CD7 expression blockers and chimeric antigen receptors for immunotherapy of T cell malignancies

Abstract

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 the treatment of cancer.

Description

[0001] The application is a divisional application of the Chinese patent application with the application date of November 22, 2017, application number 201780073071.6, and invention name “CD7 expression blocker and chimeric antigen receptor for T cell malignant tumor immunotherapy” (the corresponding PCT application with the application date of November 22, 2017 and application number (PCT / US2017 / 063048)).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 425,398, filed November 22, 2016, and U.S. Provisional Application No. 62 / 543,696, filed August 10, 2017, which are expressly incorporated by reference in their entirety for all purposes.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated 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 of the Invention

[0007] Chimeric antigen receptors (CARs) can redirect immune cells to specifically recognize and kill tumor cells. CARs are artificial multimolecular proteins composed of antibody single-chain variable regions (scFvs) connected to signaling molecules via transmembrane domains. When scFv is connected to its cognate antigen, signal transduction is triggered, resulting in CAR-expressing cytotoxic T lymphocytes killing tumor cells (Eshhar Z, Waks T, et al., PNAS USA. 90 (2): 720-724, 1993; Geiger TL, et al., J Immunol. 162 (10): 5931-5939, 1999; Brentjens RJ, et al., Nat Med. 9 (3): 279-286, 2003; Cooper LJ, et al., Blood 101 (4): 1637-1644, 2003; Imai C, et al., Leukemia. 18: 676-684, 2004). Clinical trials using autologous T lymphocytes expressing CARs have demonstrated positive responses in patients with B-cell refractory leukemias and lymphomas (see, e.g., Till BG et al., Blood 119(17):3940-3950, 2012; Maude SL et al., N Engl J Med. 371(16):1507-1517, 2014).

[0008] The development of CAR technology targeting T-cell malignancies lags far behind that of its B-cell counterpart. Novel therapies for T-cell malignancies are needed, but progress has been slow to date. Specifically, there is a lack of effective immunotherapy options, and the treatment of T-cell acute lymphoblastic leukemia (T-ALL) relies on intensive chemotherapy and hematopoietic stem cell transplantation. Although these approaches have improved morbidity and mortality, the results are far from satisfactory.

[0009] In summary, there is a clear unmet need for new treatment options for patients with T-cell malignancies. Summary of the Invention

[0011] In one aspect, the present invention provides an engineered immune cell comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain, wherein 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), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0012] 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).

[0013] 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.

[0014] 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 retention sequence, a protease 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 SEQ ID NO:8 or SEQ ID NO:9 amino acid sequence. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and a transmembrane domain sequence comprising an 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 protein 21 (TRIM21) targeting domain sequence and co-expressing a nucleic acid sequence encoding a human TRIM21 E3 ubiquitin ligase protein.

[0015] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0016] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0017] 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 still 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.

[0018] In some embodiments, 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.

[0019] In another aspect, the present invention provides an engineered immune cell comprising (i) a target binding molecule linked to a localization domain, wherein the target binding molecule is a first antibody that specifically binds to CD7; and (ii) a chimeric antigen receptor (CAR), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0020] 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).

[0021] 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.

[0022] 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 retention sequence, a protease 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 SEQ ID NO:8 or SEQ ID NO:9 amino acid sequence. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and a transmembrane domain sequence comprising an amino acid sequence of SEQ ID NO:13.

[0023] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0024] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0025] 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 still 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.

[0026] In some embodiments, 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.

[0027] In some embodiments, provided herein is a pharmaceutical composition comprising the engineered immune cells described herein and a pharmaceutically acceptable carrier.

[0028] On the other hand, provided herein is a method for producing an engineered immune cell as described herein. The method may include: (i) introducing (a) a first nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain into an immune cell, wherein the target binding molecule is a first antibody that specifically binds to CD7; and (b) a second nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7; and (ii) separating an engineered immune cell comprising the target binding molecule and the CAR connected to the localization domain, thereby producing the engineered immune cell.

[0029] In another aspect, the present invention provides a method for treating cancer in a subject (e.g., a patient) in need thereof, comprising administering a therapeutic amount of an engineered immune cell to the patient, thereby treating cancer in a subject in need thereof. In some embodiments, the engineered immune cell comprises: (i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain, wherein 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), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0030] 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).

[0031] 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.

[0032] 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 retention sequence, a protease 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 SEQ ID NO:8 or SEQ ID NO:9 amino acid sequence. In other embodiments, the localization domain comprises a transmembrane domain sequence derived from a CD8α hinge and a transmembrane domain sequence comprising an amino acid sequence of SEQ ID NO:13.

[0033] In some embodiments, the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:4.

[0034] In some embodiments, the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO:10.

[0035] 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 still 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.

[0036] In some embodiments, 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.

[0037] In some embodiments, the engineered cells are administered to the subject (e.g., patient) by intravenous infusion, intraarterial infusion, intraperitoneal infusion, direct injection into the tumor and / or post-operative perfusion of the tumor bed, implantation at the tumor site within an artificial scaffold, or intrathecal administration.

[0038] In some embodiments, the cancer is a T-cell malignancy. In one embodiment, the T-cell malignancy is early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0039] The present disclosure provides engineered immune cells and methods of using them for treating T-cell hematological malignancies. Those skilled in the art recognize that when CAR-T effector cells are used to treat T-cell leukemias, CAR T cell self-killing or cannibalism, as well as killing of normal T cells, can occur. Therefore, there is a need for engineered immune cells and treatments that minimize or eliminate T cell siblings.

[0040] The engineered immune cells and therapeutic methods described herein utilize novel CAR-T cells that are resistant to mutualism, such as engineered anti-CD7 PEBL and anti-CD7 CAR-T cells. These engineered immune cells can induce potent and durable therapeutic responses in patients with T-cell malignancies, including relapsed T-cell malignancies. These cells can effectively target and kill malignant T cells without significant T-cell mutualism. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A-Figure 1D Figure 3 illustrates CD7 expression in T-ALL. The percentage of ALL cells expressing CD7 at diagnosis, relapse, and during chemotherapy (MRD) is shown; the number of bone marrow samples studied at each stage ( Figure 1A ). Mean fluorescence intensity (MFI) of CD7 in T-ALL cells and remaining normal T cells from the same sample (n=19; P<0.0001 by paired t-test) ( Figure 1B CD7 MFI in T-ALL cells at diagnosis or relapse ("D / R") and in follow-up bone marrow samples after MRD (n=18) ( Figure 1C Flow cytometry contour plots illustrate CD7 expression in T-ALL cells (CD3-negative) and normal T cells (CD3-positive) at diagnosis, MRD, and relapse in a representative patient.

[0043] Figure 2A-2E Schematic diagram of the anti-CD7-41BB-CD3ζ construct ( Figure 2AFlow cytometric analysis of Jurkat cells transduced with GFP alone (“mock”) or GFP plus anti-CD7 CAR. The dot plots illustrate 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 Jurkat cells transduced with mimics and CAR were separated on 10% polyacrylamide gels under reducing or non-reducing conditions. The blot was probed with mouse anti-human CD3ζ antibody (8D3; BD Biosciences) and goat anti-mouse IgG (R&D Systems) coupled to horseradish peroxidase. Antibody binding was shown using Clarity Western ECL substrate (Bio-Rad). Anti-CD7 CAR induced activation markers to be expressed after connection. The bars show the mean values (± SD) of CD25 and CD69 MFI in Jurkat cells transduced with CAR and mimics 24 hours later with or without CD7+MOLT-4 cells. P values for significant differences by t-test are shown (*=0.016; ***<0.001) ( Figure 2D ). Figure 2E Provided Figure 2D Representative flow cytometry histograms of the experiments shown in .

[0044] Figure 3A-Figure 3I This indicates that the expression of anti-CD7 CAR in human peripheral blood T cells leads to mutualism, which is prevented by downregulation of CD7. The percentage of viable T cells recovered 24 hours after electroporation with or without anti-CD7 CAR mRNA (n=7) ( Figure 3A ). Viable cells were counted by flow cytometry. The percentage of viable T cells recovered 24 hours after CAR transduction with retroviral vectors (n=10) compared to cells from the same donor transduced with GFP alone ("mock") ( Figure 3B The percentage of viable T cells recovered within one week after transduction with CAR and mock transduction ( Figure 3C ). It shows Figure 3B Follow-up results for 5 of 10 experiments are shown in Figure 2. Percentage of CD107a in T cells after electroporation with or without anti-CD7 CAR mRNA ( Figure 3D ). The mean (±SD) of three repeated measurements is shown. Schematic diagram of the anti-CD7 protein expression blocker (PEBL) construct ( Figure 3ERepresentative flow cytometry histograms illustrate CD7 expression in T lymphocytes following retroviral transduction with three anti-CD7 PEBLs or transduction with GFP mimic alone (“mock”). Figure 3F ). T cells were stained with anti-CD7-PE (M-T701; BD Biosciences). The percentage of CD7 in T cells transduced with anti-CD7 PEBL-1 retrovirus or mock transduced (n=5) ( Figure 3G ). Flow cytometry dot plots illustrate downregulation of CD7 expression in T cells by PEBL transduction, along with expression of anti-CD7-41BB-CD3ζCAR 12 hours after electroporation with or without CAR mRNA. Cells were stained with biotin-conjugated goat anti-mouse F(ab')2 antibody and streptavidin-APC (Jackson ImmunoResearch). Percentage of viable T cells recovered 24 hours after electroporation of anti-CD7 CAR mRNA after transduction with anti-CD7 PEBL, compared to cells electroporated with anti-CD7 CAR mRNA but transduced with vector without CD7 PEBL (n=6) ( Figure 3I The number of viable cells was measured by flow cytometry. **, P < 0.01; ***, P < 0.001.

[0045] Figures 4A-4F We show that downregulation of CD7 by PEBL does not alter T cell phenotype, expansion, or functionality. The percentages of CD4 and CD8 cells ( Figure 4A Each symbol corresponds to a different T cell donor. Growth rate of PEBL- and mock-transduced T cells (from three donors) maintained with 200 IU / mL IL-2 for 14 days ( Figure 4B Symbols represent the mean (±SD) of three replicates. T cells transduced with PEBL and mock were electroporated with anti-CD19-41BB-CD3ζCAR mRNA or without mRNA ( Figure 4C Flow cytometry dot plots illustrate 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). Cytotoxicity of PEBL- and mock-transduced T cells against CD19+ ALL cells (OP-1) electroporated with or without anti-CD19 CAR mRNA ( Figure 4D ). The bars show the mean (± SD) of cytotoxicity at 1:1 E:T for 4 hours. Figure 4E Shown from the Figure 4D CD107a expression in T cells was performed in the same experiments as those described in . Figure 4F Shown is the production of IFNγ in T cells transduced with PEBL or mock, electroporated with or without anti-CD19 CAR mRNA, and co-cultured with OP-1 at an E:T of 1:1 for 6 hours. Bars represent the mean (± SD) of three replicates. ***, P < 0.001; ****, P < 0.0001.

[0046] Figures 5A-5F T cells whose CD7 was downregulated by PEBL acquired potent cytotoxicity against CD7+ leukemia cells after CD7 CAR expression. Cytotoxicity of anti-CD7 PEBL-transduced T cells electroporated with or without anti-CD7 CAR mRNA against CD7+ cell lines ( Figure 5A ). Data measured at 1:1 E:T for 4 hours are shown. Symbols represent the mean of 3 measurements each using T cells from 4 donors of MOLT-4, CCRF-CEM and Jurkat and 5 donors of Loucy and KG1a (P < 0.001 for each comparison). Cytotoxicity of T cells transduced with anti-CD7 PEBL electroporated with or without anti-CD7 CAR mRNA against primary leukemia cells from T-ALL patients ( Figure 5B ). Data are shown for 4 hours at the indicated E:T. Symbols refer to the mean (± SD) of 3 measurements. Figure 5C The overall specific cytotoxicity of T cells transduced with anti-CD7 PEBL or GFP alone ("mock") against 5 CD7+ cell lines after electroporation with anti-CD7 CAR mRNA is shown. T cells from 3 donors were tested at 1:1E:T in a 4-hour assay. Each symbol represents the specific percentage cytotoxicity against CD7+ cell lines after subtracting the percentage cytotoxicity obtained with the same T cells electroporated in the absence of mRNA. The horizontal bars represent the median of each group. T cells from 3 donors transduced with anti-CD7 PEBL or mocks were electroporated with or without anti-CD7 CAR mRNA ( Figure 5D ). Cytotoxicity against MOLT-4 was tested at 1:1 E:T in a 4-hour assay. The mean fluorescence intensity (MFI) of anti-CD107a-PE (H4A3; BD Biosciences) is shown. The bars represent the mean (± SD) of three replicates. T cells transduced with anti-CD7 PEBL were retrovirally transduced with anti-CD7 CAR or mock transduced and tested against primary leukemia cells from T-ALL patients ( Figure 5EEach symbol represents the mean (±SD) of three replicates. T cells transduced with mock or PEBL, sequentially transduced with or without anti-CD7 CAR, were cultured alone or in the presence of Streck-treated MOLT-4 cells and 120 IU / mL IL-2 added weekly ( Figure 5F Symbols represent the mean (±SD) percentage of cell recovery relative to the input cell number in triplicate cultures. **, P < 0.01, ***, P < 0.001; ****, P < 0.0001.

[0047] Figures 6A-6D The results showed that PEBL-transduced T cells expressing CD7-41BB-CD3ζCAR exerted anti-tumor activity in xenografts. NOD-SCID-IL2RG nude mice were intravenously (iv) infused with 1x10 6 luciferase-labeled CCRF-CEM cells. Figure 6A ) or Day 3 and Day 7 ( Figure 6B ), intravenous administration of 2x10 7 PEBL-CAR T cells. The remaining mice received T cells transduced with the mock, or received RPMI-1640 instead of cells ("control"). All mice received 20,000 IU IL-2 intraperitoneally (ip) every two days. In vivo imaging of leukemia cell growth after intraperitoneal injection of D-luciferin is shown. Figure 6B Images of the ventral side of mice on day 3, with enhanced sensitivity demonstrating CCRF-CEM engraftment in all mice. The full set of luminescent images is in Figure 14. Figure 6C Shown Figure 6A and Figure 6B Leukemic cell growth in all mice shown in , expressed as photons per second. Each symbol corresponds to the bioluminescence measurement in each mouse, which was normalized to the average of the ventral plus dorsal signals in all mice before CAR-T cell infusion. Kaplan-Meier curves show the overall survival of mice in different groups (8 per group) ( Figure 6D When the total bioluminescence signal reaches 1x10 10 The mice were euthanized at 400 nm. P values were calculated using the log-rank test.

[0048] Figures 7A-7E Figure 2 shows the activity of PEBL-CAR-T cells against ETP-ALL in a patient-derived xenograft (PDX) model. 6cells, and primary ETP-ALL cells previously expanded in NOD-SCID-IL2RG nude mice were infused intravenously (iv). Figure 7A Five mice ("control") were left untreated. The remaining five mice received a single intravenous infusion of PEBL-CAR T cells (2x10 7 In the remaining 4 mice, 2x10 6 ), and received 20,000 IU IL-2 intraperitoneally every two days; IL-2 was also administered to 2 of the 5 control mice. The black symbols (left y-axis) indicate the number of ETP-ALL cells / mL counted in the peripheral blood. The gray symbols (right y-axis) show the number of PEBL-CAR T cells. When the percentage of ETP-ALL cells in blood monocytes reached ≥80%, the mice were euthanized. The percentage of ETP-ALL in various organs of 5 untreated mice (denominator, total number of human plus mouse CD45+ cells) ( Figure 7B Blood smears of treated (PEBL-CAR#1) and untreated ETP-ALL 7 days after T cell infusion; broken cells are prominent in the blood after PEBL-CAR T cells ( Figure 7C ). Flow cytometry dot plots showing the presence of CD7+CD3-ETP-ALL cells in tissues of untreated control mice with ETP-ALL and the presence of CD7-CD3+PEBL-CAR T cells in PEBL-CAR#1 mice treated with PEBL-CAR-T cells ( Figure 7D ). ETP-ALL was not detected in treated mice (<0.01%). The events shown are normalized to the events acquired at the corresponding points shown in control mice. Spleens of treated (PEBL-CAR#1) and untreated mice ( Figure 7E ).

[0049] Figures 8A-8C The specificity and function of the anti-CD7-41BB-CD3ζCAR are shown. OP-1 (CD7-) and MOLT-4 (CD7+) ( 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 showed that anti-CD7 scFv bound to MOLT-4 but not OP-1. Jurkat cells were transduced with anti-CD7-41BB-CD3ζCAR, anti-CD19-41BB-CD3ζCAR, or a vector containing GFP alone ( Figure 8B These cells were co-cultured with CD7+ MOLT-4 or CCRF-CEM cells, or with CD7- OP-1 cells at a 1:1 E:T ratio. Target cells were labeled with Calcein Red-Orange AM (Invitrogen). After a 30-minute incubation, the percentage of cell doublets was measured by flow cytometry. Bars indicate the mean (± SD) of three replicates. Figure 8C CAR-mediated cell aggregation was shown to be inhibited by pre-incubation of target cells with a soluble form of anti-CD7 scFv. ***P < 0.001.

[0050] Figure 9A and Figure 9B Shown is the expression of anti-CD7-41BB-CD3ζ CAR in human peripheral blood T lymphocytes. Figure 9A Provided are representative flow cytometry dot plots of T lymphocytes activated for 7 days with Dynabeads human T-activator CD3 / CD28 (ThermoFisher Scientific) and IL-2 and transduced with anti-CD7 CAR. Flow cytometry dot plots illustrate GFP fluorescence and CAR expression, the latter visualized by staining with biotin-conjugated goat anti-mouse F(ab')2 antibody followed by streptavidin-APC (Jackson ImmunoResearch). Figure 9B Western blot analysis of CAR expression is shown. Cell lysates of T cells transduced with mimics and CAR were separated on 10% polyacrylamide gels under reducing or non-reducing conditions. The blot was 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 ECL substrate (Bio-Rad).

[0051] Figure 10A and Figure 10B This indicates that CD7 expression is downregulated by anti-CD7 PEBL. Flow cytometry dot plots illustrate GFP expression (x-axis), CD7 expression (y-axis, top row), and intracellular anti-CD7 PEBL-1 expression (y-axis, bottom row) ( Figure 10AT lymphocytes were retrovirally transduced with anti-CD7 PEBL-1 or a vector containing GFP alone ("mock"). T cells were stained with a phycoerythrin-conjugated anti-CD7 antibody (M-T701; BD Biosciences). Intracellular expression of PEBL-1 was tested using a PE-conjugated anti-Myc antibody (9B11; CellSignaling Technology), which binds to the sequence EQKLISEEDL (SEQ ID NO:40) incorporated into the ER binding motif. Prior to antibody labeling, cells were permeabilized with 8E reagent, a permeabilization reagent developed in our laboratory. Figure 10B RT-PCR analysis of CD7 mRNA expression is shown. cDNA derived from total mRNA extracted from Jurkat cells transduced with PEBL1-3, GFP alone ("mock"), or not transduced ("WT") was used as a template. CD7 cDNA (723 bp) was amplified using the following primers: forward, ATGGCCGGGCCTCCG (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. An 87 bp (nucleotides 676-762) region of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified in parallel as a control.

[0052] Figure 11A and Figure 11B The results showed that anti-CD7 CAR signaling triggered higher cytokine secretion in T cells in which CD7 expression was knocked down by anti-CD7 PEBL. T lymphocytes from three donors were transduced with anti-CD7 PEBL or GFP alone ("mock") and electroporated with anti-CD7-41BB-CD3ζ mRNA or without mRNA. Intracellular IFNγ ( Figure 11A ) and TNFα( Figure 11B ) expression. Bars represent the mean (±SD) of three replicate MFI measurements. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0053] Figure 12The CD7 negative T cells expressing anti-CD7-41BB-CD3 ζ CAR are shown to play anti-tumor cell toxicity for CD7+ cell lines.Shown are the results of 4-hour cytotoxicity assays performed by CD7 PEBL transduction, then by CD7-41BB-CD3 ζ or only GFP (" analogs") transduced T cells.Symbols represent the mean value (± SD) of three replicate experiments under specified E:T ratios.For all comparisons, P < 0.001.

[0054] Figures 13A-13E A functional comparison of anti-CD7-41BB-CD3ζ and anti-CD19-41BB-CD3ζ CARs is provided. Figure 13A The expression of anti-CD19 and anti-CD7 CAR (in a vector containing mCherry) in peripheral blood T cells previously transduced with anti-CD7 PEBL is shown. Flow cytometry dot plots illustrate mCherry expression by T cells and staining with biotin-conjugated goat anti-mouse F(ab')2 antibody followed by streptavidin conjugated to allophycocyanin (Jackson ImmunoResearch). Results for T cells transduced with a vector containing mCherry alone ("mock") are also shown. CD19 expression in CCRF-CEM and Jurkat cells transduced with vectors containing CD19 and GFP ( Figure 13B CD19 was detected using anti-CD19 APC (Miltenyi Biotech). 4-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 ( Figure 13C ). Symbols indicate the mean (± SD) of three replicates. P < 0.001 for data at all E:T ratios for T cells transduced with CAR and mocks. As measured by hepatocyte image analysis using the IncuCyte Zoom system (Essen Bioscience), the long-term cytotoxicity of anti-CD19 or anti-CD7 PEBL-CAR-T cells at different E:T ratios ( Figure 13D ). Symbols indicate the mean (± SD) of 3 measurements of CD19+CCRF-CEM (upper) or CD19+Jurkat cells (lower) in wells containing CAR-T cells, mock-transduced T cells, or cells without cells. Measurements were performed at 4-hour intervals. Expansion capacity of anti-CD19 and anti-CD7 PEBL-CAR-T cells co-cultured with and without CD19+Jurkat cells ( Figure 13E). Anti-CD7 PEBL-transduced T cells were sequentially transduced with anti-CD19 or anti-CD7 CAR or mCherry alone and cultured alone or in the presence of irradiated CD19+ Jurkat cells and 120 IU / mL IL-2 added weekly. Symbols indicate the mean (± SD) percentage of cell recovery relative to the input cell number in three replicate cultures.

[0055] Figures 14A-14C The results showed that PEBL-transduced T cells expressing anti-CD7-41BB-CD3ζCAR exerted anti-tumor activity in a mouse model. NOD-SCID-IL2RG nude mice were intravenously (iv) infused with 1x10 6 luciferase-labeled CCRF-CEM cells. Figure 14A ) or Day 3 and Day 7 ( Figure 14B ), intravenous administration of 2x10 7 PEBL-CAR T cells. The remaining mice received T cells transduced with the mock or received RPMI-1640 instead of cells ("control"). All mice received 20,000 IU IL-2 intraperitoneally (ip) once every two days. In vivo imaging of leukemia cell growth was performed after intraperitoneal injection of D-luciferin. Figure 14B Images of the ventral side of mice on day 3 demonstrate enhanced sensitivity in demonstrating leukemic cell engraftment in all mice. Leukemic cell growth is expressed as photons per second, normalized to the average of the ventral plus dorsal signals in all mice before CAR-T cell infusion ( Figure 14C ). Each symbol corresponds to the bioluminescence measurement in each mouse.

[0056] Figure 15A and 15B These results demonstrate that PEBL-transduced T cells expressing the anti-CD7-41BB-CD3ζ CAR exert anti-tumor activity in a mouse model and remain active against cells collected at relapse. Figure 15A Figure 2 shows the effect of intravenous infusion of luciferase-labeled CCRF-CEM cells followed by PEBL-CAR-transduced T cells, mock-transduced T cells, or T cells transduced with PEBL-CAR. Figure 6C The percentage of CCRF-CEM cells in the white blood cells of NOD-SCID-IL2RG nude mice treated intravenously with RPMI-1640 instead of cells ("control"). For "control" and "mock", the percentage of CCRF-CEM cells in the white blood cells reached 10 17-23 days after leukemia cell infusion. 10Blood was obtained from euthanized mice that had reached a bioluminescence threshold of 10 photons / second. For PEBL-CAR mice, blood was obtained via cheek puncture on day 24 after CCRF-CEM infusion. CCRF-CEM cells collected from the spleen and liver of mice treated with PEBL-CAR at the time of relapse were cultured for 2 days ( Figure 15B ). Then in 4- hour cytotoxicity assays with E: T 1: 1, using T cells transduced with PEBL-CAR or analogs initially for infusion, the cultured cells were used as targets. The same batch of CCRF-CEM cells expressing luciferase for producing xenografts were also used for comparison. After adding the BrightGlo luciferase assay system (Promega), the percentage cytotoxicity was determined by the plate measurement value of the bioluminescent signal. The bars show the mean (± SD) of three replicates; each white column and gray column corresponds to a cell from a mouse.

[0057] Figure 16 The immunophenotypic characteristics of ETP-ALL at diagnosis and after proliferation in NOD-SCID-IL2RG nude mice are provided. Flow cytometry contour plots show immunophenotypic diagnostic bone marrow samples of ETP-ALL used to develop PDX models in this study and immunophenotypic diagnostic bone marrow samples of ETP-ALL cells recovered from the spleen of one of the control mice shown in Figure 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), all from BD Biosciences; CD33-BV421 (Biolegend); CD1a-PE (Beckman Coulter). Quadrants were drawn based on staining with isotype-matched inactive antibodies conjugated to the same fluorochrome.

[0058] Figure 17 Illustrations of exemplary embodiments of the present invention are provided. DETAILED DESCRIPTION

[0060] The following is a description of example embodiments of the invention.

[0061] The present invention is based in part on the design of a chimeric antigen receptor (CAR), which is directed against CD7, a 40kDa type I transmembrane glycoprotein that is a major marker of T cell malignancies and is highly expressed in all T cell ALL situations, including early T cell progenitor acute lymphoblastic leukemia (ETP-ALL). As described herein, anti-CD7 CAR induces T cells to exert specific cytotoxicity against T cell malignancies. In addition, when anti-CD7 CAR is used in combination with the reduction of CD7 expression on effector T cells, the cytotoxicity of T cells is shown to be significantly increased. As demonstrated herein, the reduction (e.g., elimination, reduction and / or repositioning) of CD7 prevents the mutual killing effect exerted by the corresponding anti-CD7 CAR, allowing higher T cell recovery after CAR expression compared to cells retaining the target antigen (e.g., CD7), as well as more effective cytotoxicity to T leukemia / lymphoma cells.

[0062] Therefore, on the one hand, the present invention relates to an engineered immune cell, comprising: 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 specifically bound to differentiation group 7 (CD7). The CAR of the present invention is sometimes referred to herein as "anti-CD7-41BB-CD3ζ". Figure 17 An exemplary embodiment is depicted in .

[0063] As used herein, "engineered" immune cells include immune cells that have been genetically modified compared to naturally occurring immune cells. For example, engineered T cells produced according to the present methods carry nucleic acids comprising nucleotide sequences that are not naturally present in the T cells from which they are derived.

[0064] In certain embodiments, the engineered immune 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. In certain embodiments, the engineered immune cell is an engineered T cell. As used herein, the term "nucleic acid" refers to a polymer comprising a plurality of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of double-stranded polymers, "nucleic acid" can refer to any one or two chains of the molecule.

[0065] With respect to nucleic acids, the term "nucleotide sequence" refers to a series of consecutive nucleotides linked by covalent bonds, such as phosphodiester bonds (e.g., phosphodiester, alkyl and aryl phosphonates, phosphorothioate, phosphotriester bonds), and / or non-phosphodiester bonds (e.g., peptide and / or sulfamate bonds). In certain embodiments, the nucleotide sequence encoding a target binding molecule, e.g., linked to a localization domain, is a heterologous sequence (e.g., a gene from a different species or cell type).

[0066] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotides or deoxyribonucleotide monomers, and non-naturally occurring derivatives and analogs thereof. Thus, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

[0067] As will be appreciated by those skilled in the art, in some aspects, the nucleic acid further comprises a plasmid sequence. The plasmid sequence can include, for example, one or more sequences of a promoter sequence, a selectable marker sequence, or a locus targeting sequence.

[0068] As used herein, "antibody" means 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 antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies.

[0069] When referring to a protein or peptide, the term "specifically (or selectively) binds" or "specifically (or selectively) immunoreacts with..." refers to a binding reaction that determines the presence of a protein, often in a heterologous population of proteins and other biological preparations. Thus, under specified immunoassay conditions, a specified antibody binds to a specific protein at least twice the background and more typically 10 to 100 times the background. Specific binding to an antibody under such conditions requires an antibody selected for 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 can be achieved by subtracting antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998)).

[0070] In certain embodiments, the antibody that binds to CD7 is a single-chain variable fragment antibody ("scFv antibody"). scFv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology Of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. Springer-Verlag, New York, pp. 269-315. See also PCT Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. As will be appreciated by those skilled in the art, various suitable linkers can be designed and tested for optimal function, as provided in the art and as disclosed herein.

[0071] 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 an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOs: 1 and 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 with 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 with the VL sequence of SEQ ID NO: 2. 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 some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 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 some cases, the light chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding 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 shown 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 shown in SEQ ID NO:24.

[0072] 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 that each 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 NOs: 14 and 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 with 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 with the VL sequence of SEQ ID NO: 15.

[0073] 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 some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 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 some cases, the heavy chain variable region comprises 10 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in the sequence set forth in SEQ ID NO: 15.

[0074] 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: 25. 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: 26.

[0075] 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 that each 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 NOs: 16 and 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 with 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 with the VL sequence of SEQ ID NO: 17.

[0076] 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 some cases, the heavy chain variable region comprises 13 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acid substitutions in the sequence set forth in SEQ ID NO: 14. 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 some cases, the heavy chain variable region comprises 5 or fewer (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid substitutions in the sequence set forth in SEQ ID NO: 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 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 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.

[0077] In some embodiments, the scFv of the present invention comprises a variable heavy chain sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with the variable heavy chain sequence of an anti-CD7 antibody. In some embodiments, the scFv of the present invention comprises a variable light chain sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with the variable light chain sequence of an anti-CD7 antibody. For example, the anti-CD7 antibody can be any such antibody recognized by those skilled in the art.

[0078] Table 1. Amino acid sequences of the VH and VL regions of anti-CD7 scFv

[0079]

[0080]

[0081] Table 2. Nucleic acid sequences of the VH and VL regions of anti-CD7 scFv

[0082]

[0083]

[0084] The term "sequence identity" means two nucleotide sequences or two amino acid sequences, when optimally aligned, such as by using acquiescence gap weights using program GAP or BESTFIT, there is at least such as 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. For sequence comparison, usually a sequence serves as a reference sequence (such as, parental sequence), and test sequence is compared therewith. When using a sequence comparison algorithm, test sequence and reference sequence are input to a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percentage sequence identity of the test sequence relative to the reference sequence based on the program parameters specified.

[0085] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search similarity method of Pearson and Lipman, Proc. Nat'l 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). An example of an algorithm suitable for measuring percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403 (1990). The software used to perform BLAST analysis is publicly available through the National Center for Biotechnology Information (available through the NCBI web server of the National Institutes of Health). Typically, sequence comparisons can be performed using default program parameters, but custom parameters can also be used. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix as default values (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0086] As will be appreciated by one skilled 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) may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with a 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 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 3, as long as it possesses the desired function. In certain embodiments, the intracellular signaling domain of 4-1BB comprises the sequence shown in SEQ ID NO: 3 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[0087] For example, in certain embodiments, the intracellular signaling domain 4-1BB can be replaced by another intracellular signaling domain from a co-stimulatory molecule such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2. In some embodiments, the intracellular signaling domain of CAR may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2.

[0088] For example, in some cases, the intracellular signaling domain of 4-1BB may also include another intracellular signaling domain from a costimulatory molecule such as CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2. In some embodiments, the additional intracellular signaling domain may have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with the intracellular signaling domain of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2. In other embodiments, the additional intracellular signaling domain comprises at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to one or more intracellular signaling domain fragments of CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2.

[0089] For example, in some cases, the intracellular signaling domain CD3 zeta can have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity with SEQ ID NO: 4, as long as it possesses the desired function. In certain embodiments, the intracellular signaling domain CD3 zeta comprises the sequence set forth in SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0090] In some cases, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) or a portion thereof, as long as it has the desired function. The intracellular signaling domain of CAR may include a sequence having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with ITAM. In certain embodiments, the intracellular signaling domain may have at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity or 100% sequence identity with FcεRIγ, CD4, CD7, CD8, CD28, OX40 or H2-Kb, as long as it has the desired function.

[0091] In certain embodiments, anti-CD7 CAR further includes hinge and transmembrane sequence. Hinge and transmembrane sequence suitable for use in the present invention are known in the art and are provided in, for example, the publication WO2016 / 126213 incorporated by reference as a whole. In certain embodiments, the hinge sequence includes the sequence (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD) shown in SEQ ID NO:5. In certain embodiments, the transmembrane sequence includes the sequence (IYIWAPLAGTCGVLLLSLVITLYC) shown in SEQ ID NO:6. In some embodiments, the hinge and transmembrane domain of anti-CD7 CAR may include a signaling domain (e.g., a transmembrane domain) from CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, FGFR2B or another transmembrane protein.

[0092] In certain embodiments, the anti-CD7 CAR further comprises a CD8α signal peptide (MALPVTALLLPLALLLHAARP; SEQ ID NO: 7). Figure 17 A schematic diagram of an anti-CD7 CAR comprising the embodiments described herein is depicted in FIG.

[0093] In certain aspects of the invention, the chimeric antigen receptor (CAR) can bind to molecules expressed on the surface of cells, including but not limited to members of the CD1 glycoprotein family, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127, and CD137.

[0094] As described herein, when anti-CD7 CAR is used in combination with the downregulation of CD7 expression on effector T cells, the cytotoxicity of T cells is shown to be significantly increased. As demonstrated herein, the downregulation (e.g., elimination, reduction and / or repositioning) of CD7 prevents the mutual killing effect exerted by the corresponding anti-CD7 CAR, allowing for higher T cell recovery after CAR expression compared to cells retaining the target antigen (e.g., CD7), as well as more effective cytotoxicity to 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 a variety of known methods, including, for example, "intrabody" (as described in WO2016 / 126213) for CD7, RNAi or gene editing methods for CD7, such as large-range nucleases, TALEN, CRISPR / Cas9 and zinc finger nucleases.

[0095] 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. A "target binding molecule linked to a localization domain" is sometimes referred to herein as a protein expression blocker (PEBL) or in some cases, an "intrabody," as described in WO2016 / 126213, the teachings of which are incorporated by reference in their entirety. Figure 3E and Figure 17 An exemplary embodiment of a PEBL is shown in FIG.

[0096] As used herein, " connection " in the context of protein expression blockers refers to that the gene encoding the target binding molecule is directly adjacent to the one or more genes encoding one or more localization domains in the frame (for example, without a joint). Alternatively, the gene encoding the target binding molecule can be connected to the one or more genes encoding one or more localization domains by a linker sequence, for example, as described in WO2016 / 126213. As will be appreciated by those skilled in the art, variants of such linker sequences and such linker sequences are known in the art. Design methods for constructs incorporated with linker sequences and methods for assessing functionality are readily available to those skilled in the art.

[0097] 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 the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2. In certain embodiments, the scFv comprises the VH sequence set forth in SEQ ID NO: 14 and the VL sequence set forth in SEQ ID NO: 15. In certain embodiments, the scFv comprises the VH sequence set forth in SEQ ID NO: 16 and the 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 each 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 NOs: 1 and 2, respectively; the VH and VL sequences set forth in SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or the VH and VL sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17, respectively.

[0098] In some embodiments, the nucleic acid sequence of SEQ ID NO: 23 encoding the immunoglobulin heavy chain variable region of an anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 24 encoding the immunoglobulin light chain variable region of an anti-CD7 scFv are used to produce an anti-CD7 protein expression inhibitor. In other embodiments, the nucleic acid sequence of SEQ ID NO: 25 encoding the immunoglobulin heavy chain variable region of an anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 26 encoding the immunoglobulin light chain variable region of an anti-CD7 scFv are used to produce an anti-CD7 protein expression inhibitor. In certain embodiments, the nucleic acid sequence of SEQ ID NO: 27 encoding the immunoglobulin heavy chain variable region of an anti-CD7 scFv and the nucleic acid sequence of SEQ ID NO: 28 encoding the immunoglobulin light chain variable region of an anti-CD7 scFv are used to produce an anti-CD7 protein expression inhibitor.

[0099] In certain embodiments, as described herein, the antibody that binds to CD7 in the context of a CAR may be different from the antibody that binds to CD7 in the context of a target binding molecule (PEBL). By way of example only, the antibody that binds to CD7 in the context of a CAR may comprise the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 2, while the antibody that binds to CD7 in the context of a PEBL may comprise the VH sequence set forth in SEQ ID NO: 14 and the VL sequence set forth in SEQ ID NO: 15. In certain embodiments, as described herein, the antibody that binds to CD7 in the context of a CAR may be the same as the antibody that binds to CD7 in the context of a target binding molecule (PEBL).

[0100] In certain embodiments, the localization domain of PEBL comprises an endoplasmic reticulum (ER) or Golgi retention sequence; a proteomic localization sequence; 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 certain embodiments, as described herein, the localization domain comprises the endoplasmic reticulum (ER) retention peptide EQKLISEEDLKDEL (SEQ ID NO: 8), (GGGGS)4AEKDEL (SEQ ID NO: 9), or the CD8α hinge and transmembrane domain (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLA GTCGVLLLSLVITLY) (SEQ ID NO: 10), followed by KYKSRRSFIDEKKMP (SEQ ID NO: 11). Depending on the application, the localization domain can direct PEBL to a specific cellular compartment, such as the Golgi apparatus or the endoplasmic reticulum, the proteasome, or the cell membrane. ER or Golgi retention sequences comprise 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 comprise a PEST (SEQ ID NO: 22) motif.

[0101] In some embodiments, proteasome localization is achieved by connecting the scFv sequence to a protein 21 (TRIM21) targeting domain sequence containing a tripartite motif and co-expressing a nucleic acid sequence encoding human TRIM21 E3 ubiquitin ligase protein. TREVI21 binds to the Fc domain of the antibody with high affinity and can recruit ubiquitin-proteosome complexes to degrade molecules (e.g., proteins and peptides) bound to the antibody. The TRIM21 targeting domain sequence encodes an amino acid sequence selected from human immunoglobulin G (IgG) constant region (Fc) genes (such as IgG1, IgG2, or IgG4) and is used to form a fusion protein comprising scFv and Fc domains. In this embodiment, the exogenously expressed TREVI21 protein binds to the scFv-Fc fusion protein bound to the target protein (e.g., CD7) and guides the complex to the proteasome for degradation.

[0102] Details of the amino acid sequence of human TRIM21 E3 ligase protein can be found, for example, in the NCBI protein database under NCBI Ref. Seq. No. NP_003132.2. Details of the nucleic acid sequence encoding human TRIM21 E3 ligase protein can be found, for example, in the NCBI protein database under NCBI Ref. Seq. No. NM_003141.3.

[0103] In certain embodiments, the protein expression blocker is any one or more of the anti-CD7 PEBLs disclosed in WO2016 / 126213, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Thus, as described in WO2016 / 126213, the engineered immune cells described herein may comprise a PEBL (target binding molecule linked to a localization domain) that binds to CD7. The sequences of the anti-CD7 intracellular antibody components are as shown in FIG2 and Tables 1 and 2 of WO2016 / 126213. Figure 3E and Figure 17 An exemplary embodiment of an anti-CD7 PEBL is depicted in .

[0104] Table 3. Amino acid sequence information of selected components of anti-CD7 PEBL

[0105]

[0106] In some embodiments, the anti-CD7 protein expression inhibitor comprises the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and a VH-VL linker. The VH-VL linker can be (GGGGS) nlinker, wherein n can range from 1 to 6, for example, 1, 2, 3, 4, 5 or 6. In one embodiment, the anti-CD7 protein expression blocking agent comprises the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the anti-CD7 protein expression blocking agent comprises the amino acid sequence of SEQ ID NO: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 2, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocking agent further comprises a localization domain selected from any one of the sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocking agent further comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide shown in SEQ ID NO: 7. In other cases, the anti-CD7 protein expression blocking agent further comprises a CD8α hinge and transmembrane domain, such as, but not limited to, the CD8α hinge and transmembrane domain shown in SEQ ID NO: 10.

[0107] In some embodiments, the anti-CD7 protein expression inhibitor comprises the amino acid sequence of SEQ ID NO: 14, the amino acid sequence of SEQ ID NO: 15, and a VH-VL linker. The VH-VL linker can be (GGGGS) nlinker, wherein n can range from 1 to 6, for example, 1, 2, 3, 4, 5 or 6. In one embodiment, the anti-CD7 protein expression blocking agent comprises the amino acid sequence of SEQ ID NO: 14, the amino acid sequence of SEQ ID NO: 15, and the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 95% sequence identity with 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 blocking agent comprises the amino acid sequence of SEQ ID NO: 14, an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 15, and an amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 14, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocking agent further comprises a localization domain selected from any one of the sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocking agent further comprises a CD8α signal peptide, such as, but not limited to, a CD8α signal peptide shown in SEQ ID NO: 7. In other cases, the anti-CD7 protein expression blocking agent further comprises a CD8α hinge and transmembrane domain, such as, but not limited to, a CD8α hinge and transmembrane domain shown in SEQ ID NO: 10.

[0108] In some embodiments, the anti-CD7 protein expression inhibitor comprises the amino acid sequence of SEQ ID NO: 16, the amino acid sequence of SEQ ID NO: 17, and a VH-VL linker. The VH-VL linker can be (GGGGS) nLinker, wherein n can range from 1 to 5, for example, 1, 2, 3, 4, 5 or 6 (SEQ ID NO: 29). In one embodiment, the anti-CD7 protein expression blocker comprises the amino acid sequence of SEQ ID NO: 16, the amino acid sequence of SEQ ID NO: 17, and the 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 with SEQ ID NO: 16, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In certain embodiments, the anti-CD7 protein expression blocker comprises the amino acid sequence of SEQ ID NO: 16, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In other embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 16, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 17, and an amino acid sequence of SEQ ID NO: 12. In some cases, the anti-CD7 protein expression blocking agent further comprises a localization domain selected from any one of the sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 13. In some cases, the anti-CD7 protein expression blocking agent further comprises a CD8α signal peptide, such as, but not limited to, the CD8α signal peptide shown in SEQ ID NO: 7. In other cases, the anti-CD7 protein expression blocking agent further comprises a CD8α hinge and transmembrane domain, such as, but not limited to, the CD8α hinge and transmembrane domain shown in SEQ ID NO: 10.

[0109] In some embodiments, the nucleic acid sequence encoding the anti-CD7 PEBL comprises one or more nucleic acid sequences shown 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.

[0110] Table 4. Nucleic acid sequence information of selected components of TH69-based anti-CD7 PEBL

[0111]

[0112] 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.

[0113] In certain aspects of the invention, the protein expression blocker can bind to molecules expressed on the surface of cells, including but not limited to members of the CD1 glycoprotein family, CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD28, CD30, CD38, CD45, CD45RA, CD45RO, CD52, CD56, CD57, CD99, CD127 and CD137.

[0114] In some aspects of the invention, the expression of members of the CD1 glycoprotein family, 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 methods, such as, but not limited to, gene editing technologies using meganucleases, TALENs, CRISPR / Cas9, and zinc finger nucleases. For example, in some embodiments, CD7 expression is knocked out using genome editing by Cas9 / CRISPR. In other embodiments, CD5 expression is knocked out using genome editing by Cas9 / CRISPR.

[0115] As described above, it is possible to achieve downregulation of CD7 expression on effector T cells according to a variety of other known methods, including, for example, gene editing methods using meganucleases, TALEN, CRISPR / Cas9 and zinc finger nucleases. Therefore, in certain embodiments, engineered immune cells also include modified CD7 genes, which are modified to render CD7 genes or proteins non-functional. For example, the engineered immune cells of the present invention also include modified (e.g., non-functional) CD7 genes (modified using, for example, meganucleases, TALEN, CRISPR / Cas9 or zinc finger nucleases), which prevent or reduce CD7 expression, and / or otherwise damage (e.g., structurally) CD7 proteins from being recognized by anti-CD7 CARs. Methods for modifying gene expression using such methods are readily available and well known in the art.

[0116] Methods for inactivating target genes in immune cells using CRISPR / Cas6 technology are described, for example, in U.S. Patent Publication Nos. 2016 / 0272999, 2017 / 0204372, and 2017 / 0119820.

[0117] The CRISPR / Cas system is a system for inducing targeted genetic changes (genome modification). Target recognition of the Cas9 protein requires a “seed” sequence within the guide RNA (gRNA) and a conserved polynucleotide containing a protospacer sequence adjacent to a motif (PAM) sequence upstream of the gRNA binding region. Therefore, the CRISPR / Cas system can be engineered to cleave essentially 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 particularly suitable for multi-gene editing or synergistic activation of target genes. Examples of CRISPR / Cas systems for inhibiting gene expression are described in U.S. Publication No. 2014 / 0068797 and U.S. Patent Nos. 8,697,359 and 8,771,945. The system induces permanent gene destruction by introducing DNA double-strand breaks using an RNA-guided Cas9 endonuclease that triggers error-prone repair pathways to cause frameshift mutations. In some cases, other endonucleases may also be used, including but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Cs5, Csn2, Csm2, Csm3 , Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, T7, Fok1, other nucleases known in the art, homologs thereof or modified forms thereof.

[0118] CRISPR / Cas gene disruption occurs when a gRNA sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double-strand break at the target gene. In some cases, the CRISPR system comprises one or more expression vectors comprising a nucleic acid sequence encoding a Cas endonuclease and a guide nucleic acid sequence specific for a target gene. The guide nucleic acid sequence is specific for 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. Guide nucleic acid sequences include RNA sequences, DNA sequences, combinations thereof (RNA-DNA combination sequences), or sequences with synthetic nucleotides, such as peptide nucleic acids (PNA) or locked nucleic acids (LNA). Guide nucleic acid sequences can be single molecules or double molecules. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0119] In some embodiments, the engineered immune cells of the present invention can be modified by 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 the NCBI gene database under GeneID No. 924.

[0120] Commercially available kits, gRNA vectors, and donor vectors for knocking out specific target genes are available, for example, from Origene (Rockville, Md.), GenScript (Atlanta, Ga.), Applied Biological Materials (ABM; Richmond, British Colombia), BioCat (Heidelberg, Germany), and the like. For example, commercially available kits or kit components for knocking out CD7 by CRISPR include, e.g., those available under catalog numbers KN201231, KN201231G1, KN201231G2, and KN201231D (each available from OriGene), and those available under catalog numbers sc-4072847, sc-4072847-KO-2, sc-4072847-HDR-2, sc-4072847-NIC, sc-4072847HDR-2, and sc-4072847-NIC-2 (each available from Santa Cruz Biotechnology).

[0121] In some embodiments, the chimeric antigen receptors described herein can be introduced into the human CD7 gene locus using the CRISPR / Cas system.

[0122] In certain embodiments, an engineered immune cell is provided, 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 specifically binds to cluster of differentiation 7 (CD7); and ii) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain, wherein the target binding molecule is an antibody that binds to CD7, and the localization domain comprises an endoplasmic reticulum retention sequence. In certain embodiments, in the context of the CAR and in the context of the target binding molecule, the antibody that binds to CD7 comprises: a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 2; a VH sequence as shown in SEQ ID NO: 14 and a VL sequence as shown in SEQ ID NO: 15; or a VH sequence as shown in SEQ ID NO: 16 and a VL sequence as shown in SEQ ID NO: 17. As described herein, in certain embodiments, the antibody comprises a VH and a VL having a sequence that is at least 90% identical to the VH and VL sequences shown in SEQ ID NOs: 1 and 2, respectively; the VH and VL sequences shown in SEQ ID NOs: 14 and 15, respectively; or the VH and VL sequences shown in SEQ ID NOs: 16 and 17, respectively, comprising at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity. In certain embodiments, 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 (protein expression blocker or PEBL). In certain embodiments, the intracellular signaling domain of 4-1BB comprises the sequence shown in SEQ ID NO: 3. In certain embodiments, the intracellular signaling domain of CD3ζ comprises the sequence set forth in SEQ ID NO:4.

[0123] 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.

[0124] In certain embodiments, the antibody is an scFv. In certain embodiments, the scFv comprises the VH sequence set forth in SEQ ID NO: 1 and the variable light chain VL sequence set forth in SEQ ID NO: 2. In certain embodiments, the scFv comprises the VH sequence set forth in SEQ ID NO: 14 and the variable light chain VL sequence set forth in SEQ ID NO: 15. In certain embodiments, the scFv comprises the VH sequence set forth in SEQ ID NO: 16 and the 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 are each identical to the VH and VL sequences set forth in SEQ ID NOs: 1 and 2, respectively; the VH and VL sequences set forth in SEQ ID NOs: 14 and 15, respectively; or the VH and VL sequences set forth in SEQ ID NOs: 16 and 17, respectively, 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. In certain embodiments, the CAR further comprises a hinge and a transmembrane sequence.

[0125] In certain embodiments, the 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.

[0126] Table 5. Amino acid sequence information of selected components of anti-CD7 CAR

[0127]

[0128] 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, the 4-1BB intracellular signaling domain, the CD3 ζ intracellular signaling domain, and the CD8 hinge and transmembrane domain. In some embodiments, the anti-CD7 CAR further comprises a VH-VL linker, such as but not limited to (GGGGS) n linker, wherein n can range from 1 to 6, such as 1, 2, 3, 4, 5 or 6.

[0129] In some embodiments, 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, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity to 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 blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 2, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 3, an amino acid sequence of SEQ ID NO: 4, and an amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD7 protein expression blocking agent comprises an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with SEQ ID NO: 1, an amino acid sequence having at least 90% sequence identity or at least 95% sequence identity with 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 with 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.

[0130] In certain embodiments, the isolated nucleic acid of the present invention comprises one or more nucleotide sequences of Table 6. In some embodiments, the nucleic acid comprises the nucleotide sequence of a CAR component as shown in Table 6.

[0131] Table 6. Amino acid sequence information of selected components of anti-CD7 CAR

[0132]

[0133] In certain embodiments, as described herein, the nucleic acid further comprises a nucleotide sequence encoding a target binding molecule connected to the localization domain. In certain embodiments, the target binding molecule is an antibody that binds to CD7. In certain embodiments, the antibody is a 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 higher 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 higher sequence identity) to the sequence of SEQ ID NO: 2. In certain embodiments, the scFv comprises the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown 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.

[0134] In other aspects, a method of treating cancer in a subject in need thereof is provided, comprising administering to the subject a therapeutic amount of engineered immune cells having any of the embodiments described herein, thereby treating cancer in the subject in need thereof.

[0135] In certain embodiments, the method comprises administering a therapeutic amount of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to CD7, as described herein.

[0136] In certain embodiments, the method comprises administering a therapeutic amount of engineered immune cells further comprising a nucleic acid having a nucleotide sequence encoding a target binding molecule (e.g., an anti-CD7 protein expression blocker) linked to a localization domain as described herein.

[0137] 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-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous gamma-delta T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma. In certain embodiments, the T-cell malignancy is early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0138] As used herein, the term "treating" refers to combating a medical condition (eg, a condition associated with a T-cell malignancy) to the extent that the medical condition is improved according to clinically accepted criteria.

[0139] As used herein, "subject" refers to a mammal (e.g., a human, non-human primate, cattle, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse). In certain embodiments, the subject is a human. A "subject in need thereof" refers to a subject (e.g., a patient) who has or is at risk of developing a disease or condition that can be treated (e.g., improved, ameliorated, prevented) by inducing T cells to exert specific cytotoxicity against malignant T cells.

[0140] As defined herein, a "therapeutic amount" refers to an amount that, when administered to a subject, is sufficient to achieve the desired therapeutic effect (treatment of a condition associated with a T-cell malignancy) in the subject under the conditions of administration. The effective amount of an agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art, and depends on several factors, including, for example, the specific agent selected, the age, sensitivity, tolerance to the drug, and overall health of the subject.

[0141] 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.

[0142] In certain embodiments, the engineered cells are administered to the subject by intravenous infusion, intra-arterial infusion, direct injection into the tumor and / or post-operative tumor bed perfusion, implantation at the tumor site within an artificial scaffold, intrathecal administration, and intraocular administration.

[0143] In certain embodiments, the engineered cells are administered to the subject by infusion. Methods for infusing immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient amount of cells is administered to the recipient to ameliorate the symptoms of the disease. Typically, the infusion dose in a single setting is 10 7 to 10 10 cells, for example, a dose of 10 9 Cells. Infusion is a single dose of 10 9 administration in a dose of 10 cells or divided into several 10 9 The dosage of individual cells can be determined by the method of claim 1. If necessary or specified, the frequency of infusion can be daily, every 2 to 30 days or even longer intervals. The amount of infusion is generally at least 1 infusion per subject and preferably at least 3 infusions (as tolerated), or until the symptoms of the disease have improved. The cells can be infused intravenously at a rate of 50-250 ml / hour. Other suitable modes of administration include intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or postoperative tumor bed perfusion, implantation of the tumor site in an artificial stent, and intrathecal administration. Methods for adapting the present invention to such delivery modes are readily available to those skilled in the art.

[0144] 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.

[0145] In other aspects, there is also provided a method for treating cancer using an engineered immune cell according to any embodiment described herein, comprising administering a therapeutic amount of an engineered immune cell to a subject in need thereof. In certain embodiments, the cancer is a T cell malignancy. In certain embodiments, the T cell malignancy is early T cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0146] In certain embodiments, the engineered cells are administered to the subject by intravenous infusion, intraarterial infusion, intraperitoneal infusion, direct injection into the tumor and / or post-operative tumor bed perfusion, implantation at the tumor site within an artificial scaffold, and intrathecal administration.

[0147] In another aspect, a method for producing an engineered immune cell having any of the embodiments described herein is also provided, the method comprising introducing into the immune cell a nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that binds to CD7.

[0148] 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., an anti-CD7 protein expression blocker or an anti-CD7 PEBL) connected to a localization domain. In certain embodiments, the nucleotide sequence encoding the CAR and the nucleotide sequence encoding the anti-CD7 PEBL are introduced onto a single plasmid.

[0149] In various aspects, kits for producing engineered immune cells as described herein are also provided. This kit can be used to produce, for example, allogeneic or autologous T cells having anti-CD7 CAR-mediated cytotoxic activity. In some embodiments, the kit can be used to produce allogeneic effector T cells having anti-CD7 CAR-mediated cytotoxic activity. In certain embodiments, the kit can be used to produce autologous effector T cells having anti-CD7 CAR-mediated cytotoxic activity.

[0150] Thus, provided herein is a kit comprising a nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that binds to CD7. The nucleotide sequence encoding the anti-CD7 CAR can be designed according to any of the embodiments described herein. In certain embodiments, the nucleotide sequence encodes a Figure 1A Schematic diagram of the anti-CD7 CAR ("anti-CD7-41BB-CD3ζ construct").

[0151] In certain embodiments, the kit further comprises a nucleic acid having a nucleotide sequence encoding a target binding molecule as described herein linked to a localization domain (e.g., an anti-CD7 PEBL molecule described herein). The nucleotide sequence encoding the target binding molecule linked to a localization domain can be designed according to any embodiment described herein.

[0152] In certain embodiments, the nucleotide sequence encoding anti-CD7 CAR and / or the nucleotide sequence encoding anti-CD7 PEBL further comprises a sequence (e.g., a plasmid or vector sequence) that allows, for example, cloning and / or expression. For example, the nucleotide sequence can be provided as part of a plasmid to facilitate cloning into other plasmids and / or vectors (expression vectors or viral expression vectors) for, for example, transfection, transduction, or electroporation into cells (e.g., immune cells). In certain embodiments, the nucleotide sequence encoding anti-CD7 CAR and the nucleotide sequence encoding anti-CD7 PEBL are provided on a single plasmid or vector (e.g., a single construct comprising anti-CD7 CAR and anti-CD7 PEBL). In certain embodiments, the nucleotide sequence is provided on a separate plasmid or vector (expression vector or viral expression vector).

[0153] Typically, the kit is compartmentalized for ease of use and may include one or more containers with reagents. In certain embodiments, all kit components are packaged together. Alternatively, one or more individual components of the kit may be provided in separate packaging with other kit components. The kit may also include instructions for use of the kit components.

[0154] In some embodiments, an engineered immune cell is provided herein, comprising 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). In certain embodiments, the antibody is a single-chain variable fragment (scFv). In some cases, scFv comprises a heavy chain variable domain (VH) sequence shown in SEQ ID NO: 1 and a light chain variable domain (VL) sequence shown in SEQ ID NO: 2.

[0155] In some embodiments, the CAR further comprises a hinge and a transmembrane sequence, such as, but not limited to, a hinge and a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10.

[0156] In some embodiments, the engineered immune 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.

[0157] In some embodiments, the engineered immune cell further comprises a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain. In certain embodiments, the target binding molecule is an antibody that binds to CD7. In certain embodiments, the antibody is a scFv. In some embodiments, the scFv comprises the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:2. In some embodiments, the localization domain comprises an endoplasmic reticulum (ER) or Golgi apparatus retention sequence; a protein body localization sequence; 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.

[0158] In some embodiments, provided herein is an engineered immune cell comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds to cluster of differentiation 7 (CD7); (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, and the localization domain comprises an endoplasmic reticulum retention sequence, and wherein the antibody that binds to CD7 comprises the variable heavy chain (VH) sequence shown in SEQ ID NO: 1 and the variable light chain (VL) sequence shown in SEQ ID NO: 2. In some embodiments, the intracellular signaling domain of 4-1BB comprises the sequence shown in SEQ ID NO: 3 and the intracellular signaling domain of CD3ζ comprises the sequence shown in SEQ ID NO: 4.

[0159] In some embodiments, there is provided herein a method for treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutic amount of an engineered immune cell as described herein, thereby treating a cancer in a subject in need thereof. In some embodiments, the cancer is a T cell malignancy. In certain embodiments, the T cell malignancy is an early T cell progenitor acute lymphoblastic leukemia (ETP-ALL). In certain embodiments, the engineered cells are administered to the subject by intravenous infusion, intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or postoperative tumor bed perfusion, implantation of the tumor site in an artificial scaffold, intrathecal administration.

[0160] In some embodiments, provided herein are nucleic acids comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ, and an antibody that binds cluster of differentiation 7 (CD7).

[0161] In other embodiments, provided herein are engineered immune cells for treating cancer as described herein, comprising administering a therapeutic amount of engineered immune cells to a subject in need thereof. In some embodiments, the cancer is a T cell malignancy. In certain embodiments, the T cell malignancy is early T cell progenitor acute lymphoblastic leukemia (ETP-ALL). In certain embodiments, the engineered cells are administered to the subject by intravenous infusion, intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or postoperative tumor bed perfusion, implantation of the tumor site in an artificial scaffold, intrathecal administration.

[0162] In some embodiments, provided herein are methods for producing engineered immune cells as described herein. The method may include introducing into immune cells a nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises an intracellular signaling domain of 4-IBB and CD3ζ and an antibody that binds to CD7, thereby producing engineered immune cells. The method may also include introducing into immune cells a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain.

[0163] The present invention provides chimeric antigen receptors (CAR) for CD7. As demonstrated herein, the expression of anti-CD7CAR in immune cells such as effector T cells induces T cells to exert specific cytotoxicity against T cell malignancies. When the expression of CD7 on effector T cells is lowered using antibody-based molecules (protein expression blockers or PEBL) targeting CD7, it is shown that this cytotoxic effect is enhanced. Therefore, the present invention provides an immunotherapy method for treating cancer (e.g., T cell malignancies).

[0164] In some aspects, the present invention provides an engineered immune cell comprising a nucleic acid containing 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). In some embodiments, the engineered immune cells outlined herein also include nucleic acids comprising a nucleotide sequence encoding a target binding molecule (e.g., a protein expression blocker or PEBL) connected to a localization domain. A method and kit for producing such engineered immune cells are also outlined herein.

[0165] In some aspects, the present invention provides an engineered immune cell (e.g., a T cell, a natural killer (NK) cell, a NK / T cell, a monocyte, a macrophage, or a dendritic cell) comprising (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that specifically binds to CD7; (ii) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule connected to a localization domain, wherein the target binding molecule is an antibody that binds to CD7, and the localization domain comprises an endoplasmic reticulum retention sequence, and wherein the antibody that binds to CD7 comprises the variable heavy chain (VH) sequence set forth in SEQ ID NO: 1 and the variable light chain (VL) sequence set forth in SEQ ID NO: 2.

[0166] In other aspects, the present invention provides a method for treating a cancer (e.g., a T cell malignancy) in a subject in need thereof. The method comprises administering to the subject a therapeutic amount of any engineered immune cell described herein, thereby treating the cancer in the subject in need thereof. The present disclosure also describes the use of any engineered immune cell outlined herein for treating cancer.

[0167] In other aspects, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the intracellular signaling domains of 4-1BB and CD3ζ and an antibody that specifically binds to CD7.

[0168] The present invention provides embodiments including but not limited to the following:

[0169] 1. An engineered immune cell comprising:

[0170] i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, wherein the target binding molecule is a first antibody that specifically binds to CD7; and

[0171] ii) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0172] 2. The engineered immune cell according to embodiment 1, wherein the first antibody that specifically binds to CD7 is a first single-chain variable fragment (scFv).

[0173] 3. The engineered immune cell of embodiment 1 or 2, wherein the second antibody that specifically binds to CD7 is a second single-chain variable fragment (scFv).

[0174] 4. An engineered immune cell according to any one of embodiments 1 to 3, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0175] 5. An engineered immune cell according to any one of embodiments 1 to 3, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0176] 6. An engineered immune cell according to any one of embodiments 1 to 3, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0177] 7. An engineered immune cell according to any one of embodiments 1 to 4, 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 protease 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.

[0178] 8. The engineered immune cell of embodiment 7, wherein 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.

[0179] 9. The engineered immune cell of embodiment 7, wherein the localization domain comprises a transmembrane domain sequence derived from the CD8α hinge and a transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO: 13.

[0180] 10. The engineered immune cell of any one of embodiments 1 to 9, wherein the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 4.

[0181] 11. An engineered immune cell according to any one of embodiments 1 to 10, wherein the CAR further comprises a hinge and a transmembrane domain.

[0182] 12. The engineered immune cell of embodiment 11, wherein the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 10.

[0183] 13. An engineered immune cell according to any one of embodiments 1 to 12, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0184] 14. An engineered immune cell according to any one of embodiments 1 to 12, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0185] 15. An engineered immune cell according to any one of embodiments 1 to 12, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0186] 16. The engineered immune cell of any one of embodiments 1 to 15, 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.

[0187] 17. An engineered immune cell comprising

[0188] i) a target binding molecule linked to a localization domain, wherein the target binding molecule is a first antibody that specifically binds to CD7; and

[0189] ii) a chimeric antigen receptor (CAR), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0190] 18. The engineered immune cell of embodiment 17, wherein the first antibody that specifically binds to CD7 is a first single-chain variable fragment (scFv).

[0191] 19. The engineered immune cell of embodiment 17 or 18, wherein the second antibody that specifically binds to CD7 is a second single-chain variable fragment (scFv).

[0192] 20. An engineered immune cell according to any one of embodiments 17 to 19, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0193] 21. An engineered immune cell according to any one of embodiments 17 to 19, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0194] 22. An engineered immune cell according to any one of embodiments 17 to 19, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0195] 23. An engineered immune cell according to any one of embodiments 17 to 19, 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 protease 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.

[0196] 24. The engineered immune cell of embodiment 23, wherein 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.

[0197] 25. The engineered immune cell of embodiment 23, wherein the localization domain comprises a transmembrane domain sequence derived from the CD8α hinge and a transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO: 13.

[0198] 26. The engineered immune cell of any one of embodiments 17 to 25, wherein the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 4.

[0199] 27. An engineered immune cell according to any one of embodiments 17 to 26, wherein the CAR further comprises a hinge and a transmembrane domain.

[0200] 28. The engineered immune cell of embodiment 27, wherein the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 10.

[0201] 29. An engineered immune cell according to any one of embodiments 17 to 28, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0202] 30. An engineered immune cell according to any one of embodiments 17 to 28, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0203] 31. An engineered immune cell according to any one of embodiments 17 to 28, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0204] 32. An engineered immune cell according to any one of embodiments 17 to 31, 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.

[0205] 33. A pharmaceutical composition comprising the engineered immune cell of any one of embodiments 1 to 32 and a pharmaceutically acceptable carrier.

[0206] 34. A method of producing the engineered immune cell of any one of embodiments 1 to 32, the method comprising:

[0207] i) Introduction into immune cells

[0208] a) a first nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, wherein the target binding molecule is a first antibody that specifically binds to CD7; and

[0209] b) a second nucleic acid comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3 zeta intracellular signaling domain, and a second antibody that specifically binds to CD7; and

[0210] ii) isolating an engineered immune cell comprising the target binding molecule and the CAR linked to the localization domain, thereby producing the engineered immune cell.

[0211] 35. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of engineered immune cells, thereby treating cancer in the subject in need thereof,

[0212] Wherein the engineered immune cells comprise:

[0213] i) a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, wherein the target binding molecule is a first antibody that specifically binds to CD7; and

[0214] ii) a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a 4-1BB intracellular signaling domain, a CD3ζ intracellular signaling domain, and a second antibody that specifically binds to CD7.

[0215] 36. The method of embodiment 35, wherein the first antibody that specifically binds to CD7 is a first single-chain variable fragment (scFv).

[0216] 37. The method of embodiment 35 or 36, wherein the second antibody that specifically binds to CD7 is a second single-chain variable fragment (scFv).

[0217] 38. A method according to any one of embodiments 35 to 37, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0218] 39. A method according to any one of embodiments 35 to 37, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:15.

[0219] 40. A method according to any one of embodiments 35 to 37, wherein the first single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0220] 41. A method according to any one of embodiments 35 to 38, 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 protease 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.

[0221] 42. The method of embodiment 41, wherein 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.

[0222] 43. A method according to embodiment 41, wherein the localization domain comprises a transmembrane domain sequence derived from the CD8α hinge and a transmembrane domain sequence comprising the amino acid sequence of SEQ ID NO:13.

[0223] 44. The method of any one of embodiments 35 to 43, wherein the 4-1BB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 3 and wherein the CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 4.

[0224] 45. A method according to any one of embodiments 35 to 44, wherein the CAR further comprises a hinge and a transmembrane domain.

[0225] 46. A method according to embodiment 45, wherein the hinge and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 10.

[0226] 47. A method according to any one of embodiments 35 to 46, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0227] 48. A method according to any one of embodiments 35 to 46, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:14 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:15.

[0228] 49. A method according to any one of embodiments 35 to 46, wherein the second single-chain variable fragment (scFv) comprises a heavy chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:16 and a light chain variable domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:17.

[0229] 50. The method of any one of embodiments 35 to 49, 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.

[0230] 51. The method of any one of embodiments 35 to 50, wherein the engineered cells are administered to the subject by intravenous infusion, intra-arterial infusion, intraperitoneal infusion, direct injection into the tumor and / or post-operative tumor bed perfusion, implantation at the tumor site within an artificial scaffold, or intrathecal administration.

[0231] 52. The method of any one of embodiments 35 to 51, wherein the cancer is a T-cell malignancy.

[0232] 53. A method according to embodiment 52, wherein the T cell malignancy is early T cell progenitor acute lymphoblastic leukemia (ETP-ALL).

[0233] Example

[0234] Example 1: Blockade of CD7 expression in T cells to allow chimeric antigen receptors to effectively target T cell malignancies

[0235] This example illustrates the use of a novel approach to block CD7 expression, combined with second-generation CARs, to generate highly effective anti-CD7 CAR-T cells. This practical strategy provides a new treatment option for patients with high-risk T-cell malignancies, including ETP-ALL.

[0236] summary

[0237] Effective immunotherapies for T-cell malignancies are lacking. A novel approach based on the redirection of T lymphocytes with chimeric antigen receptors (CARs) was developed. 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), median CD7 expression was >99%; CD7 expression remained high at relapse (n=14) and during chemotherapy (n=54). CD7 was targeted with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes induces antagonism due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control antagonism, a novel approach based on an anti-CD7 single-chain variable fragment coupled to an intracellular retention domain (protein expression blocker, PEBL) was applied. Transduction of anti-CD7 PEBL caused almost instantaneous elimination of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7+, compared to 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, and abolished CAR-mediated cannibalism. PEBL-CAR-T cells were highly cytotoxic against CD7+ leukemic cells in vitro and were consistently more effective than CD7+ T cells that were free of cannibalism. They also showed strong anti-leukemic activity in cell line-derived and patient-derived T-ALL xenografts. The strategy described here is well-suited to existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.

[0238] introduce

[0239] Chimeric antigen receptor (CAR) expression can be used to induce T lymphocytes to specifically recognize and kill tumor cells. 1-5 Central to the effective application of this technology is the identification of a suitable target for CAR, which must be highly expressed by tumor cells and absent in normal cells, or expressed only by normal cells so that its temporary absence can be managed clinically. 6 Therefore, B-cell-derived leukemias and lymphomas can be treated with antibodies targeting CD19, which is normally expressed only by B lymphocytes. 5,7or CD22 8 CAR targeting. 9,10 Infusion of autologous T cells expressing anti-CD19 CARs produces major clinical responses in patients with B-cell refractory leukemias and lymphomas. 11-18 These exciting results provide indisputable evidence of the power of this technology and suggest the possibility of broader application in oncology.

[0240] 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 therapies in this area is particularly urgent due to the poor prognosis associated with some T-cell leukemia and lymphoma subtypes. For example, children and adolescents with early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) have the worst response to initial therapy among all ALL patients. 19-21 Intensive chemotherapy and / or allogeneic hematopoietic stem cell transplantation often fail to prevent treatment-refractory relapse; for these patients, as well as those with other high-risk characteristics such as adulthood, there is a lack of therapeutic options. 19,22-25

[0241] A major obstacle to developing CAR-T cells effective against T-cell malignancies is that the surface marker profile of malignant T cells (which typically lack CD19 or CD22 expression) largely overlaps with that of activated T lymphocytes. 19,26 CARs targeting such targets may lead to self-elimination of CAR-T cells. 27,28 This article describes the development and application of practical technologies for CAR-T cell therapy of ETP-ALL and other T-ALL cell subtypes. First, a CAR targeting CD7 was prepared. As is known, CD7 is a 40 kDa type I transmembrane glycoprotein that is a major marker of T cell malignancies. 29-32 It is highly expressed in all T-cell ALL conditions, including ETP-ALL. 19 Second, they developed a method to rapidly and effectively downregulate CD7 expression in T cells. This method was chosen because it avoids the cannibalism effect of CAR-T cell therapy, does not involve gene editing, and can be immediately translated into clinical applications.

[0242] Materials and methods

[0243] Cells and culture conditions

[0244] 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 our 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 (from the Vector Development and Production Shared Resource of St. Jude Children's Research Hospital, Memphis, Tennessee). The same vector was used to transduce CCRF-CEM and Jurkat cells with the CD19 gene cloned from RS4 cDNA; the 11B cell line (ATCC). The cell lines were maintained in RPMI-1640 (ThermoFisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0245] Peripheral blood samples were obtained from discarded anonymous platelet donations from healthy adult donors at the National University Hospital Blood Bank, Singapore. Bone marrow aspirates were obtained from ALL patients for diagnostic immunophenotyping and monitoring of treatment response. 19,26 Surplus material was banked in some experiments with approval from the Institutional Review Board of the National University of Singapore. Monocytes were isolated by centrifugation using 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).

[0246] Gene cloning and retroviral transduction

[0247] Anti-CD7 monoclonal antibody TH69 34 The single-chain variable fragment (scFv) of the anti-CD19-41BB-CD3ζ CAR was linked to the CD8α signal peptide, CD8α hinge and transmembrane domain, and the intracellular domains of 4-1BB and CD3ζ of the anti-CD19-41BB-CD3ζ CAR previously developed in our laboratory. 5 The same scFv was linked to the CD8α signal peptide and sequences encoding the endoplasmic reticulum (ER) / Golgi retention peptides EQKLISEEDLKDEL (SEQ ID NO: 8), (GGGGS)4AEKDEL (SEQ ID NO: 9), or the CD8α hinge and transmembrane domains, followed by a localization sequence (SEQ ID NO: 13). These were subcloned into MSCV vectors with or without GFP or mCherry.

[0248] Preparation of retroviral supernatants and transduction were performed as described previously. 5,35 Briefly, pMSCV retroviral vector conditioned medium was added to a polypropylene tube coated with RetroNectin (Takara, Otsu, Japan); after centrifugation and removal of the supernatant, T cells were added to the tube and left at 37°C for 12 hours; fresh viral supernatant was added on two consecutive days. T lymphocytes were maintained in RPMI-1640 containing FBS, antibiotics, and 200 IU / mL IL-2.

[0249] For CAR transient expression, anti-CD7 and anti-CD19 CAR constructs were subcloned into the EcoRI and XhoI sites of the pVAXI vector (ThermoFisher Scientific) and transcribed into mRNA using T7 mScript (CellScript, Madison, WI). For mRNA electroporation, cells were suspended in electroporation buffer (Amaxa cell line nuclear transfection agent kit V; Lonza, Basel, Switzerland) containing 200 μg of CAR mRNA and electroporated using program X-001 using Amaxa transfection agent 2b (Lonza). 36,37 Cells not electroporated with mRNA served as controls.

[0250] Detection of CAR, PEBL, and surface markers

[0251] CAR was detected using a 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 isotype-matched inactive antibodies were from BD Biosciences (San Jose, CA); CD19 (LT19) was from Miltenyi Biotech. Cell staining was analyzed using an Accuri C6, Fortessa, or LSRII flow cytometer (BD Biosciences) with Diva (BD Biosciences) or FlowJo software (FlowJo, Ashland, OR).

[0252] Western blot analysis was performed as described previously. 35In brief, cell lysates were extracted using CelLytic M cell lysis reagent (Sigma-Aldrich, Saint Louis, MO) and subsequently protein quantified using a Pierce BCA protein assay kit (ThermoFisher). Cell lysates were diluted with 4x Laemmli sample buffer (Bio-rad, Hercules, CA) and separated by electrophoresis on a 10% polyacrylamide gel under reducing or non-reducing conditions. The blots were probed with mouse anti-human CD3ζ antibody (8D3; BD Biosciences) and horseradish peroxidase-conjugated goat anti-mouse IgG (R&D Systems, Minneapolis, MN) and Clarity Western ECL substrate (Bio-Rad). Staining was visualized using a ChemiDoc touch imager (Bio-Rad).

[0253] Cell aggregation assays, cytotoxicity assays, and cytokine production

[0254] To measure intercellular aggregation, Jurkat cells were co-cultured with CD7+ or CD7- cells labeled with Calcein Red-Orange AM (ThermoFisher) for 30 minutes; cell doublets were counted by flow cytometry. In some experiments, target cells were pre-incubated for 10 minutes before co-culture with soluble anti-CD7 scFv obtained from the supernatant of Jurkat or 293T cells transduced with constructs composed of scFvs without transmembrane or signaling sequences.

[0255] 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 along with target cells at different effector: target (E:T) ratios and cultured for 4 hours at 37°C and 5% CO2. Viable target cells were counted by flow cytometry. To measure the cytotoxicity of lytic particles, anti-human CD107a-PE (H4A3; BD Biosciences) was added to the co-culture. After 1 hour, monensin (BD GolgiStop) was added and the culture was continued for another 3 hours before flow cytometric analysis.

[0256] To assess cell proliferation, T cells were cultured in RPMI-1640 containing FBS and 120 IU / mL IL-2 at 37°C and 5% CO2, either alone or in the presence of 1:1 E:T MOLT-4 cells. Target cells irradiated or treated with Streck cell preservative (Streck Laboratories, Omaha, NE) to inhibit proliferation were added to the culture every 7 days. The number of live GFP+ or mCherry+ T cells was determined by flow cytometry. In order to produce IFNγ and TNFα, target cells and effector cells of 1:1 E:T were inoculated as described above. After 1 hour, brefeldin A (BD GolgiPlug) was added to the culture and the culture was continued for another 5 hours. Subsequently, intracellular staining was performed with anti-IFNγ-PE (clone 25723.11; BD Biosciences) or anti-TNFα-PE (6401.1111; BD Biosciences), followed by flow cytometry analysis.

[0257] Xenograft models

[0258] Press on NOD.Cg-Prkdc scid IL2rg tmlWjl / SzJ (NOD / scid IL2RGnull) mice (Jackson Laboratory, Bar Harbor, ME) were fed 1×10 6 luciferase-transduced CCRF-CEM cells were injected intravenously (iv). 3 and / or 7 days later, mice were treated with 2x10 7 T cells were injected with downregulated expression of CD7 and anti-CD7 CAR. Other mice received T cells transduced with GFP alone or RPMI-1640 containing 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 Xenogen IVIS-200 system (Caliper Life Sciences, Waltham, MA) after intraperitoneal injection of D-luciferin potassium salt solution (Perkin Elmer, Waltham, MA) (2 mg per mouse). Luminescence was analyzed using Living Image 3.0 software. When the luminescence reached 1x10 10 Mice were euthanized at 400 nm (0.1 nm) photon, or earlier if signs indicating euthanasia appeared.

[0259] For patient-derived xenograft (PDX) models, primary ETP-ALL cells were intravenously injected into NOD / scid IL2RGnull cells and allowed to proliferate for 7-8 subsequent passages. ETP-ALL cells were then injected again into NOD / scid IL2RGnull cells treated with or without PEBL-CAR-T. The presence of ALL cells in peripheral blood and tissues was monitored by flow cytometry. 19,26 After erythrocyte lysis with lysis buffer (Sigma-Aldrich), cells were stained with anti-mouse CD45-PE-cyanine 7 (30-Fl 1, Biolegend), and anti-human CD45-APC-H7 (2D1), CD7-PE (M-T701), CD3 APC (SK7), CD34-peridin chlorophyll protein (8G12) (all from BD Biosciences), and CD33-brilliant violet 421 (WM53, Biolegend). Cells were analyzed using a Fortessa flow cytometer with Diva and FlowJo software.

[0260] result

[0261] CD7 confirmed as a target for CAR-T cell therapy in leukemia

[0262] In leukemic cells from diagnostic bone marrow samples obtained from 49 T-ALL patients (including 14 ETP-ALL patients), the median percentage of CD7 expression was >99% (range, 79%->99%). In only 3 cases (6.1%), CD7 was below 99%: 98% in two cases and 79% in one case ( Figure 1A High CD7 expression was also observed in samples collected from 14 patients with relapsed T-ALL ( Figure 1A The mean fluorescence intensity (MFI) of CD7 in leukemic cells at diagnosis or relapse consistently exceeded the mean fluorescence intensity 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 and 3,032 (1,301-9,582) in normal T cells (n=19; P<0.0001) ( Figure 1B ).

[0263] 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 during the course of the disease. Figure 1C and Figure 1DAs shown, CD7 remained high during treatment. These results confirm that CD7 is a target for CAR-T cell therapy in T-ALL.

[0264] Design and expression of anti-CD7 CAR

[0265] To target CD7, an anti-CD7 CAR was designed that consisted of the scFv of the anti-CD7 antibody TH69 linked to the signaling domains of 4-1BB (CD137) and CD3ζ through the hinge and transmembrane domains of CD8α. Figure 2A Retroviral transduction of this construct in Jurkat cells resulted in high expression of anti-CD7 CAR ( Figure 2B ), which appeared as monomers, dimers, and oligomers by Western blotting ( Figure 2C ).

[0266] To confirm that the TH69 scFv could bind CD7, it was produced in soluble form and tested on CD7+MOLT-4 and CD7-OP-1 cells; MOLT-4 cells were labeled and OP-1 cells were not ( Figure 8A ). In addition, when MOLT-4 cells were pre-incubated with anti-CD7 scFv supernatant, staining with anti-CD7 monoclonal antibody was significantly reduced; CD7 MFI (±SD) ranged from 31,730±1,144 to 5,987±241 (n=3). Jurkat cells expressing anti-CD7CAR formed aggregates with CD7+MOLT-4 cells, while cells transduced with GFP alone or with anti-CD19CAR did not form aggregates; in contrast, anti-CD19CAR induced cell aggregation with CD19+OP-1 cells, while anti-CD7CAR did not ( Figure 8B Preincubation of MOLT-4 or CCRF-CEM with soluble anti-CD7 scFv prevented aggregate formation ( Figure 8C ).

[0267] To determine whether the anti-CD7 CAR was functional, the levels of activation markers CD25 and CD69 were measured in Jurkat cells after 24 hours of co-culture with MOLT4. There was a significant upregulation of both activation markers in cells expressing the anti-CD7 CAR ( Figure 2D and 2E In summary, the anti-CD7-41BB-CD3ζ CAR can bind to its cognate antigen and transduce activation signals upon ligation.

[0268] Expression of anti-CD7 CAR in T cells induces mutual killing

[0269] To determine the effect of anti-CD7-41BB-CD3ζCAR in peripheral blood T lymphocytes, two different approaches 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 rate 24 hours after mRNA electroporation was 39.8% ± 13.0 (n = 7) of the recovery rate after no mRNA electroporation ( Figure 3A ); if CAR was introduced by viral transduction, the cell recovery rate was 25.1% ± 16.2% of that of T cells transduced with mock (n = 10) ( Figure 3B ); Overall, CAR expression reduced cell recovery to 31.1% ± 16.3% (n = 17) after 24 hours. Prolonged cell culture further increased the difference in the number of cells transduced with CAR and mock ( Figure 3C In the absence of target cells, CAR expression induces exocytosis of lytic granules as indicated by CD107a expression ( Figure 3D ), indicating that the impaired cell recovery rate was caused by cannibalism.

[0270] CD7 downregulation prevents T cells from killing each other and does not affect T cell function.

[0271] If poor T cell recovery is caused by cannibalism mediated by CAR binding to T cell-expressed CD7, it should be improved by downregulating CD7 before CAR expression. To test this prediction, a rapid and practical method recently developed based on the expression of anti-CD7 scFv linked to an amino acid sequence containing the ER retention domains KDEL or KKMP [anti-CD7 protein expression blocker (PEBL)] was applied. Figure 3E ). These anchor the construct to the ER / Golgi apparatus, preventing secretion or membrane expression of the targeted protein. 39,40 Three anti-CD7 PEBL constructs were tested and PEBL-1 was selected and used in the following experiments ( Figure 3E and Figure 3F CD7 surface expression was essentially abolished in all T cells transduced with this construct, whereas CD7 mRNA expression was retained ( Figure 3F 、 Figure 10A and Figure 10B ); in 5 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) ( Figure 3G When anti-CD7 CAR was expressed by electroporation in cells with downregulated CD7, anti-CD7 CAR could be clearly detected by flow cytometry ( Figure 3H By expressing CAR in CD7 knockdown cells, T cell viability was significantly improved ( Figure 3I ); in 6 paired experiments, viable cell recovery after CAR mRNA electroporation was consistently higher in T cells that had been previously transduced with anti-CD7 PEBL (P = 0.008).

[0272] After anti-CD7 PEBL transduction, the ratios of CD4 and CD8 cells were similar to those 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 explore the functional capacity of T cells transduced with anti-CD7 PEBL, the cells were engineered to express anti-CD19-CAR (Figure CA). Their ability to exert cytotoxicity, release cytotoxic granules, and secrete IFNγ in the presence of CD19+ ALL cells was tested. Figure 4D 、 4E As shown in Figures 4 and 4F, PEBL transduction and the absence of surface CD7 did not alter CAR-mediated cellular function.

[0273] The anti-CD7-41BB-CD3ζ CAR induces potent cytotoxicity against CD7+ leukemia cells.

[0274] CD7-negative T cells were prepared using anti-CD7 PEBL and electroporated with anti-CD7-41BB-CD3ζCAR mRNA. The anti-leukemic capacity of CD7-negative T cells was evaluated in co-culture with CD7+ leukemic cell lines MOLT-4, CCRF-CEM, Jurkat, Loucy, or KG1a. Figure 5A As shown, CAR expression significantly increased cytotoxicity. PEBL-CAR T cells were also very effective against primary T-ALL cells obtained from patients ( Figure 5B ).

[0275] The cytotoxicity of PEBL-CAR T cells was compared with that of residual T cells recovered after CAR electroporation in cells not transduced with PEBL. In 45 experiments performed with cells from three donors, the cytotoxicity of PEBL-CAR cells consistently exceeded that of non-PEBL T cells ( Figure 5C When compared with CD107a ( Figure 5D ), IFNγ( Figure 11A ) and TNFα( Figure 11B ) expression, the former cells also observed excellent activity. By sequentially transducing PEBL and CAR through retroviral transduction, the patient-derived T-ALL cells ( Figure 5E ) and cell lines ( Figure 12) produced potent cytotoxicity. The proliferation of anti-CD7 PEBL-CAR-T cells in the presence of CD7+ target cells was much higher than that of CAR-T cells that did not downregulate CD7 through PEBL (P<0.01) ( Figure 5F Finally, the cytotoxicity exerted by anti-CD7 PEBL-CAR T cells was compared with that of cells expressing anti-CD19-41BB-CD3ζCAR. 5 To this end, CCRF-CEM and Jurkat cells were transduced with CD19 and CAR was also expressed in cells previously transduced with anti-CD7 PEBL ( Figure 13A and 13B Anti-CD7 and anti-CD19 CAR T cells have similar short-term and long-term cytotoxicity ( Figure 13C and 13D ); the long-term proliferation capacity in the presence of CD9+CD7+ target cells was slightly lower for anti-CD7 CAR-T cells ( Figure 13E ), which may be due to the lower expression of CD7 relative to CD19 on target cells ( Figure 13B ).

[0276] Anti-leukemic activity of anti-CD7 PEBL-CAR T cells in a murine T-ALL model

[0277] To further measure the anti-tumor capacity of anti-CD7 PEBL-CAR T cells, CCRF-CEM cells were implanted into NCR / scid IL2RGnull cells. T cells retrovirally transduced with anti-CD7 PEBL and anti-CD7 CAR produced a substantial anti-leukemic effect, with a significant reduction in leukemic cell load and decreased leukemic cell growth ( Figures 6A-6C ; Figure 14A and 14B Three weeks after leukemia cell injection, the median percentage of CCRF-CEM cells in peripheral blood, as detected by flow cytometry, was 68% for control mice (n=5) and 67% for patients who received GFP-T cells alone (n=5), but they were undetectable in mice treated with anti-CD7 PEBL-CAR T cells ( Figure 15A Relapses that occurred after treatment with anti-CD7 PEBL-CAR T cells were not due to a subset of CCRF-CEM cells that lacked CD7; leukemic cells continued to express high levels of CD7 and remained highly sensitive to anti-CD7 CAR cytotoxicity, regardless of whether the CCRF-CEM cells were derived from the liver or spleen of relapsed mice or directly from primary cell cultures ( Figure 15B ).

[0278] To test PEBL-CAR T cells against primary leukemic cells in vivo, a PDX model of ETP-ALL was used. The PDX model allows the propagation of leukemic cells derived from ETP-ALL patients at diagnosis in NOD / scid IL2RGnull mice. The leukemic cells retain an immunophenotype matching that determined at diagnosis, expressing CD7, CD34, and CD33 but lacking surface CD3, CD1a, CD8, and CD5. Figure 16 ); cells cannot survive and expand in vitro and need to be injected into mice for proliferation. During CAR-T treatment, all mice had ETP-ALL in their peripheral blood ( Figure 7A ).like Figure 7B As shown, ETP-ALL cells represent the majority of leukocytes in the bone marrow, spleen, and lungs. 7 The remaining 4 mice were given 2×10 6 After 48 hours of treatment, the number of leukemia cells in the peripheral blood decreased dramatically, and PEBL-CAR-T cells became detectable in all mice ( Figure 7A In blood smears, broken cells stand out, indicating lysis of leukemic cells ( Figure 7C ). Leukemia progressed in all five control mice, and when ETP-ALL ≥ 80% of peripheral blood mononuclear cells, these mice were euthanized. 7 The mice treated with PEBL-CAR-T cells died of obvious graft-versus-host disease (GvHD) 23 days after PEBL-CAR-T cell infusion. ETP-ALL could not be detected in the blood, bone marrow, liver, spleen, lungs and brain, while PEBL-CAR T cells could be detected in all tissues ( Figure 7D and 7E ). Use 2×10 6 Four mice treated with PEBL-CAR T cells survived without signs of GvHD from 25 days (n=1) to 39 days (n=3) after infusion.

[0279] discuss

[0280] CAR-T cells can achieve lasting remission in patients with B-cell leukemia and lymphoma, but there is a lack of effective options for patients with T-cell malignancies. To bridge this gap, this article develops and describes a CAR-T cell method that can be quickly translated into clinical intervention. Targeting CD7, a widely expressed surface T cell marker, is highly stable even in T-ALL cells exposed to chemotherapy. A second-generation anti-CD7 CAR was designed. It is necessary to determine that suppressing CD7 surface expression in T cells is necessary; without it, CAR causes severe loss of T cells and cannot achieve the full functional potential of CAR-T cells. Transduction of anti-CD7 PEBL results in almost instantaneous elimination of CD7 expression. The expression of anti-CD7 CAR in such cells produces powerful anti-leukemia activity in vitro and in xenografts and PDX models of T-ALL. Therefore, by using this strategy, a large number of CAR-T cells are rapidly produced and used to exert powerful and specific cytotoxicity against T-cell malignancies (including ETP-ALL, one of the most aggressive forms).

[0281] The PEBL technology for downregulating endogenous CD7 described herein is based on the use of scFvs directed against a targeting antigen coupled to an ER / Golgi retention motif. In this way, any newly synthesized CD7 remains anchored in the ER and / or Golgi apparatus and prevents its surface expression. This approach is very effective in downregulating CD7 and inhibiting CAR-mediated mutual killing. Importantly, intracellular retention of CD7 does not alter T cell function and allows normal expansion, cytokine secretion, and cytotoxicity. This is consistent with the results of studies in CD7-deficient mice, which showed normal lymphocyte populations in lymphoid tissues. 41,42 Another approach to downregulate CD7 is to apply gene editing methods such as meganucleases, TALENs or CRISPR / Cas9. 43 To this end, a recent study reported the expression of anti-CD7 CAR in T cells in which the CD7 gene was deleted by CRISPR / Cas9. 9,44 In addition to the difference in costimulatory molecules that may have clinical impact (the CAR described herein has 4-1BB instead of CD28), 45,46 The high specificity and practicality of the PEBL strategy make it particularly attractive for current clinical applications. This approach requires simple transduction with the same viral vector carrying the CAR, either as two sequential transductions or as a single transduction with a bicistronic vector carrying both constructs. It is well-suited to established clinical-grade cell manufacturing processes and does not raise potential regulatory issues associated with off-target activity. 47,48

[0282] CD7 is a marker molecule for early T-cell differentiation; it is almost universally expressed in T-ALL, whereas in normal cells, its expression is restricted to T cells. 19,29-32 In clinical studies of the anti-CD7-ricin-A-chain immunotoxin in patients with T-cell lymphoma, the dose-limiting toxicity was vascular leak syndrome, a side effect seen with other toxin conjugates; no binding of anti-CD7 was found in endothelial cells of various tissues. 49 However, transient expression of CARs by mRNA electroporation might be considered in early studies evaluating the acute toxicity potential of anti-CD7 PEBL-CAR T cells. One concern with anti-CD7 CAR therapy is that the infused cells can cause depletion of normal T cells, leading to immunodeficiency. It is envisioned that this technology would initially be applied as a means of reducing MRD in patients with high-risk T-ALL, thereby maximizing the success of allogeneic hematopoietic stem cell transplantation. 50 In such cases, anti-CD7 CAR T cells will be eliminated through transplant adaptation and the T cell compartment will be reconstituted from donor stem cells. Outside the transplant setting, once leukemia eradication is achieved, the "suicide gene" can be activated. 51 Ultimately, this may not be a problem, as the infused anti-CD7 T cells (which retain their endogenous CD3 / TCR complex) may reconstitute a sufficiently broad T cell repertoire. To this end, it should be noted that CD4 memory and CD8 effector T cell subsets that do not express CD7 have been described in human blood lymphocytes. 52,53 Furthermore, T-ALL cells express CD7 at higher levels than normal T cells. Therefore, the CD7-dim subset may help repopulate the T cell pool even after CD7-directed therapy.

[0283] Standard treatment for T-ALL patients with high-risk disease relies primarily on intensive chemotherapy plus hematopoietic stem cell transplantation. Results are far from satisfactory and are associated with considerable morbidity and mortality. 54,55 The results presented here suggest that infusion of anti-CD7 PEBL-CAR T cells could significantly enhance or possibly replace existing chemotherapy and transplantation-based strategies. It is conceivable that CAR expression together with downregulation of the targeted antigen in T cells should also be applicable to other T cell markers such as CD3, CD2, and CD5, whose expression is ubiquitous in T cell lymphoproliferative neoplasms. Because a proportion of high-risk acute myeloid leukemia cases express CD7, 19,30,56 Therefore, it is reasonable to test the potential of anti-CD7 CAR-T cells against this leukemia subtype.

[0284] References

[0285] 1. Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA 1993;90(2):720-724.

[0286] 2. Geiger TL, Leitenberg D, Flavell RA. The TCR zeta-chain immunoreceptor tyrosine-based activation motifs are sufficient for the activation and differentiation of primary T lymphocytes. J Immunol 1999;162(10):5931-5939.

[0287] 3. Brentjens RJ, Latouche JB, Santos E et al., Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nat Med 2003;9(3):279-286.

[0288] 4. Cooper LJ, Topp MS, Serrano LM et al., T-cell clones can be rendered specific for CD 19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood 2003;101(4):1637-1644.

[0289] 5. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Campana D. Chimeric receptors with 4-IBB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004;18:676-684.

[0290] 6. Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 2015;348(6230):62-68. 7. Brentjens RJ, Santos E, Nikhamin Y et al., Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts. Clin Cancer Res 2007;13(18Pt1):5426-5435.

[0291] 8. Haso W, Lee DW, Shah NN et al., Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood 2013;121(7):1165-1174.

[0292] 9. Nadler LM, Anderson KC, Marti G et al., B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes. J Immunol 1983;131(l):244-250.

[0293] 10. Campana D, Janossy G, Bofill M et al., Human B cell development. I. Phenotypic differences of B lymphocytes in the bone marrow and peripheral lymphoid tissue. J Immunol 1985; 134(3): 1524 - 1530.

[0294] 11. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor - modified T cells in chronic lymphoid leukemia. N Engl J Med 2011; 365(8): 725 - 733.

[0295] 12. Grupp SA, Kalos M, Barrett D et al., Chimeric antigen receptor - modified T cells for acute lymphoid leukemia. N Engl J Med 2013; 368(16): 1509 - 1518.

[0296] 13. Till BG, Jensen MC, Wang J et al., CD20 - specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4 - 1BB domains: pilot clinical trial results. Blood 2012; 119(17): 3940 - 3950.

[0297] 14. Kochenderfer JN, Dudley ME, Feldman SA, et al., B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD 19 chimeric-antigen-receptor-transduced T cells. Blood 2012;119(12):2709-2720.

[0298] 15. Davila ML, Riviere I, Wang X, et al., Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia. Sci TranslMed 2014;6(224):224ra225.

[0299] 16. Maude SL, Frey N, Shaw PA, et al., Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014;371(16):1507-1517.

[0300] 17. Lee DW, Kochenderfer IN, Stetler-Stevenson M, et al., T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015;385(9967):517-528.

[0301] 18. Turtle CJ, Hanafi LA, Berger C, et al., CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016;126(6):2123-2138.

[0302] 19. Coustan-Smith E, Mullighan CG, Onciu M, et al., Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia. Lancet Oncol 2009;10(2):147-156.

[0303] 20. Zhang J, Ding L, Holmfeldt L, et al., The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012;481(7380):157-163.

[0304] 21. Inukai T, Kiyokawa N, Campana D, et al., Clinical significance of early T-cell precursor acute lymphoblastic leukaemia: results of the Tokyo Children's Cancer Study Group Study L99-15. Br J Haematol 2012;156(3):358-365.

[0305] 22. Neumann M, Heesch S, Gokbuget N, et al., Clinical and molecular characterization of early T-cell precursor leukemia: a high-risk subgroup in adult T-ALL with a high frequency of FLT3 mutations. Blood Cancer J 2012;2(1):e55.

[0306] 23. Jain N, Lamb AV, O'Brien S, et al. Early T-cell precursor acute lymphoblastic leukemia / lymphoma (ETP-ALL / LBL) in adolescents and adults: a high-risk subtype. Blood 2016;127(15):1863-1869.

[0307] 24. Marks DI, Rowntree C. Management of adults with T-cell lymphoblastic leukemia. Blood 2017;129(9):1134-1142.

[0308] 25. Campana D, Pui CH. Minimal residual disease-guided therapy in childhood acute lymphoblastic leukemia. Blood 2017;129(14):1913-1918.

[0309] 26. Coustan-Smith E, Sancho J, Hancock ML, et al. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood 2002;100:2399-2402.

[0310] 27. Mihara K, Yanagihara K, Takigahira M, et al. Activated T-cell-mediated immunotherapy with a chimeric receptor against CD38 in B-cell non-Hodgkin lymphoma. J Immunother 2009;32(7):737-743.

[0311] 28.Mamonkin M,Rouce RH,Tashiro H,Brenner MK.A T-cell-directedchimeric antigen receptor for the selective treatment of T-cellmalignancies.Blood2015;126(8):983-992。

[0312] 29.Haynes BF,Eisenbarth GS,Fauci AS.Human lymphocyte antigens:production of a monoclonal antibody that defines functional thymus-derivedlymphocyte subsets.Proc Natl Acad Sci U S A 1979;76(11):5829-5833。

[0313] 30.Vodinelich L,Tax W,Bai Y,Pegram S,Capel P,Greaves MF.A monoclonalantibody(WTl)for detecting leukemias of T-cell precursors(T-ALL).Blood 1983;62(5):1108-1113。

[0314] 31.Janossy G,Coustan-Smith E,Campana D.The reliability of cytoplasmicCD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia:astudy of 500cases.Leukemia 1989;3(3):170-181。

[0315] 32. Yeoh EJ, Ross ME, Shurtleff SA, et al., Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002;1:133 - 143.

[0316] 33. Manabe A, Coustan - Smith E, Kumagai M, et al., Interleukin - 4 induces programmed cell death (apoptosis) in cases of high - risk acute lymphoblastic leukemia. Blood 1994;83(7):1731 - 1737.

[0317] 34. Peipp M, Kupers H, Saul D, et al., A recombinant CD7 - specific single - chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells. Cancer Res 2002;62(10):2848 - 2855.

[0318] 35. Kudo K, Imai C, Lorenzini P, et al., T lymphocytes expressing a CD16 signaling receptor exert antibody - dependent cancer cell killing. Cancer Res 2014;74(1):93 - 103.

[0319] 36. Shimasaki N, Fujisaki H, Cho D, et al., A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B - cell malignancies. Cytotherapy. 2012;14(7):830 - 40.

[0320] 37. Shimasaki N, Campana D. Natural killer cell reprogramming with chimeric immune receptors. Methods Mol Biol 2013;969:203-220.

[0321] 38. Chang YH, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res 2013;73(6):1777-1786.

[0322] 39. Munro S, Pelham HR. A C-terminal signal prevents secretion of luminal ER proteins. Cell 1987;48(5):899-907.

[0323] 40. Jackson MR, Nilsson T, Peterson PA. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J 1990;9(10):3153-3162.

[0324] 41. Bonilla FA, Kokron CM, Swinton P, Geha RS. Targeted gene disruption of murine CD7. Int Immunol 1997;9(12):1875-1883.

[0325] 42. Lee DM, Staats HF, Sundy JS et al., Immunologic characterization of CD7-deficient mice. J Immunol 1998;160(12):5749-5756.

[0326] 43. Boettcher M, McManus MT. Choosing the right tool for the job: RNAi, TALEN, or CRISPR. Mol Cell 2015; 58(4): 575 - 585.

[0327] 44. Gomes - Silva D, Srinivasan M, Sharma S et al., CD7 - edited T cells expressing a CD7 - specific CAR for the therapy of T - cell malignancies. Blood. 2017 May 24. pii: blood - 2017 - 01 - 761320.

[0328] 45. Campana D, Schwarz H, Imai C. 4 - IBB chimeric antigen receptors. Cancer J 2014; 20(2): 134 - 140.

[0329] 46. Zhao Z, Condomines M, van der Stegen SJ et al., Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells. Cancer Cell 2015; 28(4): 415 - 428.

[0330] 47. Tsai SQ, Joung JK. Defining and improving the genome - wide specificities of CRISPR - Cas9 nucleases. Nat Rev Genet 2016; 17(5): 300 - 312.

[0331] 48. Cameron P, Fuller CK, Donohoue PD et al., Mapping the genomic landscape of CRISPR - Cas9 cleavage. Nat Methods 2017; 14(6): 600 - 606.

[0332] 49. Frankel AE, Laver JH, Willingham MC, Burns LJ, Kersey JH, Vallera DA. Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin. Leuk Lymphoma 1997; 26(3-4): 287-298.

[0333] 50. Leung W, Pui CH, Coustan-Smith E et al., Detectable minimal residual disease before hematopoietic cell transplantation is prognostic but does not preclude cure for children with very-high-risk leukemia. Blood 2012; 120(2): 468-72.

[0334] 51. Straathof KC, Spencer DM, Sutton RE, Rooney CM. Suicide genes as safety switches in T lymphocytes. Cytotherapy 2003; 5(3): 227-230.

[0335] 52. Reinhold U, Abken H, Kukel S et al., CD7-T cells represent a subset of normal human blood lymphocytes. J Immunol 1993; 150(5): 2081-2089.

[0336] 53. Aandahl EM, Sandberg JK, Beckerman KP, Tasken K, Moretto WJ, Nixon DF. CD7 is a differentiation marker that identifies multiple CD8 T cell effector subsets. J Immunol 2003; 170(5): 2349-2355.

[0337] 54. Raetz EA, Teachey DT. T-cell acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program 2016; 2016(l):580-588.

[0338] 55. Jabbour E, O'Brien S, Konopleva M, Kantarjian H. New insights into thepathophysiology and therapy of adult acute lymphoblastic leukemia. Cancer2015;121(15):2517-2528.

[0339] 56. Kita K, Miwa H, Nakase K et al., Clinical importance of CD7 expression in acute myelocytic leukemia. The Japan Cooperative Group of Leukemia / Lymphoma. Blood 1993;81(9):2399-2405.

[0340] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0341] While the invention has been particularly shown and described with reference to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. An expression vector comprising: a) a first nucleic acid comprising a nucleotide sequence encoding a target CD7 binding molecule linked to an endoplasmic reticulum (ER) localization domain, wherein the target CD7 binding molecule comprises a first scFv antibody that binds to endogenous CD7 in an engineered immune cell, and wherein the ER localization domain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 13, and wherein the first nucleic acid comprises a nucleotide sequence encoding a CD8α signal peptide; and b) a second nucleic acid comprising a nucleotide sequence encoding a CD7 CAR, wherein the CD7 CAR comprises a second scFv antibody that binds to CD7, a transmembrane domain, and an intracellular signaling domain; wherein the first scFv antibody and the second scFv antibody comprise: (i) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, comprising: a heavy chain (HC) complementarity determining region (CDR) 1 consisting of amino acids 31-35 of SEQ ID NO: 1, HC CDR2 consisting of amino acids 50-65 of SEQ ID NO: 1, and HC CDR3 consisting of amino acids 98-106 of SEQ ID NO: 1, and A light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, comprising: light chain (LC) CDR1 consisting of amino acids 28-38 of SEQ ID NO: 2, LC CDR2 consisting of amino acids 54-60 of SEQ ID NO: 2, and LC CDR3, which consists of amino acids 93-101 of SEQ ID NO: 2, (ii) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 14, comprising: HC CDR1 consisting of amino acids 31-35 of SEQ ID NO: 14, HC CDR2 consisting of amino acids 50-66 of SEQ ID NO: 14, and HC CDR3 consisting of amino acids 99-112 of SEQ ID NO: 14, and A light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, comprising: LC CDR1 consisting of amino acids 24-34 of SEQ ID NO: 15, LC CDR2 consisting of amino acids 50-56 of SEQ ID NO: 15, and LC CDR3 consisting of amino acids 89-97 of SEQ ID NO: 15, or (iii) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, comprising: HC CDR1 consisting of amino acids 31-35 of SEQ ID NO: 16, HC CDR2 consisting of amino acids 50-66 of SEQ ID NO: 16, and HC CDR3 consisting of amino acids 99-109 of SEQ ID NO: 16, and A light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, comprising: LC CDR1 consisting of amino acids 24-38 of SEQ ID NO: 17, LC CDR2 consisting of amino acids 54-60 of SEQ ID NO: 17, and LC CDR3, which consists of amino acids 93-101 of SEQ ID NO: 17, The binding of the endogenous CD7 to the CD7 binding domain reduces the surface expression of the endogenous CD7 in the engineered immune cells, thereby improving the effectiveness of the engineered immune cells against CD7-positive cancer cells. 2 . The expression vector according to claim 1 , wherein the amino acid sequence of the first scFv antibody is identical to the amino acid sequence of the second scFv antibody.

3. The expression vector according to claim 1, wherein the first scFv antibody comprises: a) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 2; b) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 15; or c) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO:

17.

4. The expression vector of claim 1, wherein the second scFv antibody comprises: a) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 2; b) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 14 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 15; or c) a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO:

17. 5 . The expression vector according to claim 1 , wherein the first scFv antibody and the second scFv antibody both comprise a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO:

2.

6. The expression vector of claim 1, wherein the CD7 CAR comprises a 4-1BB intracellular signaling domain and a CD3ζ intracellular signaling domain.

7. An engineered immune cell comprising the expression vector according to claim 1.

8. The engineered immune cell of claim 7, wherein the engineered immune cell is a T cell.

9. Use of an effective amount of engineered immune cells in the preparation of a medicament for treating a T-cell malignancy in a patient in need thereof, wherein the engineered immune cells comprise the expression vector according to claim 1.

10. The use according to claim 9, wherein the engineered immune cells are T cells. The use according to claim 10 , wherein the amino acid sequence of the first scFv antibody is identical to the amino acid sequence of the second scFv antibody.

12. The method according to claim 9, wherein the first scFv and the second scFv respectively comprise: a) a heavy chain variable domain consisting of the amino acids of SEQ ID NO: 1 and a light chain variable domain consisting of the amino acids of SEQ ID NO: 2; b) a heavy chain variable domain consisting of the amino acids of SEQ ID NO: 14 and a light chain variable domain consisting of the amino acids of SEQ ID NO: 15; or c) a heavy chain variable domain consisting of the amino acids of SEQ ID NO: 16 and a light chain variable domain consisting of the amino acids of SEQ ID NO:

17. The expression vector according to claim 1 , wherein the second nucleic acid further comprises a nucleotide sequence encoding a CD8α signal peptide. The expression vector according to claim 1 , wherein the CD8α signal peptide consists of the amino acid sequence of SEQ ID NO: 7.

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