Blockade of CD7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies

Abstract

To provide blockade of CD7 expression and chimeric antigen receptors for immunotherapy of T-cell malignancies.SOLUTION: The present invention provides compositions comprising an anti-CD7 chimeric activating receptor (CAR) and an anti-CD7 protein expression blocker, and methods of using such compositions in cancer therapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 425,398, filed November 22, 2016, and No. 62 / 543,696, filed August 10, 2017, which are expressly incorporated by reference in their entireties for all purposes. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 21, 2017, is named 119419-5002-WO_ST25.txt and is 20,928 bytes in size. [Background technology]

[0003] Chimeric antigen receptors (CARs) can convert immune cells to specifically recognize and kill tumor cells. CARs are artificial multimolecular proteins composed of single-chain variable regions (scFvs) of antibodies linked to signaling molecules via transmembrane domains. When the scFvs bind to their cognate antigens, signal transduction is triggered, leading to the killing of tumor cells by CAR-expressing cytotoxic T lymphocytes (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). Clinical trials using CAR-expressing autologous T lymphocytes have shown positive responses in patients with B-cell refractory leukemia and lymphoma (e.g., Non-Patent Document 6, Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Eshhar Z, Waks T, et al.PNAS USA.90(2):720-724,1993 [Non-Patent Document 2] Geiger TL,et al.J Immunol.162(10):5931-5939,1999 [Non-Patent Document 3] Brentjens RJ, et al. Nat Med.9(3):279-286,2003 [Non-Patent Document 4] Cooper LJ, et al.Blood 101(4):1637-1644,2003 [Non-Patent Document 5] Imai C,et al.Leukemia.18:676-684,2004 [Non-Patent Document 6] Till BGet al.Blood 119(17):3940-3950,2012 [Non-Patent Document 7] Maude SL,et al.N Engl J Med.371(16):1507-1517,2014 Summary of the Invention [Problem to be solved by the invention]

[0005] The development of CAR technologies for targeting T-cell malignancies has lagged considerably behind the progress made for their B-cell counterparts. Novel therapies for T-cell malignancies are needed, but progress to date has been slow. 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 the morbidity and mortality of these approaches, the outcomes have been satisfactory. It's not something that can be done.

[0006] In summary, there is a large unmet need for novel therapeutic options for patients with T-cell malignancies. [Means for solving the problem]

[0007] In one aspect, the invention provides a genetically engineered immune cell comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, where the target binding molecule is a first antibody that specifically binds CD7; and (ii) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), where the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds CD7.

[0008] In some embodiments, the first antibody that specifically binds to CD7 is a first single chain variable fragment (scFv). In certain embodiments, the second antibody that specifically binds to CD7 is a second single chain variable fragment (scFv).

[0009] In some embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0010] In some embodiments, the localization domain comprises an amino acid sequence selected from the group consisting of an endoplasmic reticulum (ER) retention sequence, a Golgi apparatus retention sequence, a proteasome localization sequence, and a transmembrane domain sequence derived 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, the localization domain comprises an endoplasmic reticulum (ER) retention sequence comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, proteasomal localization of a target binding molecule (e.g., scFv) is achieved by linking the scFv sequence to a tripartite motif-containing 21 (TRIM21) targeting domain sequence and co-expressing a nucleic acid sequence encoding the human TRIM21 E3 ubiquitin ligase protein.

[0011] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0012] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0013] In some embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1 and a heavy chain variable domain having the sequence and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In yet other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0014] In some embodiments, the engineered cell is a genetically engineered T cell, a genetically engineered natural killer (NK) cell, a genetically engineered NK / T cell, a genetically engineered monocyte, a genetically engineered macrophage, or a genetically engineered dendritic cell.

[0015] In another aspect, the invention provides an engineered immune cell comprising: (i) a target binding molecule linked to a localization domain, where the target binding molecule is a first antibody that specifically binds CD7; and (ii) a chimeric antigen receptor (CAR), where the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds CD7.

[0016] In some embodiments, the first antibody that specifically binds to CD7 is a first single chain variable fragment (scFv). In certain embodiments, the second antibody that specifically binds to CD7 is a second single chain variable fragment (scFv).

[0017] In some embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0018] In some embodiments, the localization domain comprises an amino acid sequence selected from the group consisting of an endoplasmic reticulum (ER) retention sequence, a Golgi apparatus retention sequence, a proteasome localization sequence, and a transmembrane domain sequence derived 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, the localization domain comprises an endoplasmic reticulum (ER) retention sequence comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO:13.

[0019] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0020] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0021] In some embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In yet other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0022] In some embodiments, the engineered cell is a genetically engineered T cell, a genetically engineered natural killer (NK) cell, a genetically engineered NK / T cell, a genetically engineered monocyte, a genetically engineered macrophage, or a genetically engineered dendritic cell.

[0023] In some embodiments, provided herein is a pharmaceutical composition comprising a genetically engineered immune cell as described herein and a pharma- ceutically acceptable carrier.

[0024] In another aspect, the invention provides a method of making the engineered immune cells described herein, comprising: (i) introducing into an immune cell: (a) a first nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, the target binding molecule being a first antibody that specifically binds to CD7; and (b) a second nucleic acid comprising a nucleotide sequence encoding a CAR, the CAR comprising a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7; and (ii) isolating the engineered immune cell comprising the target binding molecule linked to the localization domain and the CAR, thereby making the engineered immune cell.

[0025] In yet another aspect, the invention provides a method of treating cancer in a subject (e.g., a patient) in need of treatment, the method comprising administering a therapeutic amount of engineered immune cells to the patient, thereby treating the cancer in the subject in need of treatment. In some embodiments, the engineered immune cells comprise (i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, where the target binding molecule is a first antibody that specifically binds to CD7, and (ii) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), where the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0026] In some embodiments, the first antibody that specifically binds to CD7 is a first single chain variable fragment (scFv). In certain embodiments, the second antibody that specifically binds to CD7 is a second single chain variable fragment (scFv).

[0027] In some embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the first single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the first single chain variable fragment (scFv) comprises , a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:16, and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:17.

[0028] In some embodiments, the localization domain comprises an amino acid sequence selected from the group consisting of an endoplasmic reticulum (ER) retention sequence, a Golgi apparatus retention sequence, a proteasome localization sequence, and a transmembrane domain sequence derived 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, the localization domain comprises an endoplasmic reticulum (ER) retention sequence comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO:13.

[0029] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0030] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0031] In some embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15. In yet other embodiments, the second single chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.

[0032] In some embodiments, the engineered cell is a genetically engineered T cell, a genetically engineered natural killer (NK) cell, a genetically engineered NK / T cell, a genetically engineered monocyte, a genetically engineered macrophage, or a genetically engineered dendritic cell.

[0033] In some embodiments, the genetically engineered immune cells are administered to the subject (e.g., patient) 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, or intrathecal administration.

[0034] In some embodiments, the cancer is a T cell malignancy. In one embodiment, the T cell malignancy is early T cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0035] The present disclosure provides genetically engineered immune cells and methods of use thereof for treating T-cell hematological malignancies.Those skilled in the art recognize that CAR T-cell self-killing or homozygous killing and normal T-cell killing may occur when CAR-T effector cells are used to treat T-cell leukemia.Therefore, there is a need for genetically engineered immune cells and methods of treatment that minimize or eliminate T-cell homozygous killing.

[0036] The genetically engineered immune cells and therapeutic methods described herein include genetically engineered anti-CD7 Utilizing novel cognate killing-resistant CAR-T cells, such as PEBL cells and anti-CD7 CAR-T cells, engineered immune cells can elicit potent and sustained therapeutic effects in patients with T-cell malignancies, including recurrent T-cell malignancies. Such cells can result in efficient targeting and killing of malignant T cells without significant effector T-cell cognate killing. [Brief description of the drawings]

[0037] [Figure 1]1A-1D. Figures illustrating CD7 expression in T-ALL. Percentage of ALL cells expressing CD7 at diagnosis, relapse, and during chemotherapy (MRD), with the number of bone marrow samples tested at each stage shown (Figure 1A). CD7 mean fluorescence intensity (MFI) in T-ALL cells and residual normal T cells from the same samples (n=19, P<0.0001 by paired t-test) (Figure 1B). CD7 MFI in T-ALL cells at diagnosis or relapse ("D / R") and follow-up bone marrow samples with MRD (n=18) (Figure 1C). Flow cytometry contour plots show CD7 expression in T-ALL cells (CD3 negative) and normal T cells (CD3 positive) at diagnosis, MRD, and relapse in one representative patient (Figure 1D). [Diagram 2]2A-E. Anti-CD7 CAR design, expression and signaling. Schematic of anti-CD7-41BB-CD3ζ construct (Figure 2A). Flow cytometry analysis of Jurkat cells transduced with either GFP alone ("Mock") or GFP plus anti-CD7 CAR. Dot plots show GFP fluorescence and CAR expression after staining with biotin-conjugated goat anti-mouse F(ab')2 antibody and streptavidin-APC (Jackson ImmunoResearch) (Figure 2B). Western blot analysis of CAR expression in Jurkat cells (Figure 2C). Cell lysates of mck-transduced and CAR-transduced Jurkat cells were resolved on a 10% polyacrylamide gel under reducing or non-reducing conditions. Blotted membranes were probed with mouse anti-human CD3ζ antibody (8D3, ​​BD Biosciences) and goat anti-mouse IgG conjugated to horseradish peroxidase (R&D Systems). Antibody binding was revealed using Clarity Western EC substrate (Bio-Rad). Anti-CD7 CAR induces expression of activation markers upon ligation. Bars show the mean (±SD) of CD25 and CD69 MFI in CAR-transduced and mock-transduced Jurkat cells after 24 h in the presence or absence of CD7+ MOLT-4 cells. P values ​​by t-test are shown for significant differences (*=0.016, ***<0.001) (Figure 2D). Figure 2E presents a representative flow cytometry histogram of the experiment shown in Figure 2D. [Figure 3-1]Figures 3A-I. Expression of anti-CD7 CAR in human peripheral blood T cells results in cognate killing that is prevented by CD7 downregulation. Percentage of viable T cells recovered 24 hours after electroporation in the presence or absence of anti-CD7 CAR mRNA (n=7) (Figure 3A). Viable cells were counted by flow cytometry. Percentage of viable T cells recovered 24 hours after CAR transduction with retroviral vectors compared to cells from the same donor transduced with GFP alone ("Mock") (n=10) (Figure 3B). Percentage of viable CAR-transduced or mock-transduced T cells recovered during the week after transduction (Figure 3C). Follow-up results for 5 of the 10 experiments shown in Figure 3B. Percentage of CD107a in T cells after electroporation in the presence or absence of anti-CD7 CAR mRNA (Figure 3D). Mean (±SD) of triplicate measurements is shown. Schematic diagram of anti-CD7 protein expression blocker (PEBL) constructs (FIG. 3E). Representative flow cytometry histograms show CD7 expression in T lymphocytes following retroviral transduction of the three anti-CD7 PEBL constructs or mock-transduced GFP alone ("Mock") (FIG. 3F). [Figure 3-2]T cells were stained with anti-CD7-PE (M-T701; BD Biosciences). Percentage of CD7 expression in anti-CD7 PEBL-1 retrovirally transduced or mock transduced T cells (n=5) (Figure 3G). Flow cytometry dot plots show downregulation of CD7 expression in T cells by PEBL transduction, along with expression of anti-CD7-41BB-CD3ζ CAR 12 hours after electroporation in the presence or absence of CAR mRNA (Figure 3H). Cells were stained with biotin-conjugated goat anti-mouse F(ab')2 antibody and streptavidin-APC (Jackson ImmunoResearch). Percentage of viable anti-CD7 PEBL transduced T cells recovered 24 hours after electroporation of anti-CD7 CAR mRNA compared to cells electroporated with anti-CD7 CAR mRNA but vector-transduced without anti-CD7 PEBL (n=6) (Figure 3I). The number of viable cells was measured by flow cytometry. **, P<0.01. ***, P<0.001. [Figure 4-1] Figures 4A-F. CD7 downregulation by PEBL did not alter T cell phenotype, proliferation and functionality. Percentage of CD4 and CD8 cells 7-14 days after retroviral transduction with either anti-CD7 PEBL or GFP alone ("Mock") (Figure 4A). Each symbol corresponds to a different T cell donor. Proliferation rates of PEBL-transduced and mock-transduced T cells (from three donors) were maintained for 14 days with 200 IU / mL IL-2 (Figure 4B). Symbols represent the mean (± SD) of triplicate measurements. PEBL-transduced and mock-transduced T cells were electroporated either in the presence of anti-CD19-41BB-CD3ζ CAR mRNA or in the absence of mRNA (Figure 4C). Flow cytometry dot plots show GFP and CAR expression 12 hours after electroporation. CAR was detected using biotin-conjugated goat anti-mouse F(ab')2 antibody and streptavidin-APC (Jackson ImmunoResearch). [Figure 4-2]Cytotoxicity of PEBL- or mock-transduced T cells electroporated in the presence or absence of anti-CD19 CAR mRNA against CD19+ ALL cells (OP-1) (Figure 4D). Bars represent the mean (±SD) of cytotoxicity at 1:1 E:T for 4 hours. Figure 4E shows CD107a expression in T cells from the same experiment as described in Figure 4D. Figure 4F shows IFNγ production in PEBL- or mock-transduced T cells electroporated in the presence or absence of anti-CD19 CAR mRNA and co-cultured with OP-1 at E:T 1:1 for 6 hours. Bars represent the mean (±SD) of triplicate experiments. ***, P<0.001. ****, P<0.0001. [Figure 5-1]Figure 5A-5F. After expression of anti-CD7 CAR, T cells with CD7 downregulated by PEBL acquire potent cytotoxicity against CD7+ leukemia cells. Cytotoxicity of anti-CD7 PEBL-transduced T cells electroporated in the presence or absence of anti-CD7 CAR mRNA against CD7+ cell lines (Figure 5A). Data from a 4-hour assay in 1:1 E:T are shown. Symbols represent the mean of triplicate measurements with T cells from 4 donors for MOLT-4, CCRF-CEM and Jurkat, and 5 donors for Loucy and KG1a, respectively (P<0.001 for each comparison). Cytotoxicity of anti-CD7 PEBL-transduced T cells electroporated in the presence or absence of anti-CD7 CAR mRNA against primary leukemia cells from T-ALL patients (Figure 5B). Data from a 4-hour assay in the indicated E:T are shown. Symbols represent the mean (±SD) of triplicate measurements. Figure 5C shows the overall specific cytotoxicity of T cells transduced with either anti-CD7 PEBL or GFP alone ("Mock") against five CD7+ cell lines after electroporation with anti-CD7 CAR mRNA. T cells from three donors were tested in a 4-hour assay in 1:1 E:T. Each symbol represents the specific percent cytotoxicity against a CD7+ cell line after subtraction of the percent cytotoxicity obtained with the same T cells electroporated in the absence of mRNA. Horizontal bars indicate the median of the respective group. Anti-CD7 PEBL-transduced or mock-transduced T cells from three donors were electroporated in the presence or absence of anti-CD7 CAR mRNA (Figure 5D). Cytotoxicity against MOLT-4 was tested in a 4-hour assay in 1:1 E:T. Mean fluorescence intensity (MFI) of anti-CD107a-PE (H4A3, BD Biosciences) is shown. Bars represent the mean (±SD) of three experiments. [Figure 5-2]Anti-CD7 PEBL-transduced T cells were retrovirally transduced with either anti-CD7 CAR or mock transduced and tested against primary leukemia cells from T-ALL patients (Figure 5E). Each symbol represents the mean (±SD) of triplicate experiments. Mock-transduced or PEBL-transduced T cells sequentially transduced in the presence or absence of anti-CD7 CAR were cultured alone or in the presence of Streck-treated MOLT-4 cells and supplemented weekly with 120 IU / mL IL-2 (Figure 5F). Symbols indicate the mean (±SD) of cell recovery relative to input cell number in triplicate cultures. **, P<0.01; ***, P<0.001; ****, P<0.0001 [Figure 6] Figure 6A-6D. PEBL-transduced T cells expressing anti-CD7-41BB-CD3ζ CAR exert anti-tumor activity in xenografts. NOD-SCID-IL2RGnull mice were intravenously (iv) injected with 1x106 CCRF-CEM cells labeled with luciferase. 2x107 PEBL-CAR T cells were administered i.v. to three and five mice, respectively, on day 7 (Figure 6A), or days 3 and 7 (Figure 6B) after leukemia cell injection. The remaining mice received either mock-transduced T cells, or RPMI-1640 ("control") instead of cells. All mice received 20,000 IU of IL-2 intraperitoneally (ip) once every two days. In vivo imaging of leukemia cell proliferation after D-luciferin i.p. injection is shown. Abdominal images of mice on day 3 in Figure 6B are shown with increased sensitivity to demonstrate CCRF-CEM engraftment in all mice. The complete set of luminescence images is shown in Figure 14. Figure 6C shows leukemic cell proliferation expressed as photons per second in the mice shown in Figures 6A and 6B. Each symbol corresponds to the bioluminescence measurement in each mouse, normalized to the average of the ventral plus dorsal signal in all mice before CAR-T cell infusion. Kaplan-Meier curves show the overall survival rate of mice in the different groups (8 in each group) (Figure 6D). Mice were euthanized when the total bioluminescence signal reached 1 x 1010 photons per second. P values ​​calculated by log-rank test. [Figure 7-1] Figures 7A-E. PEBL-CAR-T cell activity against ETP-ALL in a patient-derived xenograft (PDX) model. Primary ETP-ALL cells pre-expanded in NOD-SCID-IL2RGnull mice were intravenously (iv) injected into 10 NOD-SCID-IL2RGnull mice at 2x106 cells per mouse (Figure 7A). Five mice ("control") were left untreated. The remaining five mice received a single iv infusion of PEBL-CAR T cells (2x107 for PEBL-CAR#1 and 2x106 for the remaining four mice) at the indicated time points (grey arrows) and 20,000 IU of IL-2 every two days; two of the five control mice also received IL-2. Black symbols (left y-axis) indicate the number of ETP-ALL cells / mL counted in peripheral blood. Grey symbols (right y-axis) indicate the number of PEBL-CAR T cells. Mice were euthanized when the percentage of ETP-ALL cells in blood mononuclear cells reached ≥80%. Percentage of ETP-ALL (denominator, total human + mouse CD45+ cells) in various organs of 5 untreated mice (Figure 7B). Blood smears of treated (PEBL-CAR#1) and untreated ETP-ALL 7 days after T cell infusion, smear cells were prominent in the blood after PEBL-CAR T cells (Figure 7C). [Figure 7-2] Flow cytometry dot plots show the presence of CD7+ CD3- ETP-ALL cells in tissues of untreated control mice with ETP-ALL and CD7- CD3+ PEBL-CAR T cells in PEBL-CAR#1 mice treated with PEBL-CAR-T cells (Figure 7D). No ETP-ALL (<0.01%) was detected in treated mice. The events shown were normalized to those obtained for the corresponding plots shown in control mice. Spleens of treated (PEBL-CAR#1) and untreated mice (Figure 7E). [Figure 8]8A-8C. Specificity and function of anti-CD7-41BB-CD3ζ CAR. OP-1 (CD7-) and MOLT-4 (CD7+) were incubated with supernatants from Jurkat cells transduced with anti-CD7 scFv or with vector containing GFP alone ("control") (Figure 8A). After washing, cells were incubated with biotin-conjugated goat anti-mouse F(ab')2 antibody followed by streptavidin-APC (Jackson ImmunoResearch). Flow cytometry histograms show binding of anti-CD7 scFv to MOLT-4 but not to OP-1. Jurkat cells were transduced with vector containing anti-CD7-41BB-CD3ζ CAR, anti-CD19-41BB-CD3ζ CAR, or GFP alone (Figure 8B). These cells were co-cultured with CD7+ MOLT-4 or CCRF-CEM cells, or CD7- cells OP-1 in 1:1 E:T. Target cells were labeled with calcein red-orange AM (Invitrogen). After 30 min of incubation, the percentage of cell doublets was measured by flow cytometry. Bars indicate the mean (±SD) of triplicate determinations. Figure 8C shows that pre-incubating target cells with a soluble form of anti-CD7 scFv inhibits CAR-mediated cell aggregation. *** P<0.001. [Figure 9]Figure 9A-B. Expression of anti-CD7-41BB-CD3ζ CAR in human peripheral blood T lymphocytes. Figure 9A presents representative flow cytometry dot plots of T lymphocytes activated with Dynabeads human T activator CD3 / CD28 (ThermoFisher Scientific) and IL-2 for 7 days and transduced with anti-CD7 CAR. Flow cytometry dot plots show GFP fluorescence and CAR expression, the latter revealed by staining with biotin-conjugated goat anti-mouse F(ab')2 antibody followed by streptavidin-APC (Jackson ImmunoResearch). Figure 9B shows Western blot analysis of CAR expression. Cell lysates of mock-transduced and CAR-transduced T cells were separated on a 10% polyacrylamide gel under reducing or non-reducing conditions. Blotted membranes were probed with mouse anti-human CD3ζ antibody (8D3; BD Biosciences) followed by goat anti-mouse IgG conjugated to horseradish peroxidase (R&D Systems). Antibody binding was revealed using Clarity Western EC substrate (Bio-Rad). [Figure 10]10A-10B. Downregulation of CD7 protein expression by anti-CD7 PEBL. Flow cytometry dot plots show GFP expression (x-axis), CD7 expression (y-axis, top row), and intracellular anti-CD7 PEBL-1 expression (y-axis, bottom row) (FIG. 10A). T lymphocytes were retrovirally transduced with vectors containing anti-CD7 PEBL-1 or GFP alone ("Mock"). T cells were stained with an anti-CD7 antibody conjugated to phycoerythrin (M-T701, BD Biociences). Intracellular expression of PEBL-1 was examined using a PE-conjugated anti-Myc antibody (9B11; Cell Signaling Technology) that binds to the sequence EQKLISEEDL (SEQ ID NO: 40) incorporated into an ER-binding motif. Prior to antibody labeling, cells were permeabilized with 8E reagent (permeabilization reagent developed in the applicant's laboratory). FIG. 10B shows RT-PCR analysis of CD7 mRNA expression. cDNA derived from total mRNA extracted from PEBL1-3, GFP alone ("mock") or untransduced ("WT") Jurkat cells was used as template. CD7 cDNA (723 bp) was amplified with the following primers: forward, ATGGCCGGGCCTCCCG (SEQ ID NO: 38), reverse, TCACTGGTACTGGTTGGG (SEQ ID NO: 39). Electrophoresis was performed on a 1% agarose gel using SYBR Safe Gel Stain (ThermoFisher). Template controls are also not shown. As a control, an 87 bp (nucleotides 676 to 762) region of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified in parallel. [Figure 11]11A-11B. Anti-CD7 CAR signal induced higher cytokine secretion in T cells with CD7 knockdown expression by anti-CD7 PEBL. T lymphocytes from three donors were transduced with anti-CD7 PEBL or GFP alone ("Mock") and electroporated either with anti-CD7-41BB-CD3ζ mRNA or without mRNA. Intracellular IFNγ (FIG. 11A) and TNFα (FIG. 11B) expression was measured in T cells after 6 h of coculture with MOLT4. Bars represent the mean (±SD) of triplicate MFI measurements. **, P<0.01. ***, P<0.001. ****, P<0.0001. [Figure 12] FIG. 1 shows that CD7-negative T cells expressing anti-CD7-41BB-CD3ζ CAR exerted anti-tumor cytotoxicity against CD7+ cell lines. Shown are the results of a 4-hour cytotoxicity assay performed with T cells transduced with anti-CD7 PEBL and then transduced with either CD7-41BB-CD3ζ or GFP alone ("Mock"). Symbols represent the mean (±SD) of triplicate experiments at the indicated E:T ratios. P<0.001 for all comparisons. [Figure 13-1]Figures 13A-E. Functional comparison of anti-CD7-41BB-CD3ζ and anti-CD19-41BB-CD3ζ CARs. Figure 13A shows expression of anti-CD19 and anti-CD7 CARs (in mCherry-containing vectors) in peripheral blood T cells previously transduced with anti-CD7 PEBL. Flow cytometry dot plots show mCherry expression and staining of T cells with biotin-conjugated goat anti-mouse F(ab')2 antibody followed by streptavidin conjugated to allophycocyanin (Jackson ImmunoResearch). Results with T cells transduced with vectors containing mCherry alone ("Mock") are also shown. Expression of CD19 in CCRF-CEM and Jurkat cells transduced with vectors containing CD19 and GFP (Figure 13B). CD19 was detected with anti-CD19 APC (Miltenyi Biotech). Four-hour cytotoxicity assay targeting CD19+ CCRF-CEM or CD19+ Jurkat cells with anti-CD19 or anti-CD7 PEBL-CAR-T cells at different E:T ratios (FIG. 13C). Symbols represent the mean (±SD) of triplicate determinations. P<0.001 for data with either CAR versus mock-transduced T cells at all E:T ratios. [Figure 13-2]Long-term cytotoxicity of anti-CD19 or anti-CD7 PEBL-CAR-T cells at different E:T ratios measured by live cell image analysis using an IncuCyte Zoom System (Essen Bioscience) (Figure 13D). Symbols indicate the mean (±SD) of triplicate measurements of CD19+ CCRF-CEM (top) or CD19+ Jurkat cells (bottom) in wells containing CAR-T cells, mock-transduced T cells, or no T cells. Measurements were performed at 4-h intervals. Proliferative potential of anti-CD19 and anti-CD7 PEBL-CAR-T cells with and without coculture with CD19+ Jurkat cells (Figure 13E). Anti-CD7 PEBL-transduced T cells sequentially transduced with anti-CD19 or anti-CD7 CAR or mCherry alone were cultured alone or in the presence of irradiated CD19+ Jurkat cells and supplemented weekly with 120 IU / mL of IL-2. Symbols indicate the mean (±SD) percentage of cell recovery relative to input cell number for triplicate cultures. [Figure 14-1] Figures 14A-C. PEBL-transduced T cells expressing anti-CD7-41BB-CD3ζ CAR exerted anti-tumor activity in a mouse model. NOD-SCID-IL2RGnull mice were intravenously injected with 1x106 CCRF-CEM cells labeled with luciferase. 2x107 PEBL-CAR T cells were administered intravenously to three and five mice, respectively, on day 7 (Figure 14A) or days 3 and 7 (Figure 14B) after leukemia cell injection. The remaining mice received either mock-transduced T cells or RPMI-1640 ("control") instead of cells. All mice received 20,000 IU of IL-2 intraperitoneally (ip) every 2 days. In vivo imaging of leukemia cell proliferation was performed after intraperitoneal injection of D-luciferin. In FIG. 14B, abdominal images of mice on day 3 are shown with increased sensitivity to demonstrate leukemic cell engraftment in all mice. [Figure 14-2]Leukemia cell proliferation was expressed as photons per second over time, normalized to the average sum of ventral and dorsal signals in all mice before CAR-T cell infusion (Figure 14C). Each symbol corresponds to a bioluminescence measurement for each mouse. [Figure 15] Figures 15A-B. PEBL-transduced T cells expressing anti-CD7-41BBCD3ζ CAR exerted antitumor activity in a mouse model and maintained activity against cells harvested at relapse. Figure 15A shows the percentage of CCRF-CEM cells among leukocytes in blood from NOD-SCID-IL2RGnull mice that were intravenously injected with luciferase-labeled CCRF-CEM cells and then treated intravenously with either PEBL-CAR-transduced T cells, mock-transduced T cells, or RPMI-1640 ("control") instead of cells as described for Figure 6C. For "control" and "mock", blood was obtained from euthanized mice that reached a bioluminescence threshold of 1010 photons / sec 17-23 days after leukemia cell injection. For PEBL-CAR mice, blood was obtained via cheek puncture 24 days after CCRF-CEM injection. CCRF-CEM cells harvested from spleens and livers of PEBL-CAR-treated mice at relapse were cultured for 2 days (FIG. 15B). They were then used as targets in a 4-hour cytotoxicity assay in an E:T 1:1 setup using PEBL-CAR-transduced or mock-transduced T cells originally used for infusion. Comparisons were also made with the same batch of CCRF-CEM-expressing luciferase cells used to generate the xenografts. Percent cytotoxicity was determined from plate measurements of bioluminescent signals after addition of the BrightGlo luciferase assay system (Promega). Bars indicate the mean (±SD) of triplicate measurements, with each white and grey bar corresponding to cells from one mouse. [Figure 16]FIG. 7 shows immunophenotypic characteristics of ETP-ALL at diagnosis and after expansion in NOD-SCID-IL2RGnull mice. Flow cytometry contour plots show the immunophenotyping bone marrow samples of ETP-ALL used to develop the PDX models in this study and of ETP-ALL cells recovered from the spleen of one of the control mice shown in FIG. 7. The following antibodies were used: CD7-PE, CD45-APC-H7, CD34-PerCP, CD8-BV510, CD5-PE-Cy7, CD3-PerCP (for cytoplasmic staining), CD3-V450 (for surface staining), CD33-BV421 (Biolegend), CD1a-PE (Beckman Coulter), all from BD Biosciences. Quadrants were drawn based on staining with isotype-matched non-reactive antibodies coupled to the same fluorochrome. [Figure 17] FIG. 1 is a schematic diagram of an exemplary embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] A description of an exemplary embodiment of the present invention follows.

[0039] The present invention is based in part on the design of a chimeric antigen receptor (CAR) against CD7, a 40 kDa type I transmembrane glycoprotein that is a primary marker of T cell malignancies and is highly expressed in all cases of T cell ALL, including early T cell precursor acute lymphoblastic leukemia (ETP-ALL). As described herein, anti-CD7 CARs induce T cells to exert specific cytotoxicity against T cell malignancies. Furthermore, anti-CD7 It has been shown that when CARs are used in combination with downregulation of CD7 expression on effector T cells, the cytotoxicity of T cells is significantly increased. As demonstrated herein, downregulation (e.g., removal, reduction, and / or relocalization) of CD7 inhibits the expression of the corresponding anti-CD7 Prevents the allogeneic killing effect exerted by CAR, allows for greater T cell recovery after CAR expression compared to cells bearing the target antigen (e.g., CD7), and targets T leukemia / lymphoma cells This results in more effective cytotoxicity.

[0040] Thus, in one aspect, the present invention relates to a genetically engineered immune cell comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that specifically binds to cluster of differentiation 7 (CD7). The CAR of the present invention may be referred to herein as "anti-CD7-41BB-CD3ζ". An exemplary embodiment is shown in FIG. 17.

[0041] As used herein, a "genetically engineered" immune cell includes an immune cell that is genetically modified compared to a naturally occurring immune cell. For example, a genetically 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 the nucleotide sequence is derived.

[0042] In certain embodiments, the engineered immune cells are engineered T cells, engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages, or engineered dendritic cells. In certain embodiments, the engineered immune cells are engineered T cells. As used herein, the term "nucleic acid" refers to a polymer that includes 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 double-stranded polymers, "nucleic acid" can refer to either or both strands of the molecule.

[0043] The term "nucleotide sequence" with respect to nucleic acids refers to a contiguous series of nucleotides linked by covalent bonds, such as phosphorus bonds (e.g., phosphodiester, alkyl and aryl phosphonates, phosphorothioate, phosphotriester bonds) and / or non-phosphorus bonds (e.g., peptide and / or sulfamate bonds). In certain embodiments, for example, the nucleotide sequence encoding the target binding molecule linked to the localization domain is a heterologous sequence (e.g., a gene originating from a different species or cell type).

[0044] 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 can include, for example, nucleotides that contain naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides that contain modified bases known in the art.

[0045] As will be appreciated by one of skill in the art, in some embodiments, the nucleic acid further comprises a plasmid sequence, which can include, for example, one or more of a promoter sequence, a selection marker sequence, or a gene targeting sequence.

[0046] As used herein, "antibody" refers to an intact antibody or an antigen-binding fragment of an antibody, including intact antibodies or antigen-binding fragments that have been modified or engineered, or that are human antibodies. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Antigen binding Examples of fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (eg, scFv), minibodies, and diabodies.

[0047] 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 particular antibody will bind to a particular protein at least twice background, and more commonly more than 10 to 100 times background. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular protein. For example, polyclonal antibodies can 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).

[0048] 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. In general, 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 International Application PCT / WO88 / 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 can be designed and tested for optimal function, as provided in the art and as disclosed herein.

[0049] In certain embodiments, the anti-CD7 scFv 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 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 NO:1 and SEQ ID NO:2, 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: 1. 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, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO:2. or 100% sequence identity. In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO:1. 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, or 10) substitutions in the sequence set forth in SEQ ID NO:1. In some cases, the light chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO:2. In certain cases, 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:2. 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: 23. 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: 24.

[0050] In certain embodiments, the anti-CD7 scFv comprises a variable heavy chain (heavy chain variable region or VH) and a variable light chain (light chain variable region or VL) having sequences 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 NO:14 and SEQ ID NO:15, 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: 14. 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, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 15.

[0051] In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 14. In certain cases, the heavy chain variable region comprises no more than 10 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) substitution in the sequence set forth in SEQ ID NO: 14. 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: 15. In certain cases, the heavy chain variable region comprises no more than 10 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) substitution in the sequence set forth in SEQ ID NO: 15.

[0052] In some embodiments, the nucleic acid sequence encoding the VH has at least 90% sequence identity, at least 91% sequence identity, at least In some 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:26.

[0053] In certain embodiments, the anti-CD7 scFv comprises a variable heavy chain (heavy chain variable region or VH) and a variable light chain (light chain variable region or VL) having sequences 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 NO:16 and SEQ ID NO:17, 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: 16. 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, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VL sequence of SEQ ID NO: 17.

[0054] In some cases, the heavy chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 16. In certain cases, the heavy chain variable region comprises 13 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) substitutions in the sequence set forth in SEQ ID NO: 16. In some cases, the light chain variable region comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in the sequence set forth in SEQ ID NO: 17. In certain cases, the heavy chain variable region comprises 5 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) substitutions in the sequence set forth in SEQ ID NO: 17. 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:27. 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 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:28.

[0055] In some embodiments, the scFv of the invention is directed to the variable heavy chain sequence of an anti-CD7 antibody. , 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. In some embodiments, the scFv of the invention comprises a variable light chain sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the variable light chain sequence of an anti-CD7 antibody. For example, the anti-CD7 antibody can be any such antibody recognized by one of skill in the art.

[0056] [Table 1]

[0057] [Table 2-1] [Table 2-2] [Table 2-3]

[0058] The term "sequence identity" means that two nucleotide sequences or two amino acid sequences share at least 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% or more sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. For sequence comparison, generally, one sequence serves as a reference sequence (e.g., parent sequence), to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then determines the percent identity of the test sequence(s) relative to the reference sequence based on the designated program parameters. Calculate the sequence identity.

[0059] Optimal alignment of sequences for comparison can be performed, 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. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.)), or by visual inspection (see generally, Ausubel et al., Current Protocols in Molecular Biology). One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (publicly accessible through the National Institutes of Health NCBI Internet Server). Generally, sequence comparison can be performed using default program parameters, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0060] As will be understood by one of skill in the art, in certain embodiments, any of the sequences of the various components disclosed herein (e.g., scFv, intracellular signaling domain, hinge, linker, localization sequence, and combinations thereof) 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 the specific corresponding sequence 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: 3, so long as it has the desired function. In certain embodiments, the intracellular signaling domain of 4-1BB comprises the sequence set forth in SEQ ID NO: 3 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[0061] As another example, in certain embodiments, the intracellular signaling domain 4-1BB can be replaced with another intracellular signaling domain from a costimulatory molecule such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LAF1, 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, or a combination thereof to the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LAF1, or CD2. The sequence may have identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity.

[0062] As another example, in certain cases, the intracellular signaling domain of 4-1BB can also include another intracellular signaling domain (or a portion thereof) from a costimulatory molecule such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LAF1, or CD2. In some embodiments, the additional intracellular signaling domain can have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LAF1, 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 an intracellular signaling domain fragment(s) of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LAF1, or CD2.

[0063] As another example, in certain embodiments, the intracellular signaling domain CD3ζ 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: 4, so long as it has the desired function. In certain embodiments, the intracellular signaling domain of CD3ζ comprises the sequence set forth in SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0064] In some cases, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or a portion thereof, so long as it has a desired function. The intracellular signaling domain of the CAR can 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 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 a desired function.

[0065] In certain embodiments, the anti-CD7 CAR further comprises a hinge and transmembrane sequence. Hinge and transmembrane sequences suitable for use in the present invention are known in the art and are provided, for example, in WO 2016 / 126213, the entire contents of which are incorporated herein by reference. In certain embodiments, the hinge sequence is the sequence set forth in SEQ ID NO: 5 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR In certain embodiments, the transmembrane sequence comprises the sequence set forth in SEQ ID NO:6 (IYIWAPLAGTCGVLLLSLVITLYC). In some embodiments, the hinge and transmembrane domain of the anti-CD7 CAR can comprise a signaling domain (e.g., a transmembrane domain) from 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, FGFR2B, or other transmembrane proteins.

[0066] In certain embodiments, the anti-CD7 CAR further comprises a CD8α signal peptide (MALPVTALLLPLLLHLAARP, SEQ ID NO: 7). A schematic diagram of an anti-CD7 CAR including embodiments described herein is shown in FIG.

[0067] In certain aspects of the invention, the chimeric antigen receptor (CAR) 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.

[0068] As described herein, it has been shown that when anti-CD7 CARs are used in combination with downregulation of CD7 expression on effector T cells, the cytotoxicity of T cells is significantly increased. As demonstrated herein, downregulation (e.g., removal, reduction, and / or relocalization) of CD7 prevents the cognate killing effect exerted by the corresponding anti-CD7 CAR, resulting in greater T cell recovery after CAR expression compared to cells that retain the target antigen (e.g., CD7), and more effective cytotoxicity against T leukemia / lymphoma cells. As will be appreciated 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, "intrabodies" against CD7 (as described in WO 2016 / 126213), RNAi against CD7, or gene editing methods such as, for example, meganucleases, TALENs, CRISPR / Cas9, and zinc finger nucleases.

[0069] In certain embodiments, the engineered immune cell further comprises a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain. The "target binding molecule linked to a localization domain" may be referred to herein as a protein expression blocker (PEBL) or, in some cases, as an "intrabody" as described in International Publication No. WO 2016 / 126213, the teachings of which are incorporated by reference in their entirety. An exemplary embodiment of PEBL is shown in Figure 3E and Figure 17.

[0070] As used herein, "linked" in the context of protein expression blockers refers to a gene encoding a target binding molecule directly adjacent to and in frame with one or more genes encoding one or more localization domains (e.g., without a linker). Alternatively, the gene encoding the target binding molecule may be connected to one or more genes encoding one or more localization domains via a linker sequence, for example, as described in WO 2016 / 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.

[0071] In certain embodiments, the target binding molecule is an antibody that binds to CD7. In certain embodiments, the antibody is an scFv. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 1 and a VL sequence set forth in SEQ ID NO: 2. In certain embodiments, the scFv comprises , a VH sequence set forth in SEQ ID NO: 14, and a VL sequence set forth in SEQ ID NO: 15. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 16, and a VL sequence set forth in SEQ ID NO: 17. As described herein, in certain embodiments, the scFv comprises a VH and a VL that 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 VH and VL sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, SEQ ID NO: 14 and SEQ ID NO: 15, respectively, or SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0072] In some embodiments, the anti-CD7 protein expression blocker is made using the nucleic acid sequence of SEQ ID NO: 23 encoding the immunoglobulin heavy chain variable region of the anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 24 encoding the immunoglobulin light chain variable region of the anti-CD7 scFv. In other embodiments, the anti-CD7 protein expression blocker is made using the nucleic acid sequence of SEQ ID NO: 25 encoding the immunoglobulin heavy chain variable region of the anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 26 encoding the immunoglobulin light chain variable region of the anti-CD7 scFv. In a specific embodiment, the anti-CD7 protein expression blocker is made using the nucleic acid sequence of SEQ ID NO: 27 encoding the immunoglobulin heavy chain variable region of the anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 28 encoding the immunoglobulin light chain variable region of the anti-CD7 scFv.

[0073] In certain embodiments, as described herein, an antibody 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 (PEBL). By way of example only, an antibody that binds to CD7 in the context of a CAR may comprise a VH sequence set forth in SEQ ID NO: 1 and a VL sequence set forth in SEQ ID NO: 2, while an antibody that binds to CD7 in the context of a PEBL may comprise a VH sequence set forth in SEQ ID NO: 14 and a VL sequence set forth in SEQ ID NO: 15. In certain embodiments, as described herein, an antibody that binds to CD7 in the context of a CAR may be identical to an antibody that binds to CD7 in the context of a target binding molecule (PEBL).

[0074] In certain embodiments, the localization domain of PEBL comprises an endoplasmic reticulum (ER) or Golgi apparatus retention sequence, a proteosome localization sequence, and a transmembrane domain sequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, CD16, OX40, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B. In certain embodiments, the localization domain comprises the CD8α hinge and transmembrane domain (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIWAPLAGTCGVLLLSLVITLY) (SEQ ID NO: 10) followed by the endoplasmic reticulum (ER) retention peptide EQKLISEEDLKDEL (SEQ ID NO: 8), (GGGGS)4AEKDEL (SEQ ID NO: 9), or KYKSRRSFIDEKKMP (SEQ ID NO: 11), as described herein. The localization domain can direct PEBL to a specific cellular compartment, such as the Golgi apparatus or endoplasmic reticulum, the proteasome, or the plasma membrane, depending on the application. The ER or Golgi apparatus retention sequence comprises the amino acid sequence KDEL (SEQ ID NO: 18), KKXX (wherein X is any amino acid) (SEQ ID NO: 19), KXD / E (such as KXD or KXE) (wherein X is any amino acid) (SEQ ID NO: 20), or YQRL (SEQ ID NO: 21). The proteasome localization sequence may include a PEST (SEQ ID NO: 22) motif.

[0075] In some embodiments, proteasomal localization is achieved by isolating the scFv sequence from a tripartite motif. This is achieved by co-expressing a sequence encoding the human TRIM21 E3 ubiquitin ligase protein linked to a TRIM21 targeting domain sequence. 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 an scFv domain and an Fc domain. In this embodiment, the exogenously expressed TRIM21 protein binds to the scFv-Fc fusion protein bound to the target protein (e.g., CD7) and directs the complex to the proteasome for degradation.

[0076] Details of the amino acid sequence of the human TRIM21 E3 ligase protein can be found, for example, in the NCBI protein database under NCBI Ref.Seq.No.NP003132.2. Details of the nucleic acid sequence encoding the human TRIM21 E3 ligase protein can be found, for example, in the NCBI protein database under NCBI Ref.Seq.No.NM_003141.3.

[0077] In certain embodiments, the protein expression blocker is any one or more anti-CD7 PEBL as disclosed in WO 2016 / 126213, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Thus, the genetically engineered immune cells described herein can include PEBL (a target binding molecule linked to a localization domain) that binds to CD7, as described in WO 2016 / 126213. The sequence of components of anti-CD7 intrabodies as described in FIG. 2 and Tables 1 and 2 of WO 2016 / 126213. Exemplary embodiments of anti-CD7 PEBL are illustrated in FIG. 3E and FIG. 17.

[0078] [Table 3]

[0079] In some embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 1, an amino acid sequence of SEQ ID NO: 2, and a VH-VL linker. The VH-VL linker can be a (GGGGS)n linker, where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 1, an amino acid sequence of SEQ ID NO: 2, and an amino acid sequence of SEQ ID NO: 12. In some 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: 1, an amino acid sequence of SEQ ID NO: 2, an amino acid sequence of SEQ ID NO: 12, In certain embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 2, and an amino acid sequence of SEQ ID NO: 12. 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: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 2, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocker also comprises a localization domain selected from any one of the sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocker also comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide set forth in SEQ ID NO: 7. In other cases, 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: 10.

[0080] In some embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 14, an amino acid sequence of SEQ ID NO: 15, and a VH-VL linker. The VH-VL linker can be a (GGGGS)n linker, where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6. In one embodiment, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 14, an amino acid sequence of SEQ ID NO: 15, and an amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14, an amino acid sequence of SEQ ID NO: 15, and an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 14, an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 15, and an amino acid sequence of SEQ ID NO: 12. 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: 14, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocker also comprises a localization domain selected from any one of the sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocker also comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide set forth in SEQ ID NO: 7. In other cases, 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: 10.

[0081] In some embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 16, an amino acid sequence of SEQ ID NO: 17, and a VH-VL linker. The VH-VL linker can be a (GGGGS)n linker, where n can range from 1 to 5, e.g., 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 29). In one embodiment, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 16, an amino acid sequence of SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In some 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: 16, an amino acid sequence of SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the anti-CD7 protein expression blocker comprises an amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CD7 protein expression blocker has at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:16. and an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocker also includes a localization domain selected from any one of the sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocker also includes a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide set forth in SEQ ID NO: 7. In other cases, the anti-CD7 protein expression blocker also includes a CD8α hinge and transmembrane domain, such as, but not limited to, the CD8α hinge and transmembrane domain set forth in SEQ ID NO: 10.

[0082] In some embodiments, the nucleic acid sequence encoding anti-CD7 PEBL comprises one or more nucleic acid sequences set forth in Table 4. In some embodiments, the VH domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO:23, and the VL domain of the anti-CD7 scFv of PEBL comprises the nucleotide sequence of SEQ ID NO:24. In certain embodiments, the VH domain of the anti-CD7 scFv of PEBL 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:23, and the VL domain of the anti-CD7 scFv of PEBL 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:24.

[0083] [Table 4-1] [Table 4-2]

[0084] In some embodiments, the nucleic acid sequence encoding the localization domain of the anti-CD7 protein expression blocker comprises a sequence selected from SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, or a codon-optimized variant thereof.

[0085] In certain aspects of the invention, the protein expression blocker 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.

[0086] In some aspects of the present invention, the expression of 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, or CD137 can be downregulated using gene editing techniques, including but not limited to meganuclease, TALEN, CRISPR / Cas9, or zinc finger nuclease.For example, in some embodiments, CD7 expression is knocked out using genome editing with Cas9 / CRISPR.In other embodiments, CD5 expression is knocked out using genome editing with Cas9 / CRISPR.

[0087] As described above, downregulation of CD7 expression on effector T cells can be achieved according to various other known methods, including, for example, gene editing methods using meganucleases, TALENs, CRISPR / Cas9, and zinc finger nucleases. Thus, in certain embodiments, the genetically engineered immune cells further comprise modified CD7 genes, the modification of which renders the CD7 gene or protein non-functional. By way of example, the genetically engineered immune cells of the present invention further comprise modified (e.g., non-functional) CD7 genes (e.g., modified using meganucleases, TALENs, CRISPR / Cas9, or zinc finger nucleases) that prevent or reduce the expression of CD7 and / or otherwise (e.g., structurally) prevent the CD7 protein from being recognized by anti-CD7 CAR. Methods for modifying gene expression using such methods are readily available and well known in the art.

[0088] Methods for inactivating target genes in immune cells using CRISPR / Cas6 technology are described, for example, in U.S. Patent Application Publication Nos. 2016 / 0272999, 2017 / 0204372, and 2017 / 0119820.

[0089] The CRISPR / Cas system is a system for inducing targeted genetic engineering (genomic modification). Target recognition by the Cas9 protein requires a "seed" sequence in the guide RNA (gRNA) and a conserved multi-nucleotide-containing protospacer adjacent motif (PAM) sequence upstream of the gRNA binding region. The CRISPR / Cas system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines, primary cells, and engineered cells. The CRISPR / Cas system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making the system uniquely suitable for multiple gene editing or synergistic activation of target genes. Examples of CRISPR / Cas systems used to inhibit gene expression are described in U.S. Patent Application Publication No. 2014 / 0068797 and U.S. Patent Nos. 8,697,359 and 8,771,945. This system utilizes the RNA-guided Cas9 endonuclease to induce permanent gene disruption by introducing DNA double-strand breaks, which trigger error-prone repair pathways resulting in frameshift mutations. In some cases, the following enzymes are involved in the repair: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cm Other endonucleases may also be used, including, but not limited to, r5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsxX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, T7, Fok1, other nucleases known in the art, their homologs, or their variants.

[0090] CRISPR / Cas gene disruption occurs when a gRNA sequence specific to a target gene and a Cas endonuclease are introduced into a cell to form a complex that allows the Cas endonuclease to introduce double-strand breaks into the target gene. In some cases, the CRISPR system comprises one or more expression vectors that comprise a nucleic acid sequence encoding a Cas endonuclease and a guide nucleic acid sequence specific to a target gene. The guide nucleic acid sequence is specific to a gene and targets the gene for Cas endonuclease-induced double-strand breaks. The sequence of the guide nucleic acid sequence can be within the locus of the gene. In some embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more nucleotides in length. The guide nucleic acid sequence can be an RNA sequence, a DNA sequence, a combination thereof (RNA-DNA combination sequence), or a sequence with synthetic nucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA). The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0091] In some embodiments, the genetically engineered immune cells of the present invention can be modified via the CRISPR / Cas system to inactivate the human CD7 gene.Details of the genomic structure and sequence of the human CD7 gene can be found, for example, in GeneID No.924 of the NCBI Gene database.

[0092] Commercially available kits, gRNA vectors and donor vectors for knockout of specific target genes are available from, for example, Origene (Rockville, MD), GenScript (Atlanta, GA), Applied Biological Materials (ABM; Richmond, British Colombia), BioCat (Hedelberg, Germany), etc. Commercially available kits or kit components for knockout of CD7 by PR include, for example, those available from Origene under catalog numbers KN201231, KN201231G1, KN201231G2, and KN201231D, respectively, and those available from Santa Cruz Biotechnology under catalog numbers sc-4072847, sc-4072847-KO-2, sc-4072847-HDR-2, sc-4072847-NIC, sc-4072847HDR-2, and sc-4072847-NIC-2.

[0093] In some embodiments, the chimeric antigen receptors described herein can be introduced into the human CD7 locus using the CRISPR / Cas system.

[0094] In certain embodiments, genetically engineered immune cells are provided that comprise: i) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an intracellular signaling domain of 4-1BB and CD3ζ and an antibody that specifically binds to cluster of differentiation 7 (CD7); and ii) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, the target binding molecule being an antibody that binds to CD7, the localization domain comprising an endoplasmic reticulum retention sequence. In certain embodiments, the antibody that binds to CD7 in the context of the CAR and in the context of the target binding molecule comprises a VH sequence set forth in SEQ ID NO: 1 and a VL sequence set forth in SEQ ID NO: 2, a VH sequence set forth in SEQ ID NO: 14 and a VL sequence set forth in SEQ ID NO: 15, or a VH sequence set forth in SEQ ID NO: 16 and a VL sequence set forth in SEQ ID NO: 17. As described herein, in certain embodiments, the antibody comprises a VH and a VL having sequences comprising 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 NO:1 and SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:15, respectively, or SEQ ID NO:16 and SEQ ID NO:17, respectively. In certain embodiments, an antibody 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. In certain embodiments, the intracellular signaling domain of 4-1BB comprises the sequence set forth in SEQ ID NO:3. In certain embodiments, the intracellular signaling domain of CD3zeta comprises the sequence set forth in SEQ ID NO:4.

[0095] In another aspect, a nucleic acid comprising a nucleotide sequence encoding a CAR, as described herein, is also provided, wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to CD7.

[0096] In certain embodiments, the antibody is an scFv. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 1 and a variable light chain VL sequence set forth in SEQ ID NO: 2. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 14 and a variable light chain VL sequence set forth in SEQ ID NO: 15. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO: 16 and a variable light chain VL sequence set forth in SEQ ID NO: 17. As described herein, in certain embodiments, the scFv comprises a VH and a VL having sequences that 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 NO:1 and SEQ ID NO:2, respectively, SEQ ID NO:14 and SEQ ID NO:15, respectively, or SEQ ID NO:16 and SEQ ID NO:17, respectively. In certain embodiments, the CAR further comprises a hinge and a transmembrane sequence.

[0097] In certain embodiments, an isolated nucleic acid of the invention comprises a nucleotide sequence encoding a CAR according to Table 5. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a component of a CAR according to Table 5.

[0098] [Table 5]

[0099] In some embodiments, the anti-CD7 CAR comprises the amino acid sequence of SEQ ID NO:1, the amino acid sequence of SEQ ID NO:2, a 4-1BB intracellular signaling domain, a CD3 zeta intracellular signaling domain, and a CD8 hinge and transmembrane domain. In some embodiments, the anti-CD7 CAR can also be a VH-VL linker, such as, but not limited to, a (GGGGS)n linker, where n can range from 1 to 6, e.g., 1, 2, 3, 4, 5, or 6.

[0100] In one embodiment, the anti-CD7 protein expression blocker comprises the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 10. In some 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: 1 and at least 90% sequence identity to SEQ ID NO: 2. or at least 95% sequence identity to SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 10. In some 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: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 2, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 10. In some 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:1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:2, an amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:4, and an amino acid sequence of SEQ ID NO:10. In some 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:1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:2, an amino acid sequence of SEQ ID NO:3, an amino acid sequence of SEQ ID NO:4, and an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:10.In some 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:1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:2, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:3, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:4, and an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to SEQ ID NO:10.

[0101] In certain embodiments, an isolated nucleic acid of the invention comprises one or more nucleotide sequences of Table 6. In some embodiments, the nucleic acid comprises the nucleotide sequence of a component of a CAR listed in Table 6.

[0102] [Table 6-1] [Table 6-2]

[0103] In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a target binding molecule linked to the localization domain as described herein. In certain embodiments, the target binding molecule is an antibody that binds to CD7. In certain embodiments, the antibody is an scFv. In some embodiments, the scFv comprises a VH 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:1 and a VL sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the sequence of SEQ ID NO:2. In certain embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO:1 and a VL sequence set forth in SEQ ID NO:2. In some embodiments, the VH domain of the anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO:23 and the VL domain of the anti-CD7 scFv comprises the nucleotide sequence of SEQ ID NO:24.

[0104] In other aspects, methods of treating cancer in a subject in need of treatment are also provided, comprising administering to the subject a therapeutic amount of engineered immune cells having any of the embodiments described herein, thereby treating cancer in the subject in need of treatment.

[0105] In certain embodiments, as described herein, the methods include administering a therapeutic amount of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a CAR, the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to CD7.

[0106] In certain embodiments, the methods involve the use of a polypeptide linked to a localization domain as described herein. The method includes administering a therapeutic amount of engineered immune cells that further comprise a nucleic acid having a nucleotide sequence encoding a target binding molecule (e.g., an anti-CD7 protein expression blocker).

[0107] In certain embodiments, the cancer is a T-cell malignancy, e.g., a T-cell leukemia or T-cell lymphoma, such as 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, Sezary syndrome, primary cutaneous gamma delta T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, etc. In certain embodiments, the T-cell malignancy is early T-cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0108] As used herein, the terms "treat," "treating," or "treatment" refer to alleviating a medical condition (e.g., a condition associated with a T-cell malignancy) to the extent that the condition is ameliorated according to clinically acceptable criteria.

[0109] As used herein, "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. "Subject in need of 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., improved, ameliorated, prevented) by inducing T cells to exert specific cytotoxicity against malignant T cells.

[0110] As defined herein, a "therapeutic amount" refers to an amount sufficient to achieve a desired therapeutic effect (treat a condition associated with a T-cell malignancy) in a subject when administered to the subject under the conditions of administration. The effective amount of an agent to be administered can be determined by one of skill in the art using the guidelines provided herein and other methods known in the art, and depends on several factors, including, for example, the particular agent selected, the subject's age, sensitivity, tolerance to the drug, and general health.

[0111] 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.

[0112] In certain embodiments, the genetically 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 bed following surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration, and intraocular administration.

[0113] In certain embodiments, the genetically engineered immune cells are administered by injection into the 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 the symptoms of the disease. Typically, within 10 7 ~10 10 The dose of cells is set to a single dose, e.g., 10 9 The cells are injected at a dose of 10. 9 Cell dose or several 10 9 The cells are administered in either one of two separate doses. The frequency of injections can be daily, every 2-30 days, or at longer intervals as needed or indicated. The amount of injection is generally at least one injection, preferably at least three injections per subject, as tolerated or until disease symptoms are improved. 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 at the tumor site in an artificial scaffold, and intrathecal administration. Methods for adapting the present invention to such delivery modes are readily available to those skilled in the art. It is possible to use.

[0114] 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.

[0115] In another aspect, the use of the genetically engineered immune cells having any of the embodiments described herein for treating cancer is also provided, which comprises administering a therapeutic amount of the genetically engineered immune cells to a subject in need of treatment. In certain embodiments, the cancer is a T-cell malignancy. In certain embodiments, the T-cell malignancy is early T-cell precursor acute lymphoblastic leukemia (ETP-ALL).

[0116] In certain embodiments, the genetically 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.

[0117] In another aspect, a method of producing an engineered immune cell having any of the embodiments described herein is also provided, the method comprising introducing into an immune cell a nucleic acid comprising a nucleotide sequence encoding a CAR, the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to CD7.

[0118] In certain embodiments, the method further comprises introducing into the immune cell a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., anti-CD7 protein expression blocker or anti-CD7 PEBL) linked to a localization domain. In certain embodiments, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding anti-CD7 PEBL are introduced into a single plasmid.

[0119] In various aspects, kits are also provided for producing the genetically engineered immune cells described herein. The current kits can be used, for example, to produce allogeneic or autologous T cells with anti-CD7 CAR-mediated cytotoxic activity. In some embodiments, the kits are useful for producing allogeneic effector T cells with anti-CD7 CAR-mediated cytotoxic activity. In certain embodiments, the kits are useful for producing autologous effector T cells with anti-CD7 CAR-mediated cytotoxic activity.

[0120] Thus, kits are provided herein that include a nucleic acid comprising a nucleotide sequence encoding a CAR, the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that binds to CD7. The nucleotide sequence encoding an anti-CD7 CAR can be designed according to any of the embodiments described herein. In certain embodiments, the nucleotide sequence encodes an anti-CD7 CAR according to the schematic diagram in FIG. 1A ("anti-CD7-41BB-CD3ζ construct").

[0121] In certain embodiments, the kit further comprises a nucleic acid having a nucleotide sequence encoding a target binding molecule (e.g., an anti-CD7 PEBL molecule described herein) linked to a localization domain, as described herein. The nucleotide sequence encoding the target binding molecule linked to a localization domain can be designed according to any of the embodiments described herein.

[0122] In certain embodiments, the nucleotide sequence encoding the anti-CD7 CAR and / or the nucleotide sequence encoding the anti-CD7 PEBL further comprises sequences (e.g., plasmid or vector sequences) that allow, for example, cloning and / or expression. For example, the nucleotide sequence can be, for example, transfected into a cell (e.g., an immune cell). In certain embodiments, the nucleotide sequence encoding the anti-CD7 CAR and the nucleotide sequence encoding the anti-CD7 PEBL can be provided as part of a plasmid to facilitate cloning into other plasmids and / or vectors (expression vectors or viral expression vectors) for injection, transduction, or electroporation. ... In certain embodiments, the nucleotide sequences are provided on separate plasmids or vectors (expression vectors or viral expression vectors).

[0123] Generally, the kits are compartmentalized for ease of use and may include one or more containers with 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 separate package from the other kit components. The kits may also include instructions for using the kit components.

[0124] In some embodiments, provided herein is a genetically engineered immune cell comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that binds to cluster of differentiation 7 (CD7). In certain embodiments, the antibody is a single chain variable fragment (scFv). In some cases, the scFv comprises a heavy chain variable domain (VH) sequence set forth in SEQ ID NO:1 and a light chain variable domain (VL) sequence set forth in SEQ ID NO:2.

[0125] In some embodiments, the CAR further comprises a hinge and transmembrane sequence, such as, but not limited to, a hinge and transmembrane domain comprising the amino acid sequence of SEQ ID NO:10.

[0126] In some embodiments, the engineered immune cells are engineered T cells, engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages, or engineered dendritic cells.

[0127] In some embodiments, the engineered immune cell further comprises a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to the localization domain. In certain embodiments, the target binding molecule is an antibody that binds to CD7. In certain embodiments, the antibody is an scFv. In some embodiments, the scFv comprises a VH sequence set forth in SEQ ID NO:1 and a VL sequence set forth in SEQ ID NO:2. In some embodiments, the localization domain comprises an endoplasmic reticulum (ER) or Golgi apparatus retention sequence, a proteosome localization sequence, and a transmembrane domain sequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B.

[0128] In some embodiments, provided herein is a genetically engineered immune cell comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an intracellular signaling domain of 4-1BB and CD3ζ and an antibody that binds to cluster of differentiation 7 (CD7); and (ii) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, wherein the target binding molecule is an antibody that binds to CD7, wherein the localization domain comprises an endoplasmic reticulum retention sequence, and wherein the antibody that binds to CD7 comprises a variable heavy chain (VH) sequence set forth in SEQ ID NO: 1 and a variable light chain (VL) sequence set forth in SEQ ID NO: 2. In some embodiments, the intracellular signaling domain of 4-1BB comprises the sequence set forth in SEQ ID NO: 3, and the intracellular signaling domain of CD3ζ comprises the sequence set forth in SEQ ID NO: 4.

[0129] In some embodiments, provided herein is a method of treating cancer in a subject in need of treatment, comprising administering to the subject a therapeutic amount of the genetically engineered immune cells described herein, thereby treating the cancer in the subject in need of treatment. In some embodiments, the cancer is a T-cell malignancy. In certain embodiments, the T-cell malignancy is early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). In certain embodiments, the genetically 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 after surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration.

[0130] In some embodiments, provided herein is a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to cluster of differentiation 7 (CD7).

[0131] In other embodiments, provided herein is a method for treating cancer comprising administering a therapeutic amount of the genetically engineered immune cells to a subject in need of treatment. In some embodiments, the cancer is a T-cell malignancy. In certain embodiments, the T-cell malignancy is early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). In certain embodiments, the genetically 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 after surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration.

[0132] In some embodiments, methods are provided herein for producing engineered immune cells as described herein. The methods can include introducing a nucleic acid comprising a nucleotide sequence encoding a CAR into an immune cell, the CAR comprising an intracellular signaling domain of 4-1BB and CD3ζ and an antibody that binds to CD7, thereby producing an engineered immune cell. The methods can further include introducing a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain into the immune cell.

[0133] The present invention provides a chimeric antigen receptor (CAR) against CD7. As demonstrated herein, expression of anti-CD7 CAR in immune cells, such as effector T cells, induces the T cells to exert specific cytotoxicity against T cell malignancies. This cytotoxic effect was shown to be enhanced when expression of CD7 on effector T cells was downregulated using an antibody-based molecule (protein expression blocker or PEBL) that targets CD7 for downregulation. Thus, the present invention provides an immunotherapeutic method for treating cancer, e.g., T cell malignancies.

[0134] In some aspects, the present invention provides engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that binds to cluster of differentiation 7 (CD7). In some embodiments, the engineered immune cells outlined herein also comprise a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., protein expression blocker or PEBL) linked to a localization domain. Methods and kits for producing such engineered immune cells are also outlined herein.

[0135] In some aspects, the invention provides a nucleic acid comprising (i) a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an intracellular signaling domain of 4-1BB and CD3ζ and an antibody that specifically binds to CD7; and (ii) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, the nucleic acid comprising: The target binding molecule is an antibody that binds to CD7, and the localization domain comprises an endoplasmic reticulum retention sequence, and the antibody that binds to CD7 comprises a nucleic acid comprising a variable heavy chain (VH) sequence set forth in SEQ ID NO:1 and a variable light chain (VL) sequence set forth in SEQ ID NO:2.

[0136] In another aspect, the present invention provides a method for treating cancer (e.g., T-cell malignancies) in a subject in need of treatment. The method comprises administering to the subject a therapeutic amount of any of the genetically engineered immune cells described herein, thereby treating the cancer in the subject in need of treatment. The present disclosure also describes the use of any of the genetically engineered immune cells outlined herein to treat cancer.

[0137] In another aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that specifically binds to CD7. EXAMPLES

[0138] Example 1: Blockade of CD7 expression on T cells for effective chimeric antigen receptor targeting of T cell malignancies This example illustrates the novel blockade of CD7 expression in combination with a second generation CAR, resulting in highly potent anti-CD7 CAR-T cells. This practical strategy offers a new treatment option for patients with high-risk T-cell malignancies, including ETP-ALL. overview

[0139] Effective immunotherapies for T-cell malignancies are lacking. A novel approach based on chimeric antigen receptor (CAR)-transformed T lymphocytes was devised. CD7 was chosen as a target due to its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL), the median CD7 expression was >99%, and CD7 expression remained high at relapse (n=14) and during chemotherapy (n=54). CD7 was targeted by a second-generation CAR (anti-CD7-41BB-CD3ζ), but due to the presence of CD7 on the T cells themselves, CAR expression in T lymphocytes triggered cognate killing. To downregulate CD7 and control cognate killing, a novel method based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain (protein expression blocker, PEBL) was applied. Transduction of anti-CD7 PEBL resulted in a virtually immediate suppression of surface CD7 expression in all transduced T cells, with 2.0% ± 1.7% being CD7+ versus 98.1% ± 1.5% of mock-transduced T cells (n = 5, P < 0.0001). PEBL expression did not impair T cell proliferation, IFNγ and TNFα secretion, or cytotoxicity, nor did it abrogate CAR-mediated cognate killing. PEBL-CAR-T cells were highly cytotoxic to CD7+ leukemia cells in vitro and were consistently more potent than CD7+ T cells that escaped cognate killing. They also demonstrated robust anti-leukemia activity in cell line-derived and patient-derived T-ALL xenografts. The strategy described here fits well into existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies. Introduction

[0140] T lymphocytes can be directed to specifically recognize and kill tumor cells through expression of chimeric antigen receptors (CARs) 1-5Central to the effective application of this technology is the identification of a suitable target for CAR. The target must be highly expressed by tumor cells and should not be present in normal cells or its temporary absence should be clinically manageable. Should be expressed only by certain normal cells 6 Thus, B-cell derived leukemias and lymphomas are usually expressed exclusively by B lymphoid cells. 9,10 , CD19 5,7 or CD22 8 Infusion of autologous T cells expressing anti-CD19 CAR in patients with B-cell refractory leukemia and lymphoma resulted in major clinical responses 11-18 These exciting results provide clear evidence of the power of this technology and suggest the possibility of broader applications in oncology.

[0141] The development of CAR-T cell therapies for T cell malignancies has lagged far behind that of their B cell counterparts. The need for effective treatments in this field is particularly urgent due to the poor prognosis associated with several T cell leukemia and lymphoma subtypes. For example, children and adolescents with early stage T cell precursor acute lymphoblastic leukemia (ETP-ALL) have the poorest response to initial treatment among all ALL patients. 19-21 Intensive chemotherapy and / or allogeneic hematopoietic stem cell transplantation often do not prevent refractory relapse, leaving these patients, and those with other high-risk features such as adult age, lacking treatment options. 19,22-25 .

[0142] A major obstacle to the development of effective CAR-T cells for T-cell malignancies is that the surface marker profile of malignant T cells, which generally lack CD19 or CD22 expression, largely overlaps with that of activated T lymphocytes. 19-26 CARs directed against such targets are likely to lead to self-elimination of CAR-T cells. 27,28The development and application of practical technology for CAR-T cell therapy of ETP-ALL and other T-ALL cell subtypes is described herein. First, a CAR against CD7 was created. As is recognized, CD7 is a 40 kDa type I transmembrane glycoprotein that is a primary marker for T-cell malignancies. 29-32 , highly expressed in all cases of T-cell ALL, including ETP-ALL 19 Second, a method was developed to rapidly and effectively downregulate CD7 expression in T cells, which was selected because it avoids the homozygous killing effect of CAR-T cell therapy, does not involve gene editing, and can be readily translated into clinical applications. material and method

[0143] Cells and culture conditions

[0144] The leukemia cell lines Jurkat, CCRF-CEM, Loucy, MOLT4, and KG1a were obtained from the American Type Culture Collection (ATCC; Rockville, MD). The B-lineage ALL cell line OP-1 was developed in the applicant's laboratory. 33 CCRF-CEM cells were transduced with a murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) retroviral vector containing the firefly luciferase gene (obtained from the Vector Development and Production Shared Resource at St. Jude Children's Research Hospital, Memphis, TN). The same vector was used to transduce CCRF-CEM and Jurkat cells with the CD19 gene cloned from the cDNA of the RS4;11 B cell line (ATCC). Cell lines were maintained in RPMI-1640 (ThermoFisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0145] Peripheral blood samples were obtained from discarded anonymous by-products of platelet donations from healthy adult donors at the National University Hospital Blood Bank, Singapore. Bone marrow aspirates were collected from ALL patients for diagnostic immunophenotyping and monitoring of treatment response. 19,26 Some experiments were approved by the Institutional Review Board of the National University of Singapore. The stored surplus material was used. Mononuclear cells were isolated by centrifugation on a Lymphoprep density step (Axis-Shield, Oslo, Norway) and washed twice in RPMI-1640. T cells were enriched with Dynabeads human T activator CD3 / CD28 (ThermoFisher) and cultured in RPMI-1640, 10% FBS, 1% penicillin-streptomycin, and interleukin-2 (IL-2; 120 IU / mL; Proleukin, Novartis, Basel, Switzerland).

[0146] Gene cloning and retroviral transduction

[0147] Single-chain variable fragment (scFv) of the anti-CD7 monoclonal antibody TH69 34 was linked to the CD8α signal peptide, the CD8α hinge and transmembrane domains, and the intracellular domains of 4-1BB and CD3ζ of the anti-CD19-41BB-CD3ζ CAR previously developed in the Applicant's laboratory. 5 The same scFvs were also linked to sequences encoding the CD8α hinge and transmembrane domains followed by the CD8α signal peptide and the endoplasmic reticulum (ER) / Golgi retention peptides EQKLISEEDLKDEL (SEQ ID NO:8), (GGGGS)4AEKDEL (SEQ ID NO:9), or a localization sequence (SEQ ID NO:13), and were subcloned into the MSCV vector in the presence or absence of GFP or mCherry.

[0148] Retroviral supernatant preparation and transduction were performed as previously described. 5,35Briefly, pMSCV retroviral vector-conditioned medium was added to RetroNectin (Takara, Otsu, Japan)-coated polypropylene tubes, and after centrifugation and removal of the supernatant, T cells were added to the tubes and left at 37°C for 12 h, and fresh viral supernatant was added for 2 consecutive days thereafter. T lymphocytes were maintained in RPMI-1640 containing FBS, antibiotics, and 200 IU / mL IL-2.

[0149] For transient CAR expression, anti-CD7 and anti-CD19 CAR constructs were subcloned into the EcoRI and Xhol sites of the pVAXI vector (ThermoFisher Scientific) and transcribed into mRNA using T7 mScript (CellScript, Madison, WI). 36 For mRNA electroporation, cells were suspended in electroporation buffer (Amaxa Cell Line Nucleofector Kit V; Lonza, Basel, Switzerland) containing 200 μg of CAR mRNA and electroporated with an Amaxa Nucleofector 2b (Lonza) using program X-001. 36,37 Cells electroporated in the absence of mRNA were used as a control.

[0150] Detection of CAR, PEBL, and surface markers

[0151] CAR was detected with biotin-conjugated goat anti-mouse F(ab')2 antibody (Jackson ImmunoResearch, West Grove, PA), followed by allophycocyanin (APC)-conjugated streptavidin (Jackson ImmunoResearch). Phycoerythrin (PE) or APC-conjugated anti-CD7 (M-T701), CD4 (RPA-T4), CD8 (RPA-T8), CD3 (SK7), and non-reactive isotype-matched antibodies were obtained from BD Biosciences (San Jose, CA), and CD19 (LT19) was obtained from Miltenyi Biotech. Cell staining was performed using Accuri C6, Fortessa, or LSRII flow cytometers (BD Biosciences) with Diva (BD Biosciences) or Flow Jo software (FlowJo, Ashland, OR). Analyzed.

[0152] Western blotting was performed as previously described. 35 Briefly, cell lysates were extracted using CelLytic M cell lysis reagent (Sigma-Aldrich, Saint Louis, MO) prior to protein quantification with the Pierce BCA Protein Assay Kit (ThermoFisher). Cell lysates were diluted with 4× Laemmli sample buffer (Bio-rad, Hercules, CA) and resolved on 10% polyacrylamide gels by electrophoresis under reducing or non-reducing conditions. Blotted membranes were probed with mouse anti-human CO3 antibody (8D3; BD Biosciences), goat anti-mouse IgG horseradish peroxidase conjugate (R&D Systems, Minneapolis, MN), and Clarity Western ECL substrate (Bio-Rad). Staining was visualized using a ChemiDoc Touch Imager (Bio-Rad).

[0153] Cell aggregation assay, cytotoxicity assay and cytokine production

[0154] To measure cell-cell aggregation, Jurkat cells were cocultured with calcein red orange AM (ThermoFisher)-labeled CD7+ or CD7- cells for 30 min, and cell doublets were counted by flow cytometry. In some experiments, target cells were preincubated for 10 min before coculture with soluble anti-CD7 scFv obtained from the supernatant of Jurkat or 293T cells transduced with constructs consisting of scFv without transmembrane or signaling sequences.

[0155] To test cytotoxicity, target cells were labeled with calcein red-orange AM and placed in 96-well round-bottom plates (Corning Costar, Corning, NY). T cells were added with target cells at different effector:target (E:T) ratios and cultured for 4 h at 37 °C and 5% CO2. Viable target cells were counted by flow cytometry. 38 To measure exocytosis of lytic granules, anti-human CD107a-PE (H4A3; BD Biosciences) was added to the co-cultures. After 1 h, monensin (BD GolgiStop) was added and culture was continued for another 3 h before flow cytometry analysis.

[0156] To assess cell proliferation, T cells were cultured alone or in the presence of MOLT-4 cells 1:1 E:T in RPMI-1640 containing FBS and 120 IU / mL IL-2 at 37 °C and 5% CO2. To inhibit proliferation, irradiated or Streck cell preservative (Streck Laboratories, Omaha, NE)-treated target cells were added to the cultures every 7 days. Viable GFP+ or mCherry+ T cells were counted by flow cytometry. For IFNγ and TNFα production, target and effector cells were plated as above in 1:1 E:T. After 1 h, Brefeldin A (BD GolgiPlug) was added to the cultures and the cultures were continued for another 5 h. Subsequently, intracellular staining with anti-IFNγ-PE (clone 25723.11; BD Biosciences) or anti-TNFα-PE (6401.1111; BD Biosciences) was performed before flow cytometry analysis.

[0157] Xenograft model

[0158] CCRF-CEM cells were transduced with luciferase and NOD.Cg-Prkdc scid IL2rg tm1Wjl / SzJ (NOD / scid IL2RGnull) mice (Jackson Laboratory, Bar Harbor, ME) were incubated at 1 × 10 6 After 3 and / or 7 days, mice were injected intravenously with 100 μg / mL of 10 ... 2 x 10 per mouse 7Mice received T cells with downregulated CD7 and anti-CD7 CAR expression in T cells. Other mice received GFP alone or T cells transduced in RPMI-1640 with 10% FBS instead of T cells. All mice received 20,000 IU of IL-2 intraperitoneally (ip) every 2 days. Tumor burden was determined using a Xenoge IVIS-200 system (Caliper Life Sciences, Waltham, MA) after intraperitoneal injection (2 mg per mouse) of aqueous D-luciferin potassium salt (Perkin Elmer, Waltham, MA). Luminescence was analyzed with Living Image 3.0 software. Luminescence was measured at 1 × 10 per second. 10 Mice were euthanized when photons were reached or earlier if physical signs justifying euthanasia appeared.

[0159] For patient-derived xenograft (PDX) models, primary ETP-ALL cells were injected intravenously into NOD / scid IL2RGnull mice and expanded for the next 7-8 generations. ETP-ALL cells were then re-injected into NOD / scid IL2RGnull mice that had been treated with PEBL-CAR-T cells or left untreated. Peripheral blood and tissues were monitored for the presence of ALL cells by flow cytometry. 19,26 After red blood cells were lysed with lysis buffer (Sigma-Aldrich), cells were stained with anti-mouse CD45-PE-Cyanine 7 (30-F11, Biolegend), and anti-human CD45-APC-H7 (2D1), CD7-PE (M-T701), CD3-APC (SK7), CD34-Peridinin Chlorophyll Protein (8G12) (all from BD Biosciences), and CD33-Brilliant Violet 421 (WM53, Biolegend). Cells were analyzed on a Fortessa flow cytometer using Diva and FlowJo software.

[0160] result

[0161] Validating CD7 as a target for CAR-T cell therapy in leukemia

[0162] In leukemic cells from diagnostic bone marrow samples obtained from 49 T-ALL patients (including 14 with ETP-ALL), the median percentage of CD7 expression was >99% (range, 79% to >99%). In only three cases (6.1%), CD7 was <99%, 98% in two, and 79% in one (Figure 1A). High CD7 expression was also observed in samples collected from 14 relapsed T-ALL patients (Figure 1A). The mean fluorescence intensity (MFI) of CD7 in leukemic cells at diagnosis or relapse always exceeded the MFI measured in residual normal T cells in the same samples. The median (range) MFI was 20,617 (4,105-66,674) in T-ALL cells versus 3,032 (1,301-9,582) in normal T cells (n=19, P<0.0001) (Fig. 1B).

[0163] To determine whether chemotherapy affects CD7 expression, bone marrow samples containing minimal residual disease (MRD) collected during treatment were examined. In all 54 samples (from 21 patients), >99% of the residual leukemic cells were CD7+ (Figure 1A). In 18 patients, CD7 levels were monitored throughout the course of disease. As shown in Figure 1C and Figure 1D, CD7 remained high during treatment. These results demonstrate that CD7 is a valid target for CAR-T cell therapy in T-ALL.

[0164] Anti-CD7 CAR design and expression

[0165] To target CD7, the anti-CD7 antibody TH was conjugated through the hinge and transmembrane domains of CD8α to the signaling domains of 4-1BB (CD137) and CD3ζ. We designed an anti-CD7 CAR (Figure 2A) consisting of 69 scFvs. Retroviral transduction of this construct in Jurkat cells resulted in high expression of the anti-CD7 CAR (Figure 2B), which appeared as monomers, dimers, and oligomers by Western blotting (Figure 2C).

[0166] To confirm that TH69 scFv can bind CD7, it was produced in a soluble form and tested on CD7+ MOLT-4 and CD7- OP-1 cells; it labeled MOLT-4 cells but not OP-1 (Figure 8A). Furthermore, when MOLT-4 cells were preincubated with anti-CD7 scFv supernatant, staining with anti-CD7 monoclonal antibody was significantly reduced, and CD7 MFI (±SD) changed from 31,730±1,144 to 5,987±241 (n=3). Jurkat cells expressing anti-CD7 CAR formed aggregates with CD7+ MOLT-4 cells, but cells transduced with GFP alone or anti-CD19 CAR did not; conversely, anti-CD19 CAR induced cell aggregation with CD19+ OP-1 cells, but anti-CD7 CAR did not (Figure 8B). Preincubation of MOLT-4 or CCRF-CEM with soluble anti-CD7 scFv prevented aggregate formation (Fig. 8C ).

[0167] To determine whether the anti-CD7 CAR was functional, the levels of activation markers CD25 and CD69 were measured in Jurkat cells after 24 h of coculture with MOLT4. There was a clear upregulation of both activation markers in cells expressing the anti-CD7 CAR (Figure 2D and Figure 2E). In summary, the anti-CD7-41BB-CD3ζ CAR is able to bind its cognate antigen and transmits an activation signal upon ligation.

[0168] Expression of anti-CD7 CAR in T cells triggers cognate killing

[0169] To determine the effect of anti-CD7-41BB-CD3ζ CAR in peripheral blood T lymphocytes, two different methods were used to express it: retroviral transduction (Figure 9A) and mRNA electroporation. However, it significantly reduced T cell viability. The mean (±SD) T cell recovery after 24 hours of mRNA electroporation was 39.8% ± 13.0 (n = 7) recovery after electroporation in the absence of mRNA (Figure 3A), and when the CAR was introduced by viral transduction, the cell recovery was 25.1% ± 16.2% (n = 10) recovery of mock-transduced T cells (Figure 3B), and overall, CAR expression reduced the cell recovery to 31.1% ± 16.3% (n = 17) after 24 hours. Long-term cell culture further increased the number difference between CAR-transduced and mock-transduced cells overall (Figure 3C). CAR expression in the absence of target cells induced exocytosis of lytic granules revealed by CD107a expression (Figure 3D), suggesting that the impairment of cell recovery was caused by cognate killing.

[0170] Downregulation of CD7 prevents T cell cognate killing and does not affect T cell function

[0171] If the poor T-cell recovery was caused by cognate killing mediated by CAR binding to CD7 expressed by T cells, it should be improved by downregulating CD7 before CAR expression. To test this prediction, we applied a recently developed rapid and practical method based on the expression of anti-CD7 scFvs linked to amino acid sequences containing the ER retention domains KDEL or KKMP [anti-CD7 protein expression blocker (PEBL)] (Figure 3E). These anchor the constructs to the ER / Golgi apparatus and prevent secretion or membrane expression of the target protein. 39,40 Three anti-CD7 PEBL constructs were tested, and PEBL-1 was the PEBL-1 of choice for subsequent experiments (Figure 3E and Figure 3C). F). CD7 surface expression was essentially abolished in all T cells transduced with this construct, while CD7 mRNA expression was retained (Fig. 3F, Fig. 10A and Fig. 10B), and in five experiments, 98.1% ± 1.5% of mock-transduced T cells were CD7+, compared with 2.0% ± 1.7% of anti-CD7 PEBL-transduced T cells (P<0.0001) (Fig. 3G). When anti-CD7 CAR was expressed by electroporation in cells with downregulated CD7, its expression was clearly detectable by flow cytometry (Fig. 3H). By expressing the CAR in cells with CD7 knockdown, T cell viability was significantly improved (Fig. 3I), and in six paired experiments, viable cell recovery after CAR mRNA electroporation was consistently superior in T cells that had been pre-transduced with anti-CD7 PEBL (P=0.008).

[0172] After anti-CD7 PEBL transduction, the ratio of CD4 to CD8 cells was similar to that of mock-transduced cells (Figure 4A). The absence of CD7 expression on the surface membrane did not affect T cell survival in culture (Figure 4B). To further probe the functional capacity of anti-CD7 PEBL-transduced T cells, cells were engineered to express anti-CD19-CAR (Figures A-C). Their ability to exert cytotoxicity, release cytotoxic granules, and secrete IFNγ in the presence of CD19+ ALL cells was tested. As shown in Figures 4D, E, and F, PEBL transduction and the lack of surface CD7 did not alter CAR-mediated cell function.

[0173] Anti-CD7-41BB-CD3ζ CAR induces potent cytotoxicity against CD7+ leukemia cells

[0174] CD7-negative T cells were prepared with anti-CD7 PEBL and electroporated with anti-CD7-41BB-CD3ζ CAR mRNA. Their anti-leukemic capacity was evaluated in coculture with the CD7+ leukemic cell lines MOLT-4, CCRF-CEM, Jurkat, Loucy or KG1a. As shown in Figure 5A, cytotoxicity was dramatically increased by CAR expression. PEBL-CAR T cells were also highly effective against primary T-ALL cells obtained from patients (Figure 5B).

[0175] The cytotoxicity of PEBL-CAR T cells was compared to that of residual T cells recovered after CAR electroporation in cells not transduced with PEBL. In 45 experiments with cells from three donors, the cytotoxicity of PEBL-CAR cells consistently exceeded that of non-PEBL T cells (Figure 5C). Superior activity of PEBL-CAR cells was also observed when comparing expression of CD107a (Figure 5D), IFNγ (Figure 11A) and TNFα (Figure 11B). Expression of PEBL and CAR by sequential retroviral transduction also resulted in potent cytotoxicity against patient-derived T-ALL cells (Figure 5E) and cell lines (Figure 12). Proliferation of anti-CD7 PEBL-CAR-T cells in the presence of CD7+ target cells was much higher (P<0.01) than that of CAR-T without CD7 downregulation by PEBL (Figure 5F). Finally, we compared the cytotoxicity exerted by anti-CD7 PEBL-CAR T cells with that exerted by anti-CD19-41BB-CD3ζ CAR T cells against the same target cells. 5The cytotoxicity of T cells expressing CD19 was compared with that of T cells expressing CD19. For this purpose, CCRF-CEM cells and Jurkat cells were transduced with CD19, and both CARs were also expressed in cells pre-transduced with anti-CD7 PEBL (Figure 13A and Figure 13B). Anti-CD7 and anti-CD19 CAR T cells had similar short-term and long-term cytotoxicity (Figure 13C and Figure 13D), and the long-term proliferation capacity in the presence of CD19+ CD7+ target cells was slightly lower for anti-CD7 CAR-T cells (Figure 13E), which could be explained by the lower expression of CD7 on target cells relative to CD19 (Figure 13B).

[0176] Anti-leukemic activity of anti-CD7 PEBL-CAR T cells in a mouse model of T-ALL

[0177] To further evaluate the anti-tumor capacity of anti-CD7 PEBL-CAR T cells, NOD / scid IL2RGnull mice were transplanted with CCRF-CEM cells. T cells retrovirally transduced with anti-CD7 PEBL and anti-CD7 CAR produced a striking anti-leukemic effect, with a marked reduction in leukemic cell burden and reduced leukemic cell proliferation (Figures 6A-C, 14A and 14B). Three weeks after leukemic cell infusion, the median percentage of CCRF-CEM cells in peripheral blood by flow cytometry was 68% (n=5) in control mice and 67% (n=5) in mice that received GFP-only T cells, whereas anti-CD7 It was not detected in mice treated with PEBL-CAR T cells (Figure 15A). Relapses occurring after anti-CD7 PEBL-CAR T cell therapy were not due to a CD7-depleted CCRF-CEM cell subset; leukemic cells continued to express high levels of CD7 and remained highly sensitive to anti-CD7 CAR cytotoxicity, regardless of whether CCRF-CEM cells were derived from the liver or spleen of relapsed mice or directly from the original cell culture (Figure 15B).

[0178] To test PEBL-CAR T cells against primary leukemia cells in vivo, a PDX model of ETP-ALL was used. The PDX model allows the growth of leukemia cells derived from patients with ETP-ALL at diagnosis in NOD / scid IL2RGnull mice. The leukemia cells expressed CD7, CD34, CD33, and were absent of surface CD3, CD1a, CD8, and CD5, retaining the immunophenotypic match determined at diagnosis (Figure 16), and the cells could not survive or grow ex vivo and needed to be injected into mice for growth. All mice had ETP-ALL in the peripheral blood at the time of CAR-T treatment (Figure 7A). As shown in Figure 7B, ETP-ALL cells represented the majority of leukocytes in the bone marrow, spleen liver, and lungs. After PEBL-CAR T cell administration (2x10 in one mouse), 7 pcs, and the remaining 4 are 2×10 6 After 2 × 10 transplantation, the number of leukemic cells in the peripheral blood was dramatically reduced, while PEBL-CAR-T cells became detectable in all mice (Figure 7A). Blood smears showed prominent smear cells, suggesting leukemic cell lysis (Figure 7C). Leukemia progressed in all five control mice, which were euthanized when ETP-ALL represented >80% of peripheral blood mononuclear cells. 7 Mice treated with 2 × 10 PEBL-CAR-T cells died of apparent graft-versus-host disease (GvHD) 23 days after PEBL-CAR-T cell infusion. ETP-ALL was not detected in blood, bone marrow, liver, spleen, lung, and brain, whereas PEBL-CAR T cells were detectable in all tissues (Figure 7D and Figure 7E). 6 Four mice treated with PEBL-CAR T cells are alive and without signs of GvHD 25 (n=1) to 39 (n=3) days after infusion. Consideration

[0179] Although remissions for patients with B-cell leukemias and lymphomas can be achieved with CAR-T cells, for patients with T-cell malignancies, effective options are lacking. To fill this gap, a CAR-T cell approach was developed and described herein that could be rapidly translated into clinical intervention. CD7, a widely expressed surface T-cell marker that is highly stable even in chemotherapy-exposed T-ALL cells, was targeted. A second generation anti-CD7 CAR was designed. Suppression of CD7 surface expression on T cells was determined to be essential, and without it, the CAR would have caused severe T-cell depletion and the full functional potential of the CAR-T cells would not have been achieved. Transduction of anti-CD7 PEBL resulted in virtually immediate suppression of CD7 expression. Expression of the anti-CD7 CAR on such cells was shown to be effective in vitro and in xenografts of T-ALL and P-ALL. This resulted in potent anti-leukemic activity in the DX model. Thus, using this strategy, large numbers of CAR-T cells were rapidly generated and used to exert stable and specific cytotoxicity against T-cell malignancies, including one of the most aggressive forms of ETP-ALL.

[0180] The PEBL technique described here to downregulate endogenous CD7 is based on the use of scFvs against target antigens coupled with ER / Golgi retention motifs. In this way, any newly synthesized CD7 remains anchored in the ER and / or Golgi, and its surface expression is prevented. This method was remarkably effective in downregulating CD7 and suppressing CAR-mediated allogeneic killing. Importantly, intracellular retention of CD7 did not alter T cell function, allowing normal proliferation, cytokine secretion, and cytotoxicity. This is consistent with the results of studies using CD7-deficient mice, which displayed normal lymphocyte populations in lymphoid tissues. 41,42 Another approach to downregulate CD7 would be to apply gene editing methods such as meganucleases, TALEN, or CRISPR / Cas9. 43To this end, a recent study reported an anti-CD7 CAR expressed in T cells with CRISPR / Cas-mediated CD7 gene deletion. 9,44 In addition to differences in costimulatory molecules (the CARs described herein have 4-1BB instead of CD28) that may have clinical impact. 45,46 The high specificity and practical nature of the PEBL strategy make it particularly attractive for current clinical applications. The method requires simple transduction with the same viral vector carrying the CAR, either as two sequential transductions or a single transduction with a bicistronic vector carrying both constructs. The transduction fits well into established clinical-grade cell manufacturing processes and does not raise possible regulatory concerns related to off-target activity. 47,48 .

[0181] CD7 is a molecule characteristic of early T cell differentiation and is nearly ubiquitously expressed in T-ALL, whereas in normal cells its expression is restricted to T cells. 19,29-32 In clinical trials with anti-CD7-ricin A-chain immunotoxins in patients with T-cell lymphoma, the dose-limiting toxicity was a vascular leak syndrome, a side effect seen with other toxin conjugates, and anti-CD7 binding was not seen on endothelial cells in a variety of tissues. 49 Nevertheless, transient expression of CAR by mRNA electroporation may be considered in early studies evaluating the potential for acute toxicity of anti-CD7 PEBL-CAR T cells. A concern with anti-CD7 CAR therapy is the depletion of normal T cells by the infused cells, which could lead to immunodeficiency. One could envision a first application of this technology as a means to reduce MRD in high-risk T-ALL patients, thus maximizing the success of allogeneic hematopoietic stem cell transplantation. 50 In such cases, anti-CD7 CAR T cells would be eliminated by the transplant-conditioned and donor stem cell-reconstituted T cell compartment. Outside the transplant setting, the "suicide gene" can be activated once leukemia eradication is achieved. 51Finally, infused anti-CD7 T cells (which retain their endogenous CD3 / TCR complexes) may reconstitute a sufficiently broad T cell repertoire that this may not be an issue. To this end, subsets of CD4 memory T cells and CD8 effector T cells in human blood lymphocytes that do not express CD7 have been described. 52,53 It should be noted that T-ALL cells express CD7 at higher levels than normal T cells, and thus the CD7-dim subset may help reconstitute the T-cell repertoire even after CD7-targeted therapy.

[0182] Standard treatment for T-ALL relies primarily on intensive chemotherapy plus hematopoietic stem cell transplantation for patients with high-risk disease. The results are unsatisfactory and show significant morbidity and mortality. 54,55 The findings presented herein suggest that infusion of anti-CD7 PEBL-CAR T cells may significantly augment or perhaps replace existing chemotherapy- and transplantation-based strategies. These results suggest that CAR expression accompanied by downregulation of target antigens in T cells should also be applicable to other T cell markers such as CD3, CD2, and CD5, whose expression is predominant in T cell lymphoproliferative neoplasms. As a proportion of high-risk acute myeloid leukemias express CD7, 19,30,56 , it is also warranted to test the potential of anti-CD7 CAR-T cells for this leukemia subtype. References

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[0239] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0240] While the present invention has been particularly shown and described with reference to illustrative embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention contained in the appended claims.

Claims

1. i) a first recombinant nucleic acid molecule comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, said target binding molecule being a first antibody or fragment thereof that specifically binds to CD7; ii) a second recombinant nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a second antibody or fragment thereof that specifically binds to CD7.

2. A combination of nucleic acids described in claim 1, wherein the first antibody or fragment thereof is a first single chain variable fragment (scFv) and the second antibody or fragment thereof is a second single chain variable fragment (scFv).

3. The nucleic acid combination of claim 1 or 2, wherein the first antibody or fragment thereof comprises a heavy chain variable region (VH) comprising a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:1, and a light chain variable region (VL) comprising a light chain (LC) CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

2.

4. The nucleic acid combination of claim 1 or 2, wherein the first antibody or fragment thereof comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:14, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

15.

5. The nucleic acid combination of claim 1 or 2, wherein the first antibody or fragment thereof comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:16, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

17.

6. A nucleic acid combination described in any one of claims 1 to 3, wherein the first antibody or fragment thereof comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

2.

7. A combination of nucleic acids described in any one of claims 1 to 3 or claim 6, wherein the first antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 1 and a VL having the amino acid sequence of SEQ ID NO:

2.

8. A combination of nucleic acids described in any one of claims 1, 2 or 4, wherein the first antibody or fragment thereof comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

15.

9. A combination of nucleic acids described in any one of claims 1, 2, 4, or 8, wherein the first antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 14 and a VL having the amino acid sequence of SEQ ID NO:

15.

10. A combination of nucleic acids described in any one of claims 1, 2 or 5, wherein the first antibody or fragment thereof comprises a VH having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16 and a VL having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

17.

11. A combination of nucleic acids described in any one of claims 1, 2, 5 or 10, wherein the first nucleic acid or a fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO: 16 and a VL having the amino acid sequence of SEQ ID NO:

17.

12. The nucleic acid combination of any one of claims 1 to 11, wherein the localization domain comprises an amino acid sequence selected from the group consisting of an endoplasmic reticulum (ER) retention sequence, a Golgi apparatus retention sequence, a proteasome localization sequence, and a transmembrane domain sequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B.

13. The nucleic acid combination described in claim 12, wherein the localization domain comprises an endoplasmic reticulum (ER) retention sequence having the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

9.

14. A combination of nucleic acids described in claim 12, wherein the membrane-spanning domain sequence comprises the amino acid sequence of SEQ ID NO:

13.

15. A nucleic acid combination according to any one of claims 1 to 14, wherein the first recombinant nucleic acid molecule further comprises a nucleotide sequence encoding a CD8α signal peptide.

16. A combination of nucleic acids according to any one of claims 1 to 15, wherein the CAR comprises a 4-1BB intracellular signaling domain and a CD3ζ intracellular signaling domain.

17. The nucleic acid combination of claim 16, wherein the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

4.

18. A nucleic acid combination described in claim 16 or 17, wherein the CAR further comprises a hinge and a membrane-spanning domain.

19. The nucleic acid combination described in claim 18, wherein the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:

10.

20. The nucleic acid combination of any one of claims 1 to 19, wherein the second antibody or fragment thereof comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:1, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

2.

21. The nucleic acid combination of any one of claims 1 to 19, wherein the second antibody or fragment thereof comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:14, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

15.

22. The nucleic acid combination of any one of claims 1 to 19, wherein the second antibody or fragment thereof comprises a VH comprising HC CDR1, HC CDR2 and HC CDR3 of SEQ ID NO:16, and a VL comprising LC CDR1, LC CDR2 and LC CDR3 of SEQ ID NO:

17.

23. A combination of nucleic acids described in any one of claims 1 to 22, wherein the first recombinant nucleic acid further comprises a nucleotide sequence encoding a CD8α signal peptide.

24. A combination of nucleic acids described in claim 1 or 2, wherein the amino acid sequence of the first antibody or fragment thereof and the amino acid sequence of the second antibody or fragment thereof are identical.

25. An expression vector comprising a combination of nucleic acids described in any one of claims 1 to 24.

26. An engineered cell comprising a combination of nucleic acids according to any one of claims 1 to 24, or an expression vector according to claim 25.

27. The engineered cell of claim 26, wherein the engineered cell is an engineered T cell, an engineered natural killer (NK) cell, an engineered NK / T cell, an engineered monocyte, an engineered macrophage, or an engineered dendritic cell.

28. A pharmaceutical composition for treating cancer in a subject in need of treatment, comprising an engineered cell according to claim 26 or 27 and a pharma- ceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the cancer is a T-cell malignant tumor.

30. The pharmaceutical composition of claim 29, wherein the T cell malignant tumor is early T cell precursor acute lymphoblastic leukemia (ETP-ALL).

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