the localization domain comprises an endoplasmic reticulum (ER) retention peptide of Table 5. In certain embodiments, the localization domain comprises a proteasome localization sequence shown in Table 5. The localization domain can target PEBL to a specific intracellular compartment, such as the Golgi or endoplasmic reticulum, the proteasome, or the plasma membrane, depending on the application.

[0138] In some embodiments, proteasomal localization is achieved by linking the scFv sequence to a tripartite motif containing 21 (TRIM21) targeting domain sequence and co-expressing a sequence encoding the human TRIM21 E3 ubiquitin ligase protein. TRIM21 can bind with high affinity to the Fc domain of an antibody and recruit the ubiquitin-proteosome complex to degrade molecules (e.g., proteins and peptides) bound to the antibody. The TRIM21 targeting domain sequence encodes an amino acid sequence selected from a group of human immunoglobulin G (IgG) constant region (Fc) genes, such as IgG1, IgG2, or IgG4, and is used to form a fusion protein comprising the scFv and Fc domains. In this embodiment, the exogenously expressed TRIM21 protein is , binds to an scFv-Fc fusion protein bound to a target protein (e.g., CD7) and targets the complex to the proteasome for degradation.

[0139] Details of the amino acid sequence of the human TRIM21 E3 ligase protein can be found in the NCBI protein database, for example under NCBI reference sequence NP_003132.2. Details of the nucleic acid sequence encoding the human TRIM21 E3 ligase protein can be found in the NCBI protein database, for example under NCBI reference sequence NM_003141.3.

[0140] In some embodiments, PEBL also comprises hinge and transmembrane domain sequences from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In some embodiments, PEBL comprises the hinge and transmembrane domains of CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ ... The hinge and transmembrane domain of the TCRα polypeptide comprises a hinge and transmembrane domain selected from the group consisting of a CD3ε hinge and transmembrane domain, a CD3γ hinge and transmembrane domain, a CD3δ hinge and transmembrane domain, a TCRα hinge and transmembrane domain, a CD32 hinge and transmembrane domain, a CD64 hinge and transmembrane domain, a VEGFR2 hinge and transmembrane domain, a FAS hinge and transmembrane domain, and a FGFR2B hinge and transmembrane domain.

[0141] In certain embodiments, the PEBL comprises one or more components shown in Table 5.

[0142] [Table 5]

[0143] 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 is (G4S) n linker, wherein n is 1 to 6, e.g. For example, the number of sequences may range from 1, 2, 3, 4, 5, or 6. In one embodiment, the CD7 PEBL comprises an amino acid sequence of SEQ ID NO:32, an amino acid sequence of SEQ ID NO:33, and an 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:32, an amino acid sequence of SEQ ID NO:33, and an amino acid sequence of SEQ ID NO:79. In certain embodiments, the CD7 PEBL comprises an 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 an amino acid sequence of SEQ ID NO:79. In other embodiments, the anti-CD7 protein expression blocker 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.

[0144] 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 is (G4S) nlinker, where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, CD7 PEBL comprises an amino acid sequence of SEQ ID NO: 34, an amino acid sequence of SEQ ID NO: 35, and an amino acid sequence of SEQ ID NO: 79. In some embodiments, 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, CD7 PEBL comprises an 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, 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.

[0145] 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 is (G4S) nlinker, where n can range from 1 to 5, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, CD7 PEBL comprises an amino acid sequence of SEQ ID NO:36, an amino acid sequence of SEQ ID NO:37, and an amino acid sequence of SEQ ID NO:79. In some embodiments, 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 of SEQ ID NO:37, and an amino acid sequence of SEQ ID NO:79. In certain embodiments, CD7 PEBL comprises an amino acid sequence of 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. In other embodiments, 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.

[0146] In some instances, the CD7 PEBL also comprises a localization domain selected from any one of the sequences set forth in SEQ ID NOs: 72-77. In some instances, the CD7 PEBL also comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide set forth in SEQ ID NO: 80. In other instances, the anti-CD7 protein expression blocker also 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.

[0147] In one embodiment, CD7 PEBL encoded by the bicistronic vector described herein comprises a sequence of SEQ ID NO:24 and a proline at the N-terminus. In some embodiments, CD7 PEBL comprises a sequence of SEQ ID NO:25. The N-terminal proline residue results from 2A cleavage. In some embodiments, CD7 PEBL encoded by the bicistronic vector described herein comprises a sequence of SEQ ID NO:26 and a proline at the N-terminus. In some embodiments, CD7 PEBL comprises a sequence of SEQ ID NO:27.

[0148] In some embodiments, the engineered immune cells of the invention comprise CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises a sequence of SEQ ID NO:24 and a proline or a sequence of SEQ ID NO:25 at the N-terminus. In some embodiments, the engineered immune cells are CD4+ T cells comprising CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises a sequence of SEQ ID NO:24 and a proline or a sequence of SEQ ID NO:25 at the N-terminus. In some embodiments, the engineered immune cells are CD8+ T cells comprising CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises a sequence of SEQ ID NO:24 and a proline or a sequence of SEQ ID NO:25 at the N-terminus. In some embodiments, the engineered immune cells are CD3+ T cells comprising CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises a sequence of SEQ ID NO:24 and a proline or a sequence of SEQ ID NO:25 at the N-terminus.

[0149] In some embodiments, the engineered immune cells of the invention comprise CD7 PEBL encoded by a bicistronic vector such that CD7 PEBL comprises a sequence of SEQ ID NO:26 and a proline or a sequence of SEQ ID NO:27 at the N-terminus. In some embodiments, the engineered immune cells are CD4+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises a sequence of SEQ ID NO:26 and a proline or a sequence of SEQ ID NO:27 at the N-terminus. In some embodiments, the engineered immune cells are CD8+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises a sequence of SEQ ID NO:26 and a proline or a sequence of SEQ ID NO:27 at the N-terminus. In some embodiments, the engineered immune cells are CD3+ T cells comprising CD7 PEBL encoded by a bicistronic vector, wherein CD7 PEBL comprises a sequence of SEQ ID NO:26 and a proline or a sequence of SEQ ID NO:27 at the N-terminus.

[0150] 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 comprises 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 comprises at least 90% sequence identity to SEQ ID NO: 24. PEBL binds to CD7 and contains at least 90% sequence identity to SEQ ID NO:26.

[0151] In some embodiments, the polynucleotide encoding CD7 PEBL comprises one or more of the nucleic acid sequences shown in Table 6.

[0152] 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 a specific embodiment, the PEBL anti-CD7 The VH domain of the scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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.

[0153] In some embodiments, the VH domain of the PEBL anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO: 40, and the VL domain of the PEBL anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO: 41. The VH domain of the scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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.

[0154] In some embodiments, the VH domain of the PEBL anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO: 42, and the VL domain of the PEBL anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO: 43. The VH domain of the scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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 PEBL anti-CD7 scFv comprises a nucleotide sequence having at least 90% sequence identity (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.

[0155] [Table 6]

[0156] In certain embodiments of the invention, PEBL is capable of binding 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.

[0157] 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.

[0158] 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.

[0159] In some embodiments, the engineered immune cells of the invention comprise 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 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 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 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. A CD3+ T cell comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of PEBL. Also provided herein are populations comprising such cells.

[0160] In some embodiments, the engineered immune cells of the invention comprise 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 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 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 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. A CD3+ T cell comprising a CD7 PEBL encoded by a bicistronic construct comprising a nucleic acid sequence of PEBL. Also provided herein are populations comprising such cells. Chimeric antigen receptor that binds CD7 In some embodiments, the CAR of the present invention comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antigen binding domain (e.g., single chain variable fragment or scFv) that specifically binds to CD7. The CD7 CAR of the present invention may be referred to herein as "anti-CD7-41BB-CD3ζ". In some embodiments, the CAR also comprises a CD8α hinge domain and a transmembrane domain, such as, but not limited to, the amino acid sequence of SEQ ID NO:84.

[0161] As one of skill in the art will appreciate, in certain embodiments, any of the amino acid sequences of the various components (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 the specific corresponding sequences disclosed herein. For example, in certain embodiments, the intracellular signaling domain 4-1BB 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: 85, so 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.

[0162] As another example, in certain embodiments, the intracellular signaling domain 4-1BB may be replaced with 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 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, at least 100% sequence identity, at least 101% sequence identity, at least 102% sequence identity, at least 103% sequence identity, at least 104% sequence identity, at least 105% sequence identity, at least 106% sequence identity, at least 107% sequence identity, at least 108% sequence identity, at least 109% sequence identity, at least 110% sequence identity, at least 112% sequence identity, at least 113% sequence identity, at least 114% sequence identity, at least 115% sequence identity, at least 116% sequence identity, at least 117% sequence identity, at least 118% sequence identity, at least 119% sequence identity, at least 120% sequence identity, at least 121% sequence identity, at least 122% sequence identity, at least 123% sequence identity, at least 124% sequence identity, at least 125% sequence identity, at least 126% sequence identity, at least 127% sequence identity, at least 128% sequence identity, at least 129% sequence identity, at least 130% sequence identity, at least 131% sequence identity, at least 132% sequence identity, at least 133% sequence identity, at least It may have 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.

[0163] As another example, in certain instances, the intracellular signaling domain of 4-1BB 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 to the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. In other embodiments, the additional intracellular signaling domain comprises 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 one or more intracellular signaling domain fragments of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2.

[0164] As another example, in certain embodiments, the intracellular signaling domain CD3zeta 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 to SEQ ID NO: 86, so long as it has the desired function. In certain embodiments, the intracellular signaling domain of CD3zeta comprises the amino acid sequence set forth in SEQ ID NO: 86.

[0165] In some instances, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or a portion thereof, so long as it has the desired function. The intracellular signaling domain of a CAR can comprise a 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% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the ITAM. In certain embodiments, the intracellular signaling domain can 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 to FcεRIγ, CD4, CD7, CD8, CD28, OX40, or H2-Kb, so long as it has the desired function.

[0166] In certain embodiments, the anti-CD7 CAR further comprises a hinge domain and / or a transmembrane domain. Hinge and transmembrane domains suitable for use in the present invention are known in the art and are provided, for example, in publication WO2016 / 126213, which is incorporated by reference in its entirety. In some embodiments, the hinge and transmembrane domain of the anti-CD7 CAR are selected from the group consisting of CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TC It comprises a signaling domain (e.g., hinge and transmembrane domains) from Ra, CD32, CD64, VEGFR2, FAS, FGFR2B, or another transmembrane protein.

[0167] In certain embodiments, the anti-CD7 CAR further comprises a CD8α signal peptide. A schematic diagram of an anti-CD7 CAR including embodiments described herein is shown in Figure 17 of US2018 / 0179280.

[0168] In some embodiments, the chimeric antigen receptor (CAR) can be bound to a molecule expressed on the surface of a cell, 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.

[0169] In certain embodiments, an isolated polynucleotide of the 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.

[0170] [Table 7]

[0171] 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 (G4S) n linkers, such as, but not limited to, VH-VL linkers, where n can range from 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 CD3ζ intracellular signaling domain.

[0172] 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 shown in Figures 42-45.

[0173] In some embodiments, the engineered immune cells of the invention comprise a CD7 CAR encoded by a bicistronic vector, such that the CD7 CAR is represented by SEQ ID NO: 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 generated by cleavage of the 2A autocleaving 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 generated by cleavage of the 2A autocleaving 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 generated by cleavage of the 2A autocleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO:29. In some embodiments, the engineered immune cells are CD3+ T cells that 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 generated by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 29. Also provided herein are populations comprising such cells.

[0174] In some embodiments, the engineered immune cells of the 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 generated by cleavage of the 2A autocleaving 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 generated by cleavage of the 2A autocleaving 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 generated by cleavage of the 2A autocleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. In some embodiments, the engineered immune cells are CD3+ T cells that 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 generated by cleavage of the 2A self-cleaving peptide, or the CD7 CAR comprises the sequence of SEQ ID NO: 31. Populations of such cells are also provided herein.

[0175] 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 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 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 comprise a CD7 CAR comprising 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 a 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 a 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 a sequence of SEQ ID NO: 30. Also provided herein are populations of such cells.

[0176] In certain embodiments, the isolated polynucleotide of the CD7 CAR of the 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 as shown in Table 8.

[0177] [Table 8]

[0178] 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 an intracellular signaling domain of 4-1BB, and an intracellular signaling domain of CD3ζ. In certain embodiments, the polynucleotide also comprises a nucleic acid sequence of a CD8 signal peptide.

[0179] In certain embodiments, the antigen-binding domain is an anti-CD7 scFv. In some embodiments, the VH sequence of the scFv comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO: 38, and the VL sequence comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO: 39.

[0180] In some embodiments, the VH sequence of the scFv is a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO:40. and the VL sequence comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO: 41. In some embodiments, the VH sequence of the scFv comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO: 42, and the VL sequence comprises a nucleic acid sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the sequence of SEQ ID NO: 43.

[0181] In some embodiments, a polynucleotide encoding a CD7 CAR comprises, from the 5' to the 3' end, the nucleic acid sequence of an antigen binding domain that binds to CD7, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98. In some embodiments, a polynucleotide encoding a CD7 CAR comprises, from the 5' to the 3' end, SEQ ID NO: 81, the nucleic acid sequence of an antigen binding domain that binds to CD7, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0182] In some embodiments, the CD7 CAR 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:4 and binds to CD7. In some embodiments, the CD7 CAR 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:4.

[0183] In some embodiments, the CD7 CAR 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:5 and binds to CD7. In some embodiments, the CD7 CAR comprises 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:5.

[0184] In some embodiments, the engineered immune cells of the invention comprise a CD7 CAR encoded by a bicistronic construct or a dual promoter construct comprising a nucleic acid sequence of CD7 CAR having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) 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 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 CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the engineered immune cells are CD3+ T cells that contain CD7 PEBL encoded by a bicistronic construct that contains a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO:4. Also provided herein are populations comprising such cells.

[0185] In some embodiments, the engineered immune cells of the invention comprise a CD7 CAR encoded by a bicistronic construct or dual promoter construct comprising a nucleic acid sequence of CD7 CAR having at least 90% (e.g., 90%, 91%, 92%, 94%, 94%, 95%, 96%, 97%, 98%, 99% or more) 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 CD7 PEBL having at least 90% sequence identity to SEQ ID NO:5. 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:5. In some embodiments, the engineered immune cells are CD3+ T cells that comprise CD7 PEBL encoded by a bicistronic construct that comprises a nucleic acid sequence of CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 5. Also provided herein are populations that comprise such cells. Engineered immune cells expressing bicistronic vectors In certain embodiments, an engineered immune cell is provided that comprises a bicistronic construct, as exemplified herein, comprising: (i) a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular signaling domain of 4-1BB and CD3ζ, and an antigen binding domain that specifically binds CD7; (ii) a polynucleotide encoding a target binding molecule linked 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; and (iii) a nucleic acid sequence encoding a 2A self-cleaving peptide or an IRES sequence.

[0186] In certain embodiments, in the context of a CAR, and in the context of an antigen binding domain for CD7, an antigen binding domain that binds to CD7 comprises a VH sequence set forth in SEQ ID NO: 32 and a VL sequence set forth in SEQ ID NO: 33, a VH sequence set forth in SEQ ID NO: 34 and a VL sequence set forth in SEQ ID NO: 35, or a VH sequence set forth in SEQ ID NO: 36 and a 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 having sequences that each comprise 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 and VL sequences set forth in SEQ ID NOs: 32 and 33, respectively, SEQ ID NOs: 34 and 35, respectively, or SEQ ID NOs: 36 and 37, respectively. In certain embodiments, the antigen binding domain that binds to CD7 in the context of a CAR may be different from an antibody that binds to CD7 in the context of a target binding molecule (Protein Expression Blocker or PEBL), as described herein.

[0187] In some embodiments, an engineered immune cell comprising a bicistronic construct comprising a nucleic acid construct comprises, from the 5' end to the 3' end, a polynucleotide encoding a target binding molecule linked to a localization domain (e.g., CD7 PEBL), where the target binding molecule binds to CD7, an IRES sequence, and a polynucleotide encoding a chimeric antigen receptor for CD7 (e.g., CD7 CAR). In some examples, the engineered immune cell comprises a nucleic acid construct comprising SEQ ID NO: 11. In some embodiments, provided herein is a nucleic acid construct comprising SEQ ID NO: 11. In other embodiments, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:11. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:11.

[0188] In some embodiments, the engineered immune cell comprising a bicistronic construct comprising a nucleic acid construct comprises, 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 linked to a localization domain, where the target binding molecule binds to CD7 (e.g., PEBL for CD7). In some examples, the engineered immune cell comprises a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 12 or FIG. 26. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 12 or FIG. 26. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 12 or FIG. 26. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 12 or FIG. 26.

[0189] In some embodiments, the engineered immune cell comprising a bicistronic construct comprising a nucleic acid construct comprises, from the 5' end to the 3' end, a polynucleotide encoding a chimeric antigen receptor for CD7 (e.g., CD7 CAR), a nucleic acid sequence encoding a 2A self-cleaving peptide, and a polynucleotide encoding a target binding molecule linked to a localization domain where the target binding molecule binds to CD7 (e.g., CD7 PEBL). In some examples, the engineered immune cell comprises a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 13 or FIG. 27. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 13 or FIG. 27. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 13 or FIG. 27. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 13 or FIG. 27.

[0190] In some embodiments, the engineered immune cells comprising a bicistronic construct comprising a nucleic acid construct comprises, from its 5' to 3' end, a polynucleotide encoding a target binding molecule linked to a localization domain, where the target binding molecule binds to CD7, a nucleic acid sequence encoding a 2A self-cleaving peptide, and a polynucleotide encoding a chimeric antigen receptor for CD7.

[0191] In some embodiments, the engineered immune cells comprising a bicistronic construct comprising a nucleic acid construct comprise, 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 2A self-cleaving peptide, and a polynucleotide encoding a target binding molecule linked to a localization domain, where the target binding molecule binds to CD7 (e.g., CD7 PEBL). In some examples, the engineered immune cells comprise a nucleic acid construct comprising at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 14-16. In some examples, the engineered immune cells comprise a nucleic acid construct comprising any one of the nucleic acid sequences of SEQ ID NOs: 14-16. In some embodiments, the engineered immune cells comprise a nucleic acid construct that comprises 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 cells comprise a nucleic acid construct that comprises 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 cells comprise a nucleic acid construct that comprises 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.

[0192] 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 2A self-cleaving peptide, and a polynucleotide encoding a target binding molecule linked to a localization domain, wherein the target binding molecule binds to CD7. In some examples, the promoter is selected from an MSCV promoter, a PGK promoter, an EF1α promoter, and an EFS promoter. In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 14 or a sequence depicted in Figures 28A-28B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or a sequence depicted in Figures 28A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or a sequence depicted in Figures 28A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 14 or the sequence shown in Figures 28A-B.

[0193] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 15. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or a sequence depicted in Figures 29A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or a sequence depicted in Figures 29A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or a sequence depicted in Figures 29A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 15 or a sequence depicted in Figures 29A-B.

[0194] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 16 or a sequence depicted in Figures 30A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or a sequence depicted in Figures 30A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or a sequence depicted in Figures 30A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 16 or a sequence depicted in Figures 30A-B.

[0195] In some embodiments, the engineered immune cells or populations thereof described herein comprise at least 10% CD7 CAR+ / CD7 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% C D7 CAR+ / CD7 negative T cells, at least 50% of CD7 CAR+ / CD7 negative T cells, at least 55% of CD7 CAR+ / CD7 negative T cells, at least 60% of CD7 CAR+ / CD7 negative T cells, at least 65% of CD7 CAR+ / CD7 negative T cells, at least 70% of CD7 CAR+ / CD7 negative T cells, at least 75% of CD7 CAR+ / CD7 negative T cells, at least 80% of CD7 CAR+ / CD7 negative T cells, at least 85% of CD7 CAR+ / CD7 negative T cells, at least 90% of CD7 CAR+ / CD7 negative T cells, at least 95% of CD7 CAR+ / CD7 negative T cells, at least 96% of CD7 CAR+ / CD7 negative T cells, at least 97% of CD7 CAR+ / CD7 negative T cells, at least 98% of CD7 CAR+ / CD7 negative T cells, at least 99% of CD7 CAR+ / CD7 negative T cells, or 100% CD7 CAR+ / CD7 negative T cells. In some embodiments, the engineered immune cells outlined herein comprise a population of substantially purified CD7 CAR / CD7 negative T cells, such cells expressing any one of the bicistronic constructs described. Engineered immune cells expressing dual promoter vectors In some embodiments, an engineered immune cell is provided that comprises a recombinant retroviral vector comprising (a) a first promoter operably linked to a first polynucleotide encoding any of the CARs described herein, and (b) a second promoter operably linked to a second polynucleotide encoding any of the PEBLs described herein. In some embodiments, the engineered immune cell comprises any of the recombinant retroviral vectors described herein that comprise a promoter driving CAR expression and another promoter driving PEBL expression.

[0196] In some embodiments, the engineered immune cells comprise 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 cells comprise 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 cells comprise 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 cells comprise 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 cells comprise 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 cells comprise 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 cells comprise 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 examples, the engineered immune cells are engineered CD4+ T cells or a population thereof or a population comprising thereof. In some examples, the engineered immune cells are engineered CD8+ T cells or a population thereof or a population comprising thereof.

[0197] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 17 or a sequence depicted in Figures 31A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or a sequence depicted in Figures 31A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or a sequence depicted in Figures 31A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 17 or a sequence depicted in Figures 31A-B.

[0198] In some examples, 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 a sequence depicted in Figures 32A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or a sequence depicted in Figures 32A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 18 or a sequence depicted in Figures 32A-B.

[0199] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO: 19 or a sequence depicted in Figures 33A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or a sequence depicted in Figures 33A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or a sequence depicted in Figures 33A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO: 19 or a sequence depicted in Figures 33A-B.

[0200] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO:20 or a sequence depicted in Figures 34A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:20 or a sequence depicted in Figures 34A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:20 or a sequence depicted in Figures 34A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:20 or a sequence depicted in Figures 34A-B.

[0201] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO:21 or a sequence depicted in Figures 35A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:21 or a sequence depicted in Figures 35A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:21 or a sequence depicted in Figures 35A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:21 or a sequence depicted in Figures 35A-B.

[0202] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO:22 or a sequence depicted in Figures 36A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:22 or a sequence depicted in Figures 36A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:22 or a sequence depicted in Figures 36A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:22 or a sequence depicted in Figures 36A-B.

[0203] In some examples, the engineered immune cell comprises a polynucleotide comprising SEQ ID NO:23 or a sequence depicted in Figures 37A-B. In one embodiment, provided herein is an engineered CD4+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:23 or a sequence depicted in Figures 37A-B. In some embodiments, provided herein is an engineered CD8+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:23 or a sequence depicted in Figures 37A-B. In some embodiments, provided herein is an engineered CD3+ T cell or population thereof comprising a nucleic acid construct comprising SEQ ID NO:23 or a sequence depicted in Figures 37A-B.

[0204] In some embodiments, the engineered immune cells or populations thereof described herein comprise at least 10% CD7 CAR+ / CD7 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% CD7 CAR+ / CD7 negative T cells, at least 90% CD7 CAR+ / CD7 negative T cells, at least 95% CD7 CAR+ / CD7 negative T cells, at least 96% CD7 CAR+ / CD7 negative T cells, at least 97% CD7 CAR+ / CD7 negative T cells, at least 98% CD7 CAR+ / CD7 negative T cells, at least 99% CD7 CAR+ / CD7 negative T cells, or 100% CD7 CAR+ / CD7 negative T cells. In some embodiments, the engineered immune cells outlined herein comprise a substantially purified population of CD7 CAR / CD7 negative T cells, such cells expressing any one of the described dual promoter constructs. CD7 CAR+ engineered immune cells with reduced expression of endogenous CD7 In some embodiments, the engineered immune cells described herein express CD7 CAR and have reduced 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, the reduced expression of CD7 refers to the downregulation or partial downregulation of surface CD7 by cells. In some cases, reduced expression includes a decrease in expression level of at least 5% (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% or more) compared to the expression level of a wild type or non-engineered cell. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction. In some embodiments, the engineered immune cells outlined herein comprise a substantially purified population of CD7 CAR / CD7 negative T cells.

[0205] In some embodiments, the engineered immune cells described herein express CD7 PEBL having at least 90% sequence identity to SEQ ID NO:24. In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO:25. In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO:26. In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO:27.

[0206] 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.

[0207] 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.

[0208] In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 25 and express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0209] 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.

[0210] In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the engineered immune cells express CD7 PEBL having at least 90% sequence identity to SEQ ID NO: 27 and express a CD7 CAR having at least 90% sequence identity to SEQ ID NO: 31.

[0211] In certain embodiments, the engineered immune cells are engineered T cells, engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages, engineered leukocytes ... 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. Also provided is a population of any one of the engineered cells described herein.

[0212] In some embodiments, provided herein are populations of engineered immune cells (e.g., CD3+ T cells, CD4+ T cells, or CD8+ T cells) that comprise 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) CD7 CAR positive cells, endogenous CD7 negative cells. In some embodiments, provided herein are populations of engineered immune cells that comprise 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) CD7 CAR positive cells, endogenous CD7 negative CD4+ T cells. In some embodiments, provided herein are populations of engineered immune cells that comprise 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) CD7 CAR positive cells, endogenous CD7 negative CD8+ T cells. Such populations of cells can be produced from peripheral blood mononuclear cells (PBMCs), purified CD4+ T cells, purified CD8+ T cells, or populations comprising purified CD4+ T cells and purified CD8+ T cells.

[0213] In some embodiments, the engineered immune cells described herein are cultured to generate a highly pure population of CD7 CAR-T cells with reduced expression of endogenous CD7. The level of purity 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 level 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.

[0214] In some embodiments, the engineered immune cells of the present invention comprise CD45RO+ cells. In some embodiments, the engineered immune cells comprise CCR7- cells. In some embodiments, the engineered immune cells comprise central memory T cells. In some embodiments, the engineered immune cells comprise effector memory T cells. In some embodiments, the engineered immune cells comprise effector T cells. In some embodiments, the engineered immune cells comprise naive T cells.

[0215] In some examples, the population of engineered immune cells includes effector memory T cells, central memory T cells, effector T cells, and naive T cells. In some embodiments, the population of engineered immune cells includes 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 population of engineered immune cells includes about 40% or more effector memory T cells.

[0216] In some examples, the population of engineered immune cells comprises PD1 negative cells. In some examples, the population of engineered immune cells 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.

[0217] 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 CD7-expressing cancer cells.

[0218] In some embodiments, the cells described herein containing the bicistronic expression vector can be used to generate a population of CD7 CAR+ / CD7-negative T cells. The CD7 CAR+ / CD7-negative T cells can be expanded and enriched over time. The CD7 CAR+ / CD7-negative T cells can be generated from cells including, but not limited to, bulk PBMCs, purified T cells including CD4+ T cells and CD8+ T cells, and purified CD3+ T cells. The CD7 CAR+ / CD7-negative T cells can be generated from T EM cell, T CM The antibodies can be used to generate various subsets of T cells, including T cells, Teff cells, and naive T cells.

[0219] In another aspect, methods are also provided for producing engineered immune cells (e.g., engineered CD3+ T cells, engineered CD4+ T cells, and engineered CD8+ T cells) having any of the embodiments described herein, comprising introducing any of the bicistronic or dual promoter constructs of the invention into immune cells. In some embodiments, the engineered immune cells are derived from immune cells obtained from a subject receiving the engineered immune cells as a therapy. In some embodiments, the engineered immune cells are derived from immune cells obtained from a donor, and the resulting engineered immune cells are administered to the subject as a therapy.

[0220] In various aspects, a kit is also provided for producing the engineered immune cells described herein.The kit can be used, for example, to produce allogeneic or autologous T cells with anti-CD7 CAR-mediated cytotoxic activity.In some embodiments, the kit is useful for producing allogeneic effector T cells with anti-CD7 CAR-mediated cytotoxic activity.In certain embodiments, the kit is useful for producing autologous effector T cells with anti-CD7 CAR-mediated cytotoxic activity.

[0221] Thus, provided herein is a kit comprising any one of the bicistronic or dual promoter constructs described herein.

[0222] In certain embodiments, the bicistronic construct further comprises sequences (e.g., plasmid or vector sequences) that allow, for example, cloning and / or expression. For example, the nucleotide sequence may 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 (immune cells).

[0223] Typically, the kits are compartmentalized for ease of use and may include one or more containers containing reagents. In certain embodiments, all of the kit components are packaged together. Alternatively, one or more individual components of the kit may be provided in a package separate from the other kit components. The kits may also include instructions for using the kit components. It can be rare. Administration of engineered immune cells In other aspects, methods of treating cancer in a subject in need thereof are also provided, comprising administering to the subject a therapeutic amount of engineered immune cells having any of the embodiments described herein, thereby treating the cancer in the subject in need thereof.

[0224] In certain embodiments, the method comprises administering a therapeutic amount of engineered immune cells comprising a bicistronic viral construct comprising a polynucleotide comprising a nucleic acid sequence encoding a CAR and a polynucleotide comprising a nucleic acid sequence encoding a PEBL. In various embodiments, the method comprises administering a therapeutic amount of any one of the engineered immune cells described herein comprising a recombinant retroviral vector comprising (a) a first promoter operably linked to a first polynucleotide encoding a CD7 chimeric antigen receptor (CD7 CAR) as outlined herein, and (b) a second promoter operably linked to a second polynucleotide encoding a CD7 protein expression blocker (CD7 PEBL) as outlined herein.

[0225] In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO:11 or FIG. 25 is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO:12 or FIG. 26 is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO:13 or FIG. 27 is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or a population thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 14 or Figures 28A-28B is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or a population thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 15 or Figures 29A-29B is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or a population thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising a sequence as shown in SEQ ID NO: 16 or Figures 30A-30B is administered to a subject with cancer. In some embodiments, a therapeutic amount of an engineered immune cell or population thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising SEQ ID NO:17 or the sequence shown in Figures 31A-31B is administered to a subject with cancer.In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising SEQ ID NO: 18 or the sequence depicted in Figures 32A-B is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising SEQ ID NO: 19 or the sequence depicted in Figures 33A-B is administered to a subject with cancer. In some embodiments, a therapeutic amount of engineered immune cells or populations thereof (e.g., engineered CD3+ T cells, engineered CD4+ T cells, or engineered CD8+ T cells) comprising a nucleic acid construct comprising SEQ ID NO: 20 or the sequence depicted in Figures 34A-B is administered to a subject with cancer. In some embodiments, SEQ ID NO: 21. or a nucleic acid construct comprising a sequence as shown in Figures 35A-B is administered to a subject with cancer. 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 a sequence as shown in SEQ ID NO:22 or Figures 36A-B is administered to a subject with cancer. 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 a sequence as shown in SEQ ID NO:23 or Figures 37A-B is administered to a subject with cancer.

[0226] 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 CD7 PEBL of SEQ ID NO: 25 is administered to a subject with 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 CD7 PEBL of SEQ ID NO: 27 is administered to a subject with cancer.

[0227] 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 with 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 with cancer.

[0228] 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 and a CD7 CAR of SEQ ID NO: 29 is administered to a subject with 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 and a CD7 CAR of SEQ ID NO: 29 is administered to a subject with cancer.

[0229] 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 and a CD7 CAR of SEQ ID NO: 31 is administered to a subject with 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 and a CD7 CAR of SEQ ID NO: 31 is administered to a subject with cancer.

[0230] 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) is administered to a subject with cancer, wherein the engineered immune cells comprise SEQ ID NO:95.

[0231] In certain embodiments, the cancer is a T-cell malignancy, e.g., a T-cell leukemia or T-cell lymphoma, e.g., T-cell acute lymphoblastic leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous gamma delta T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, angioimmunoblastic T-cell lymphoma, unspecified mycosis fungoides, pulmonary leukemia ... In certain embodiments, the T-cell malignancy is early T-cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0232] In some embodiments, the engineered immune cells are autologous to the subject in need of treatment, e.g., cancer treatment. In other embodiments, the engineered immune cells are allogeneic to the subject in need of treatment.

[0233] In certain embodiments, the engineered immune cells are administered to the subject by intravenous infusion, intra-arterial infusion, direct injection into the tumor and / or perfusion of the tumor matrix following surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration, and intraocular administration.

[0234] In certain embodiments, the engineered immune cells are administered by injection into a subject. Methods for injecting immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient to ameliorate disease symptoms. Typically, within 10 7 ~10 10 Dosage of cells, e.g., 10 9 A dose of 10 cells is injected in one injection. The injection is performed in one 9 as a cell dose or in multiple doses of 10 9 The cells are administered as a dose of cells. The frequency of injections can be daily, every 2-30 days, or at longer intervals as needed or required. The amount of injection is generally at least one injection per subject, and preferably at least three injections as tolerated or until the symptoms of the disease are improved. The cells can be injected intravenously at a rate of 50-250 ml / hour. Other suitable modes of administration include intra-arterial injection, intraperitoneal injection, direct injection into the tumor and / or perfusion of the tumor bed after surgery, implantation of an artificial scaffold at the tumor site, intrathecal administration. Methods for adapting the present invention to such delivery modes are readily available to those skilled in the art.

[0235] 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.

[0236] Also provided in another aspect is a use of an engineered immune cell having any of the embodiments described herein for treating cancer, comprising administering a therapeutic amount of the engineered immune cell to a subject in need of cancer treatment. In certain embodiments, the cancer is a T-cell malignancy. In certain embodiments, the T-cell malignancy is early stage T-cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0237] In certain embodiments, the engineered immune cells are administered to the subject by intravenous infusion, intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or perfusion of the tumor bed following surgery, implantation at the tumor site in an artificial scaffold, and intrathecal administration. EXAMPLES

[0238] Example 1: Use of a bicistronic expression vector to block CD7 expression in chimeric antigen receptor T cells This example shows blocking CD7 expression in anti-CD7 CAR-T cells using a bicistronic expression construct. method cell culture 293T cells (ATCC CRL-3216) were maintained in DMEM (Gibco) containing 10% FBS (Hyclone), 100 U / mL penicillin, and 100 ug / mL streptomycin (Gibco). Jurkat clone E6-1 cells (ATCC TIB-152) and NALM6 clone G5 cells (CRL-3273) were maintained in DMEM (Gibco) containing 10% FBS (Hyclone). The cells were maintained in RPMI1640 (Gibco) containing 1x S (Hyclone), 100 U / mL penicillin, 100 ug / mL streptomycin (Gibco) and 1x GlutaMAX (Gibco). Production of lentivirus 293T cells were co-transfected with lentiviral transfer vectors and Virapower packaging plasmid mix (Invitrogen) at a ratio of 1:3 using Lipofectamine 2000 (Invitrogen). The transfection medium was replaced with fresh DMEM (Gibco) containing 10% FBS (Hyclone) 6 h after transfection. After 48 h, the viral supernatant was collected, passed through a 0.45 μM filter, and then concentrated 100-fold using a Lenti-X Concentrator (Clontech). The concentrated lentiviral stock was stored at -150 °C until use. Retrovirus production 293T cells were co-transfected with retroviral transfer vectors and pEQ and pRDF packaging plasmids using X-tremeGENE 9 DNA transfection reagent (Roche). Transfection medium was replaced with fresh DMEM (Gibco) containing 10% FBS (Hyclone) 6 h post-transfection. After 24 and 48 h, viral supernatants were collected and passed through a 0.45 μM filter. Retroviruses were used fresh or stored at -150°C until use. Lentivirus titration in 293T cells 293T cells were transduced with different amounts of lentivirus in the presence of 5 μg / mL polybrene (Sigma). After 15 h overnight culture, the transduction medium was removed and cells were treated with 10 U / mL DNaseI (New England Biolabs) in fresh medium for 15 min at 37°C. The medium was then replaced with fresh DMEM containing 10% FBS for further culture. At > 72 h post-transduction, transduced cells were harvested for analysis. Viral titers were determined using flow cytometry and RT-qPCR.

[0239] Transducing unit (TU) titers were calculated from flow cytometry data using the equation: [TU / mL = (number of 293T cells per sample x %CAR+ cells) ÷ viral load (mL)]. TU titers were calculated using samples within the linear range of %CAR+ cells and viral load.

[0240] Integral unit (IU) titers were calculated from genomic DNA RT-qPCR data using the equation: [IU / ml = (number of 293T cells per sample x number of proviral gene copies per genome) ÷ viral load (mL)]. IU titers were calculated using samples within the linear range of proviral gene copy number and viral load. Lentiviral transduction of Jurkat cells Lentivirus was added directly to Jurkat cells with or without 8 μg / mL polybrene (Sigma). A complete medium change was performed after 2 days to remove lentivirus from the culture. Transduced cells were harvested for analysis ≥ 2 days after transduction. Primary T cell culture Frozen human primary peripheral blood mononuclear cells (PBMCs) (ATCC catalog number PCS-800 -011 and Stemcell Technologies catalog number 70025) were thawed, allowed to recover overnight and maintained at 1 million cells / mL in RPMI1640 (Gibco) containing 10% FBS (Hyclone), 100U / mL penicillin, 100ug / mL streptomycin (Gibco) and 1x GlutaMAX (Gibco), TexMACS medium (Miltenyi Biotec) supplemented with 3% human AB serum (Sigma), or serum-free TexMACS medium. Medium was supplemented with 120IU / mL interleukin-2 (Miltenyi Biotec) every 2-3 days.

[0241] PBMCs were either cultured in bulk without further selection or purified for T cells after overnight collection. CD4+ and CD8+ T cells were separated using CD4 Microbeads (Miltenyi Biotec) and CD8 Microbeads (Miltenyi Biotec). CD3+ T cells were separated using CD3 Microbeads (Miltenyi Biotec).

[0242] T cells were activated with either T Cell TransAct (Miltenyi Biotec) or Dynabeads Human T-Activator CD3 / CD28 (Gibco) for T cell expansion and activation according to the manufacturer's recommendations. Dynabeads were added at a bead-to-cell ratio of 1:1. Beads were depleted after 4 days. Lentiviral transduction of primary T cells Primary T cells were transduced 1–4 days after activation. Static transduction was performed, where lentivirus was added directly to T cells. A complete medium change was performed after 2 days to remove lentivirus from the cultures. Transduced cells were analyzed by flow cytometry ≥3 days after transduction. Retroviral transduction of primary T cells Retronectin-based retroviral transduction was performed on primary T cells. Untreated tissue culture plates were coated with 2.5 μg / cm2 RetroNectin Recombinant Human Fibronectin Fragment (Takara) according to the manufacturer's recommendations. Retroviral supernatant was added to the Retronectin-coated plates and centrifuged at 1000×g for 2 h at 32° C. The viral supernatant was then removed from the wells. Wells were rinsed with medium before adding T cells. Transduced cells were analyzed by flow cytometry ≥3 days after transduction. Flow cytometry Antibody staining and washing are performed with staining buffer (1xPBS pH 7.4, 0.2% BSA, 0.02% sodium azide). Cells are incubated with antibodies for 15 minutes on ice and washed three times. The following antibodies were used for staining: anti-mouse F(ab')2-biotin (Jackson Immunoresearch 115-066-072), streptavidin-APC (Jackson Immunoresearch 016-130-084), anti-human CD7-PE (BD 555361), anti-human CD3 eFluor780 (eBioscience 47-0037-42). Cells stained with anti-mouse F(ab')2-biotin were blocked with 3 μg of mouse IgG1 isotype control antibody (BioXCell) for 5 min before adding the remaining antibodies. DAPI was used at 1 μg / mL for live / dead discrimination. Stained cells were collected on an Invitrogen Attune NxT flow cytometer and analyzed with FlowJov10 software. Real-time quantitative PCR (RT-qPCR) 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 NucleoSpin RNA Kit (Macherey-Nagel) and cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific). All kits were used according to the manufacturer's recommendations. RT-qPCR was performed using iTaq PCR on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). This was carried out using Universal SYBR Green Supermix (Bio-Rad).

[0243] The primers used were as follows:

[0244] RPPH1-F: 5'-GAGGGAAGCTCATCAGTGGG-3' (SEQ ID NO: 87) RPPH1-R: 5'-CATCTCCTGCCCAGTCTGAC-3' (SEQ ID NO: 88) WPRE-F: 5'-CCTTTCCGGGACTTTCGCTTT-3' (SEQ ID NO: 89) WPRE-R: 5'-GCAGAATCCAGGTGGCAACA-3' (SEQ ID NO: 90) TH69CD7CAR-F: 5'-GCAGCCTTTCATGAGACCAG-3' (SEQ ID NO: 91) TH69CD7CAR-R: 5'-TGCCCAGGTTCAGCTCATTA-3' (SEQ ID NO: 92) TH69CD7PEBL-F: 5'-ACCTGCCGCATACAAGGATA-3' (SEQ ID NO: 93) TH69CD7PEBL-R: 5'-CCACTGTGCAGACTAGAGGT-3' (SEQ ID NO: 94) All assay primers had primer efficiencies between 90% and 110%.

[0245] Fold changes of all genes were normalized to housekeeping genes using the equation [fold change = 2^-(Ct(target gene)-Ct(housekeeping gene))]. Copy numbers of target genes were normalized to the genomic copy number of RNaseP in 293T cells. Western blot Cells were lysed with RIPA buffer (Pierce) and protease inhibitors (Pierce). Protein quantification of cell lysates was performed using the Bradford Coomassie protein assay kit (Thermo Scientific) according to the manufacturer's recommendations. Western blots were performed using an automated Western blot system, Simple Western Wes (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 Compass for Simple Western software. IFNγ secretion Effector CAR-T cells were 6 CAR-T cells were resuspended at a cell density of 100,000 cells / mL and seeded at 100,000 per well in 96-well round-bottom plates. 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 spun down and supernatants were collected and stored at -150°C. Supernatants were assessed for IFNγ secretion using the ELISA MAX Standard Set Human IFN-γ kit (Biolegend) according to the manufacturer's recommendations. Cytotoxicity assay The target cells were 6The cells were resuspended at a cell density of 100,000 cells / mL and loaded with 0.4 μg / ml calcein red-orange AM (Invitrogen) for 10 min. The loaded cells were then washed three times to remove excess calcein. 100,000 target cells per well were seeded 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 h, DAPI was added to all wells and cells were collected by flow cytometer. The number of remaining live target cells was counted in all wells. The percentage of cytotoxicity was calculated by the following formula: Percentage of cytotoxicity = [(SE) / S]*100% S = remaining viable target cells in target cells only control wells E = remaining viable target cells after co-culture with effector T cells in experimental wells result Primary T cells were transduced with various retroviruses expressing (1) PEBL, (2) CAR, (3) PEBL and CAR sequentially, (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 blot for β-actin, Myc-tagged PEBL, CAR and endogenous CD3ζ expression (Figure 1B).

[0246] Dual promoter lentiviral constructs were prepared to express anti-CD7 CAR and anti-CD7 PEBL from a single vector. As shown in Figures 2A-2F, the general format of the dual promoter construct from the 5' end to the 3' end included first promoter-anti-CD7 CAR-second promoter-anti-CD7 PEBL. Promoters tested include MSCV promoter, EFS promoter, PGK promoter, and EF1a promoter. Nucleic acid sequences of exemplary dual promoter constructs are provided as SEQ ID NOs: 19-23 and are shown in Figures 33A-33B, 34A-34B, 35A-35B, 36A-36B, and 37A-37B. Such constructs encoded 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.

[0247] The dual promoter lentiviral vector was transduced into cells to produce cells with partial downregulation of surface CD7 expression and low expression of anti-CD7 CAR. The percentage of cells expressing CAR remained low after extended time of culture. Figure 3 shows the expression of CAR (y-axis) and CD7 (x-axis) in cells 5 days post-transduction and 14 days post-transduction. The figures show the expression of cells (e.g., healthy donor cells, including healthy donor lymphocytes) transduced with exemplary dual promoter constructs provided as SEQ ID NOs: 18-23 and shown in Figures 32A-32B, 33A-33B, Figures 34A-34B, Figures 35A-35B, Figures 36A-36C, Figures 37A-37D, Figures 38A-38E, Figures 39A-39F, Figures 40A-40G, Figures 41A-41G, Figures 42A-42G, Figures 43A-43H, Figures 44A-44H, Figures 45A-45G, Figures 46A-46H, Figures 47A-47H, Figures 48A-48G, Figures 49A-49H, Figures 50A-50G, Figures 51A-51H, Figures 52A-52H, Figures 53A-53H, Figures 54A-54H, Figures 55A-55H, Figures 56A-56H, Figures 57A-57H, Figures 58A-58H, Figures 59A-59H, Figures 60A-60H, Figures 61A-61H, Figures 62A-62H, Figures 63A-63H, Figures 64A-64H, Figure 35A-35B, 36A-36B, and 37A-37B. As an example, cells transduced with a dual promoter lentiviral vector containing MSCV promoter-CD7(TH69)CAR-EF1a promoter-CD7(TH69)PEBL produced a population of cells including 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%) 5 days after transduction. Fourteen days after transduction, MSCV promoter-CD7(TH69)CAR-EF1a promoter-CD7(TH69)PEBL-transduced cells contained populations of cells including CD7 CAR-negative / CD7-negative cells (42.6%), CD7 CAR+ / CD7-negative cells (0.14%), CD7 CAR-negative / CD7+ cells (54.9%), and CD7 CAR+ / CD7+ cells (2.37%).

[0248] To optimize the expression of the single promoter of CD7 CAR and CD7 PEBL, bicistronic lentiviral constructs were generated. Exemplary schematic diagrams of such constructs are provided in Figure 4A, Figure 4B, and Figure 4C. Figure 4A shows a schematic diagram of an exemplary bicistronic construct comprising MSCV promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 14. Figure 4B shows a schematic diagram of an exemplary bicistronic construct comprising EF1a promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 15. Figure 4c shows a schematic diagram of an exemplary bicistronic construct comprising EFS promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL, such as that of SEQ ID NO: 16.

[0249] Cells transduced with MSCV promoter-anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL lentivirus generated a population of CD7 CAR+ / CD-negative T cells. Figure 5 shows the proliferation and accumulation of CD7 CAR+ / CD7-negative T cells 0-9 days after transduction. For example, on day 0, 10.9% of 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 the cells were CD7 CAR-negative / CD7-negative cells, 63.7% were CD7 CAR+ / CD7-negative cells, 6.25% were CD7 CAR-negative / CD7+ cells, and 2.57% were CD7 CAR+ / CD7+ cells. On day 9, 16.1% of the cells were CD7 CAR-negative / CD7-negative cells, 83.7% were CD7 CAR+ / CD7-negative cells, 0.012% were CD7 CAR-negative / CD7+ cells, and 0.095% were CD7 CAR+ / CD7+ cells.

[0250] Cells transduced with anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL bicistronic lentiviral vectors with different promoters produced CD7 CAR+ / CD7-negative cells that enriched in culture over time. Figure 6 shows the increase in the percentage of CD7 CAR+ / CD7-negative cells 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% were CD7 CAR+ / CD7-negative cells, 0.44% were CD7 CAR-negative / CD7+ cells, and 1.06% were CD7 CAR+ / CD7+ cells 5 days after transduction. By 14 days after transduction, 25.0% of CD7 CAR-negative / CD7- cells and 73.8% In contrast, 5 days after transduction, cells were transduced with a lentiviral vector containing an EF1a promoter upstream of the anti-CD19 CAR, and 34.7% of the 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. Also, 14 days after transduction, 47.5% of the 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, accumulation of CD19 CAR-expressing cells was barely detectable over time.

[0251] To evaluate the consistency and reproducibility of the single promoter bicistronic vector, cells were transduced with two independent lots 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 was also detected (Figure 7B). Figure 7A shows flow cytometry analysis of CD7 CAR and CD7 expression in transduced cells. The first lot showed 53.8% CD7 The first lot produced a population of transduced cells that contained CAR+ / CD7-negative cells and 46.1% CD7-negative / CD7-negative cells. The second lot produced a population of transduced cells that contained 65.5% CD7 CAR+ / CD7-negative cells and 34.5% CD7-negative / CD7-negative cells. Untransduced cells were noted to contain 98.6% CAR-negative / CD7+ cells.

[0252] It should be noted that the single promoter bicistronic vectors described herein have been successfully used to generate CD7 PEBL-CAR-T cells from different starting cells including bulk PBMCs, purified T cells including CD4+ and CD8+ T cells, and purified CD3+ T cells. Additionally, Dynabeads® Human T-Activator CD3 / CD28 (Gibco) and T Various T cell activation reagents were used, including Cell TransAct™ (Miltenyi Biotec). Figure 8 shows the increase in the percentage of CD7 CAR+ / CD7-negative T cells when anti-human CD7(TH69)CAR-P2A-anti-human CD7(TH69)PEBL transduced cells were cultured over time. Comparable proliferation of CD7 CAR+ / CD7-negative T cells was detected between the different starting cell types and between the two activation reagents.

[0253] The cells described herein (e.g., CD7 CAR+ / CD7-negative T cells) were generated from purified CD4+ positively selected and CD8+ positively selected T cells cultured in either 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. The total fold change of transduced cells 11 days after cell activation was higher in serum-supplemented medium (Figure 9A and Figure 9B).

[0254] Purified CD4+ and CD8+ selected T cells transduced with CD7CAR-P2A-CD7PEBL lentivirus on different days (days 1-4) after activation generated highly pure populations of CAR+ T cells (Figure 10A and Figure 10B). Cells transduced on different days expanded and proliferated during the manufacturing process. Figure 10B shows that transduced T cells showed approximately 5-fold to 10-fold proliferation on average when cells were transduced on days 1, 2, 3, or 4 after activation.

[0255] 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 with low MOI had a low percentage of CD7 CAR+ / CD7-negative T cells early in the transduction process, which increased to match the percentage of CD7 CAR+ / CD7-negative T cells obtained with higher MOI transduction (see, e.g., FIG. 11). For example, in T cells from donor 1 transduced with lentivirus at MOI 3, there 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. In T cells from donor 1 transduced with lentivirus at MOI 10, there 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. CAR+ / CD7-negative was 17.7% and CD7 CAR+ / CD7-negative was 83.7% 9 days after transduction.

[0256] 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, genomic DNA was extracted, and vector copy number (VCN) was determined by RT-qPCR analysis. Higher MOI correlated with higher VCN (Figure 12B), whereas the percentage of CD7 CAR+ T cells was similar at MOI 5 and 10 (Figure 12A).

[0257] Expression of various surface markers was measured in primary T cells transduced with MSCV-CD7CAR-P2A-CD7PEBL lentivirus 11 days after activation. Figure 13A shows CD7 CAR and endogenous CD7 expression in transduced cells from three different donors. Expression of CD3 compared to CD14 / CD19 / CD56 is shown in Figure 13B. Expression of CD4 and CD8 is shown in Figure 13C. Transduced cells were shown to be capable of expressing T EM cells, TCM cells, T eff Figure 13D shows that the transduced T cells generated distinct subsets of T cells, including naive T cells as determined by CD45RO and CCR7 expression. Figure 13D shows expression of PD-1 and TIM-3 on the transduced T cells.

[0258] The response of transduced PEBL-CAR T cells against CD7+ Jurkat cells and CD7-negative Nalm6 cells was determined by IFNγ secretion (Figure 14A) and cytotoxicity (Figure 14B). IFN-g secretion was measured in culture supernatants of PEBL-CAR T cells co-cultured with Jurkat or Nalm6 cells at the indicated E:T ratios for 24 h (mean ± SD for technical replicates). PEBL-CAR-T cells demonstrated a target-specific functional response, as IFNγ was secreted by PEBL-CAR-T cells when cultured with CD7+ Jurkat cells but not with Nalm6 cells. Furthermore, PEBL-CAR-T cells killed CD7+ Jurkat cells but not CD7-negative Nalm6 cells in a cytotoxicity assay.

[0259] This example shows the generation and expansion of PEBL-CAR-T cells produced using a CD7 CAR-P2A-CD7 PEBL bicistronic lentiviral vector. Such cells demonstrated antigen-specific T cell functional responses, such as IFNγ secretion and specific toxicity against CD7+ target cell lines. PEBL-CAR-T cells demonstrated high purity of CD7-negative CAR+ T cells.

[0260] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0261] Although the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention contained in the appended claims.

Claims

1. 1. A bicistronic retroviral vector comprising: (a) a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR) comprising at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 28 to 31; (b) a second polynucleotide encoding an internal ribosome entry site (IRES) or a ribosomal codon skipping site; and (c) a third polynucleotide encoding an anti-CD7 Protein Expression Blocker (PEBL) comprising at least 90% sequence identity to an amino acid sequence of SEQ ID NOs:24-27; A bicistronic retroviral vector, wherein the first polynucleotide is operably linked to the second polynucleotide, which is operably linked to the third polynucleotide.

2. The bicistronic retroviral vector of claim 1, wherein the anti-CD7 CAR comprises an amino acid sequence of any one of SEQ ID NOs: 28 to 31.

3. The bicistronic retroviral vector composition of claim 1 or 2, wherein the anti-CD7 PEBL comprises the amino acid sequence of any one of SEQ ID NOs: 24 to 27.

4. 4. The bicistronic retroviral vector composition of any one of claims 1 to 3, wherein 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.

5. 4. The bicistronic retroviral vector composition of any one of claims 1 to 3, wherein 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.

6. The bicistronic retroviral vector of any one of claims 1 to 5, wherein the IRES is derived from encephalomyocarditis virus (EMCV) or an enterovirus.

7. The bicistronic retroviral vector of any one of claims 1 to 5, wherein the ribosomal codon skipping site comprises a 2A self-cleaving peptide.

8. 8. The bicistronic retroviral vector of claim 7, wherein the 2A self-cleaving peptide is selected from the group consisting of F2A peptide (foot and mouth disease virus 2A peptide), E2A peptide (equine rhinitis A virus 2A peptide), P2A peptide (porcine teschovirus-12A peptide), and T2A peptide (torsea signa virus 2A).

9. A bicistronic retroviral vector according to any one of claims 1 to 6, comprising at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

12.

10. 7. The bicistronic retroviral vector of any one of claims 1 to 5 and 6, comprising at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

13.

11. The bicistronic retroviral vector of claim 10 comprising the nucleic acid sequence of SEQ ID NO:

13.

12. The biosynthetic vector according to any one of claims 1 to 11, further comprising a promoter element. Strontous retroviral vectors.

13. The bicistronic retroviral vector of claim 12, wherein 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.

14. The bicistronic retroviral vector of claim 13, wherein the promoter element comprises at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6-10.

15. The bicistronic retroviral vector of claim 13 or 14, wherein the promoter element comprises any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6 to 10.

16. The bicistronic retroviral vector of any one of claims 12 to 15, comprising at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14 to 16.

17. The bicistronic retroviral vector according to any one of claims 12 to 16, comprising any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 14 to 16.

18. The bicistronic retroviral vector of any one of claims 1 to 18, wherein the retroviral vector is a lentiviral vector.

19. An engineered immune cell comprising a bicistronic retroviral vector according to any one of claims 1 to 18.

20. 20. The engineered immune cell of claim 19, wherein the engineered immune cell is an allogeneic T cell.

21. 21. The engineered immune cell of claim 20, wherein the engineered immune cell is an autologous T cell.

22. 22. The engineered immune cell of claim 20 or 21, wherein the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell and expresses the anti-CD7 CAR.

23. 20. A pharmaceutical composition comprising the engineered immune cells of claim 19 and a pharma- ceutically effective carrier.

24. 23. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of the engineered immune cell of any one of claims 19 to 21, or the pharmaceutical composition of claim 23.

25. 20. A method of producing an engineered immune cell, comprising transducing an immune cell with a bicistronic retroviral vector according to any one of claims 1 to 18, and harvesting the engineered immune cell.

26. 26. The method of claim 25, 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. method.

27. 27. The method of claim 25 or 26, wherein the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell and expresses the anti-CD7 CAR.

28. 1. A recombinant retroviral vector comprising: (a) a first promoter element operably linked to a first polynucleotide encoding an anti-CD7 chimeric antigen receptor (CAR) comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:30; (b) a second promoter element operably linked to a second polynucleotide encoding an anti-CD7 Protein Expression Blocker (PEBL) comprising at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:

26.

29. 29. The recombinant retroviral vector of claim 28, wherein the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:

30.

30. 30. The recombinant retroviral vector of claim 28 or 29, wherein the anti-CD7 PEBL comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:

26.

31. The recombinant retroviral vector of any one of claims 28 to 30, wherein 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.

32. The recombinant retroviral vector of any one of claims 28 to 31, wherein the first promoter element and / or the second promoter element comprises at least 90% sequence identity to any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10.

33. The recombinant retroviral vector of any one of claims 28 to 32, wherein the first promoter element and / or the second promoter element comprises any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 to 10.

34. 34. The recombinant retroviral vector of any one of claims 28 to 33, wherein the first promoter and the second promoter share less than 95% sequence identity.

35. 35. The recombinant retroviral vector of any one of claims 28 to 34, wherein the first promoter element operably linked to the first polynucleotide is 5' to the second promoter element operably linked to the second polynucleotide.

36. 35. The recombinant retroviral vector of any one of claims 28 to 34, wherein the second promoter element operably linked to the second polynucleotide is 5' to the first promoter element operably linked to the first polynucleotide.

37. The recombinant retroviral vector of any one of claims 28 to 35, comprising at least 90% sequence identity to any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 18 to 23.

38. The recombinant retroviral vector of any one of claims 28 to 37, wherein the retroviral vector is a lentiviral vector.

39. An engineered immune cell comprising a recombinant retroviral vector according to any one of claims 28 to 38.

40. 40. The engineered immune cell of claim 39, wherein the engineered immune cell is an allogeneic T cell.

41. 41. The engineered immune cell of claim 40, wherein the engineered immune cell is an autologous T cell.

42. 41. The engineered immune cell of claim 40, wherein the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell and expresses the anti-CD7 CAR.

43. A pharmaceutical composition comprising an engineered immune cell according to any one of claims 39 to 42 and a pharma- ceutically effective carrier.

44. 44. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of the engineered immune cell of any one of claims 39-41, or the pharmaceutical composition of claim 43.

45. 11. A method of producing an engineered immune cell, comprising transducing an immune cell with a recombinant retroviral vector according to any one of claims 28 to 38 and harvesting said engineered immune cell.

46. 46. ​​The method of claim 45, 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.

47. 47. The method of claim 45 or 46, wherein the engineered immune cell has reduced CD7 surface expression compared to a corresponding immune cell and expresses the anti-CD7 CAR.

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