Modified invariant natural killer T cells expressing chimeric antigen receptors and uses thereof

Abstract

This disclosure relates to CD7 + Modified invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells (e.g., CD3 + iTCRVα24-Jα18 + or CD3 + Vα24 + ), and pharmaceutical compositions, methods of preparation, and therapeutic uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 313,186, filed February 23, 2022, which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to modified invariant natural killer T (NKT) cells expressing CD7 chimeric antigen receptors (e.g., iNKT cells, type I NKT cells), pharmaceutical compositions, methods of preparation, and methods for identifying and determining whether or not a CD7 chimeric antigen receptor is expressed. + The therapeutic use of the cells or pharmaceutical compositions thereof for the treatment or prevention of cancer. [Background technology]

[0003] Natural killer T (NKT) cells are a subset of T cells that exhibit characteristics of both conventional T cells and natural killer (NK) cells. NKT cells are typically CD4+ T cells that have undergone T cell receptor (TCR) gene rearrangement in the thymus. + CD8 + They arise from cortical thymocytes. NKT cells are traditionally defined as CD1d-restricted, lipid antigen-reactive T cells and are classified as type I and type II NKT cells (Godfrey et al., Immunity 2018;48(3):453-73; Dhodapkar and Kumar, J Immunol. 2017;198(3):1015-21). Based on their TCR repertoire, antigen specificity, and CD1d dependency, NKT cells have also been divided into three categories: type I, type II, and type III NKT cells (Godfrey et al., Nat Rev Immunol. 2004;4(3):231-37).

[0004] Type I or invariant NKT (iNKT) cells express the invariant TCR α chain (Vα14-Jα18 in mice and Vα24-Jα18 in humans) and a limited number of non-invariant TCR β chains (Vβ8.2, Vβ7, and Vβ2 in mice, Vβ11 in humans) (Godfrey et al., Nat Immunol. 2010;11(3):197-206; Krovi and Gapin, Front Immunol. 2018;9(6):1939). iNKT (type I NKT, used interchangeably throughout) cells also recognize the glycosphingolipid α-galactosylceramide (αGalCer) antigen when presented by major histocompatibility complex (MHC) class I-like CD1d molecules (Kawano et al., Science. 1997;278(5343):1626-1629). Type II NKT cells have a more diverse and less defined TCR repertoire and recognize non-αGalCer molecules (e.g., sulfatide) presented by CD1d molecules. Type III NKT or NKT-like cells have a diverse TCR repertoire and recognize CD1d-independent molecules.

[0005] The use of iNKT cells co-expressing a chimeric antigen receptor (CAR-iNKT) with interleukin-15 (IL-15), or allogeneic hematopoietic stem cell engineered iNKT (HSC-iNKT), or bispecific single chain antibodies stabilizing the invariant T cell receptor (iTCR)-CD1d complex, has recently yielded promising antitumor outcomes in patients with neuroblastoma and in mice engrafted with human brain lymphoma or multiple myeloma (Rotolo et al., Cancer Cell. 2018;34(4):596-610; Xu et al., Clin Cancer Res. 2019;25(23):7126-7138; Heczey et al., Nat Med. 2020;26(11):1686-1690; Zhu et al., Cell Stem Cell. 2019(25):542-557; Li et al., Cell Rep Med. 2021;2(11):100449; Lameris et al., Nat Cancer. 2020;1(11):1054-1065). Advantages of CAR-iNKT versus CAR-modified conventional T (CAR-T) cells include: 1) CAR-iNKT can infiltrate tumors more efficiently, inhibit tumor-associated macrophages (TAMs), and may be more effective in solid tumors; 2) iNKT cells have unique functional characteristics and metabolic properties that may confer adaptability and longevity to the nutrient-poor tumor microenvironment (TME); and 3) CAR-iNKT do not cause graft-versus-host disease (GVHD) and can be used as an allogeneic off-the-shelf therapy (Song et al., J Clin Invest. 2009;119(6):1524-1536; Khurana et al., Front Immunol., 2021;12:700374; Nair and Dhodapkar, Front Immunol. 2017;8:1178; Delfanti et al., Sci Immunol. 2022;7(74):eabn6563. doi:10.1126 / sciimmunol.abn6563).

[0006] Since human iNKT cells in peripheral blood account for approximately 0.01-1%, expanding iNKT cells for therapeutic use has been a challenge. The most common method is to stimulate isolated iNKT cells with irradiated PBMC negative fraction supplemented with αGalCer, IL-2, IL-7, and IL-15, and expand them with αGalCer-pulsed autologous dendritic cells (DCs) or autologous PBMCs (Poels et al., Int J Mol Sci. 2021;22:1096; Tian et al., J Clin Invest. 2016;126(6):2341-2355). Isolated iNKT cells stimulated with OKT3 anti-CD3 monoclonal antibody, irradiated autologous PBMCs, and IL-2 have been used in clinical trials to treat advanced melanoma (Exley et al., Clin Cancer Res. 2017;23(14):3510-3519). Isolated iNKT cells stimulated with αGalCer-pulsed autologous PBMCs, IL-2, and IL-21 and engineered to express the GD2 CAR have been used in clinical trials to treat neuroblastoma (Heczey et al., Nat Med. 2020;26(11):1686-1690). Alternatively, isolated iNKT cells were stimulated with autologous PBMCs, anti-CD3 / CD28 beads, and IL-15 (Rotolo et al., Cancer Cell. 2018;34(10):596-610).

[0007] Adoptive transfer of CAR-T cells against tumor antigens has emerged as a powerful strategy in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or multiple myeloma (MM) (June and Sadelain, N Engl J Med. 2018:379(1):64-73). However, a major obstacle in the development of CAR-T therapy for T-cell malignancies is the fratricide of CAR-T cells through self-killing due to shared expression of many targetable antigens (e.g., CD1a, CD2, CD5, and CD7) between CAR-T cells and malignant T cells, resulting in insufficient numbers of CAR-T cells for infusion (Scherer et al., Front Oncol. 2019;9:126; Bayon-Calderon et al., Int J Mol Sci. 2020;21(20):7685).

[0008] CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genomic disruption of CD7 and / or TCR alpha chain (TRAC) expression offers an alternative approach that allows the expansion of CD7 CAR-transduced T cells from allogeneic healthy donors without extensive autoantigen-driven fratricide. Edited CD7 CAR-T cells generated robust cytotoxicity against malignant T cell lines and primary tumors and were protective in mouse xenograft models of acute T cell leukemia (T-ALL) (Gomes-Silva et al., Blood. 2017;130(3):285-296; Cooper et al., Leukemia. 2018;32(9):1970-1983). However, the safety and clinical implications of CRISPR-based gene editing remain under scrutiny. Studies have reported that CRISPR-Cas9 induces large-scale DNA deletions and chromosomal rearrangements, activation of the p53 tumor suppressor protein and selection of p53 mutations, chromothripies (i.e., chromosome shattering), and large structural variants at on- and off-target sites in vivo, which can be transmitted to subsequent generations (Sheridan C. Nat Biotechnol. 2021;39(8):897-899; 2022;40(1):5-8; Urnov FD. Nat Genet. 2021;53(6):768-769; Hoijer et al., Nat Commun. 2022;13(1):627). Moreover, a recent study documented high frequency of chromosome losses and shortening in genetically engineered T cells following CRISPR-Cas9 transfection (Nahmad et al., Nat Biotechnol. 2022;40(12):1807-1813). Summary of the Invention [Problem to be solved by the invention]

[0009] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease. T-ALL subtypes include ETP (early thymic precursor), Pro-T, Pre-T, cortical and mature T-ALL (Bayon-Calderon et al., Int J Mol Sci. 2020;21(20):7685). T-ALL represents approximately 25% and 15% of all newly diagnosed ALL cases in pediatric and adult patients, respectively. Intensive chemotherapy as the standard frontline therapy for T-ALL has increased the cure rate to approximately 85%. However, adult T-ALL still shows a dismal outcome and has a significantly lower survival rate than pediatric T-ALL. Children and adolescents with ETP ALL have the poorest response to initial therapy (Cordo et al., Blood Cancer Discov. 2020;24(2);19-31; Fleischer et al., J Hematol Oncol. 2019;12(1);141; Raetz and Teachey, Hematology Am Soc Hematol Educ Program. 2016;2016(1):580-588). Therefore, novel therapies are urgently needed for refractory or relapsed and high-risk patients with T-ALL. [Means for solving the problem]

[0010] The present disclosure provides a method for the production of CD7 + Invariant natural killer T (iNKT) cells or type I NKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the CD7 CAR-modified iNKT cells disclosed herein are + iTCR(Vα24-Jα18) + iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + CD4 + cells, CD3+ iTCR(Vα24-Jα18) + CD8 + cells, CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - cells, CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + cells, or a mixture thereof).

[0011] The present disclosure further relates to methods and compositions (e.g., pharmaceutical compositions) that use the CD7 CAR-modified iNKT cells disclosed herein. In some embodiments, the CD7 CAR-modified iNKT cells disclosed herein are, for example, + They may be useful as therapeutic agents in the treatment or prevention of cancer (e.g., T-cell leukemia and lymphoma, or acute myeloid leukemia). In some embodiments, the iNKT cells disclosed herein may be isolated (e.g., from a biological sample, e.g., from a patient or donor), cultured, modified to express a CD7 CAR, and expanded into a cell population. In some embodiments, the CD7 CAR-modified iNKT cells disclosed herein are present in and / or used in a pharmaceutical composition.

[0012] Also provided is a pharmaceutical composition comprising the iNKT cells disclosed herein. In one aspect, the present disclosure provides an isolated CD3 + iTCR(Vα24-Jα18) + The present invention relates to a pharmaceutical composition comprising an iNKT cell and a pharma- ceutical acceptable carrier. In some embodiments, the cell is a CD3 + iTCR(Vα24-Jα18) + CD4 + cells, CD3 + iTCR(Vα24-Jα18) + CD8 + cells, CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 -Cells, and CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + each optionally isolated from a biological sample of a subject or donor.

[0013] In one embodiment, the present disclosure provides a method for treating or preventing CD7 + The present invention relates to a method of treating or preventing cancer, comprising administering to a subject a therapeutically effective amount of CD7 CAR-modified iNKT cells according to any of the embodiments disclosed herein, or a pharmaceutical composition comprising a therapeutically effective amount of CD7 CAR-modified iNKT cells and a pharma- ceutically acceptable carrier.

[0014] In other embodiments, the present disclosure provides a method for treating or preventing CD7 + 1. A method of preparing a therapy for treating or preventing cancer, comprising: a) One or more CD3 + iTCR(Vα24-Jα18) + isolating iNKT cells from a biological sample; and b) one or more of the CD3 + iTCR(Vα24-Jα18) + Culturing the iNKT cells in a growth medium to express the CD7 CAR and generate an expanded cell population. The present invention relates to a method comprising the steps of:

[0015] In some embodiments, the method further comprises administering to said patient one or more CD3 CARs to express said CD7 CARs. + iTCR(Vα24-Jα18) + In some embodiments, the modification comprises introducing one or more polynucleotides encoding a CD7 CAR into the one or more cells.

[0016] In some embodiments, the CD7 CAR modified iNKT cells are further modified to include a CAR, a T cell receptor (TCR), a TCR mimic antibody (TCRm), an exogenous cytokine, a growth factor, an antibody or antigen-binding fragment, or any combination thereof, wherein the antibody or antigen-binding fragment optionally includes a bispecific T cell engager (BiTE).

[0017] In any of the embodiments, the cancers for which the isolated and engineered iNKT cells to express a CD7 CAR can be used to treat or prevent include those that express a CD7 CAR. + Cancer or CD7 + malignant tumors; for example, T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes including early thymic precursor (ETP)-ALL (ETP-ALL), Pro-T-ALL, Pre-T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) subsets and PTCL subtypes including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ALCL), Primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); CD7 + Other malignant tumors or CD7 + CD1d + These include, but are not limited to, malignant tumors (e.g., T-ALL).

[0018] Other aspects or advantages of the present disclosure can be better understood through the following description of the drawings, detailed description of the disclosure, examples, and claims. [Brief description of the drawings]

[0019] [Figure 1]1 is a flow cytometry plot showing CD1d and NGFR expression in luciferase-expressing tumor cell lines used in this disclosure. Tumor cells were transduced with humanized firefly luciferase and cytoplasmic domain deleted nerve growth factor receptor (NGFR expressing bidirectional lentiviral vector) (hfflucN). K562, chronic myeloid leukemia (CML); K562CD19, CD19 stably transfected K562; Daudi and Raji, B cell lymphoma; Nalm6, B cell precursor leukemia; HL60, KG1, Mo7e, Molm13, MV4:11, THP1, and U937, acute myeloid leukemia (AML). Data partially used in PCT / US20 / 47253. [Figure 2A] Graph showing luciferase-based cytotoxicity of iNKT1(auto) and iNKT1(allo) cells at different effector / target (E / T) ratios against luciferase-expressing CD1d- (Daudi, Nalm6) and CD1d+ (Molm13, THP1, U937) target cells preincubated overnight with DMSO or αGalCer. iNKT1(auto) and iNKT1(allo) were generated upon secondary stimulation or expansion by co-culture of iNKT1 cells with irradiated autologous peripheral blood mononuclear cell (PBMC) negative fraction, αGalCer, IL-2, and IL-15 in 24-well plates, or with irradiated allogeneic PBMC, and Epstein-Barr virus transformed B (EBV-B) cells, αGalCer, IL-2, IL-7, and IL-15 in T25 flasks. For primary stimulation, isolated iNKT1 cells from blood donor 1 were co-cultured with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15 in 48-well plates. [Figure 2B]Graph showing cytotoxicity of iNKT2(autologous) and iNKT2(allogeneic) cells at different E / T ratios against luciferase-expressing CD1d- (Daudi, Nalm6) and CD1d+ (KG1, Molm13, MV4:11, THP1) target cells preincubated with DMSO or αGalCer. iNKT2(autologous) and iNKT2(allogeneic) were generated upon secondary stimulation or expansion of iNKT2 cells with irradiated autologous PBMC negative fraction, αGalCer, IL-2, and IL-15 in 24-well plates, or with irradiated allogeneic PBMC, and EBV-B cells, αGalCer, IL-2, IL-7, and IL-15 in T25 flasks. For primary stimulation, isolated iNKT2 cells from blood donor 2 were co-cultured with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15 in 48-well plates. [Figure 3A] Graph showing specific lysis of CD1d- (K562) and CD1d+ (U937) target cells preincubated with DMSO or αGalCer by iNKT1 cells grown in T25 flasks with irradiated allogeneic PBMC and EBV-B cells, IL-2, IL-7 and IL-15, along with αGalCer or anti-CD3 antibody (OKT3). [Figure 3B] Graph showing specific lysis of CD1d- (K562, Daudi) and CD1d+ (HL60, KG1, Molm13, MV4:11, U937) target cells preincubated with DMSO or αGalCer by iNKT2 cells grown in T25 flasks with irradiated allogeneic PBMC and EBV-B cells, IL2, IL7 and IL-15, along with αGalCer or anti-CD3 antibody (OKT3). [Figure 3C]Graph showing specific lysis of CD1d- (Daudi, Raji) and CD1d+ (MV4:11, U937) target cells preincubated with DMSO or αGalCer by iNKT12 cells (αGalCer) and iNKT12 cells (OKT3) grown in T25 flasks with αGalCer or anti-CD3 antibody (OKT3) with irradiated allogeneic PBMCs, and EBV-B cells, IL-2, IL-7, and IL-15. For primary stimulation, isolated iNKT12 cells from blood donor 12 were co-cultured in 48-well plates with irradiated autologous PBMC negative fraction, αGalCer, IL-2, and IL-15. iNKT2 cells and iNKT1 grown in T25 flasks with αGalCer, irradiated allogeneic PBMCs, and EBV-B cells, IL-2, IL-7, and IL-15 were used as controls. An E / T ratio of 15:1 in iNKT1 was used. [Figure 4A] Graph showing specific lysis of CD1d+ (THP1, U937) target cells preincubated with DMSO or αGalCer at different E / T ratios by iNKT1, iNKT2, iNKT11 and iNKT12 grown in T25 flasks with αGalCer, irradiated allogeneic PBMC, and EBV-B cells, IL2, IL-7 and IL-15. [Figure 4B] Flow cytometry analysis plots showing the percentage of CD3+iTCR+ cells in expanded iNKT1 and iNKT2 cells (αGalCer or OKT3). iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to set the gates. [Figure 5A]Graph showing cytotoxicity of iNKT45(autologous), iNKT45(allogeneic), iNKT46(autologous), and iNKT46(allogeneic) cells against luciferase-expressing CD1d- (Nalm6) and CD1d+ (MV4:11, U937) target cells preincubated with DMSO or αGalCer. iNKT45(autologous) or iNKT46(autologous) and iNKT45(allogeneic) or iNKT46(allogeneic) refer to the expansion of iNKT cells in 24-well plates using irradiated autologous PBMC negative fraction or irradiated allogeneic PBMC and EBV-B cells, respectively, all supplemented with αGalCer, IL-2, and IL-15. iNKT cells were then transferred to T75 flasks for further expansion. Isolated iNKT45 and iNKT46 cells from blood donors 45 and 46 were first stimulated with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15 in 48-well plates. [Figure 5B] Flow cytometry analysis plots showing the percentage of CD3+iTCR+ cells in iNKT45 and iNKT46 cells after primary stimulation. iTCR, Vα24-Jα18. Isotype antibody was used as background control to set the gates. [Figure 5C] Flow cytometry analysis plots showing the percentage of CD3+iTCR+ cells in iNKT45 (autologous), iNKT45 (allogeneic), iNKT46 (autologous), and iNKT46 (allogeneic) cells after secondary stimulation (i.e., expansion). iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to set the gates. [Figure 6]Graph showing cytotoxicity of CD19 CAR-modified iNKT47 and iNKT48 cells against luciferase-expressing CD19- (K562) and CD19+ (Daudi, Nalm6, Raji), and DMSO- or αGalCer-treated CD1d- (Mo7e) and CD1d+ (Molm13, U937) target cells. Isolated iNKT47 and iNKT48 were modified with CD19 CAR or CD19 CAR / GFP, both supplemented with αGalCer, IL2, and IL-15, and expanded with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells. [Figure 7] Graph showing IFN-γ production in enzyme-linked immunosorbent assay (ELISA) following recognition of CD19+ B-cell malignancies (Daudi, Nalm6, Raji), and DMSO or αGalCer-treated CD1d- (Mo7e) and CD1d+ (THP1) target cells by CD19 CAR-modified iNKT47 and iNKT48 grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15. [Figure 8] Flow cytometry analysis plots showing the percentage of CD3+iTCR+ cells in CD19 CAR modified iNKT47 and iNKT48 cells grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15. iTCR, Vα24-Jα18. Isotype antibody was used as background control to set the gates. [Figure 9] Flow cytometry analysis plots showing the percentage of CAR+ or CAR+GFP+ cells in CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47 and iNKT48 cells grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2, and IL-15. [Figure 10]Graph showing cytotoxicity of CD19 CAR-modified or CD19 CAR / GFP-modified iNKT50 cells against luciferase-expressing CD19- (K562, HL60, KG1, Molm13, THP1) and CD19+ (K562CD19, Daudi, Nalm6, Raji), and DMSO- or αGalCer-treated CD1d- (Mo7e) and CD1d+ (MV4:11, U937) target cells. Isolated iNKT50 cells were modified with lentiviral CD19 CAR or CD19 CAR / GFP and expanded with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2, and IL-15. [Figure 11A] Flow cytometry analysis plots showing the percentage of CD3+iTCR+ cells in CD19 CAR modified iNKT50 or mock cells grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15. iTCR, Vα24-Jα18. Isotype antibody was used as background control to set the gates. [Figure 11B] Flow cytometry analysis plots showing the percentage of CAR+ or CAR+GFP+ cells in CD19 CAR-modified or CD19 CAR / GFP-modified iNKT50 or mock cells grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2, and IL-15. [Figure 12] Graph showing the proliferation rate of CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47, iNKT48, and iNKT50 cells, or unmodified iNKT45 and iNKT46 cells grown with irradiated autologous PBMC negative fraction and total PBMC, or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2, and IL-15. [Figure 13A]Graph showing overnight (16 h) cytotoxicity of thawed CD19 CAR-modified iNKT47 and iNKT48 grown with irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2, and IL15 against CD19- (K562) and CD19+ (K562CD19, Daudi, Nalm6, Raji) target cells. CD19 CAR-modified iNKT47 (allogeneic) and iNKT48 (allogeneic) were thawed and cultured in 24-well plates in human T cell medium supplemented with IL-2 and IL-15 for 5 days. [Figure 13B] Figure 1 shows the overnight (16 h) cytotoxicity of thawed CD19 CAR-modified iNKT47 cells grown with irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2 and IL-15 against luciferase-expressing Molm13 and THP1 cells in the presence of 7DW8-5 or αGalCer at different concentrations from 200 ng / mL to 3.12 ng / mL. CD19 CAR-modified iNKT47 (allogeneic) cells were thawed and cultured in 24-well plates in human T cell medium supplemented with IL-2 and IL-15 for 14 days. E / T ratio, 10 / 1. [Figure 13C] Graph showing IFN-γ production of iNKT47 CD19 CAR (allogeneic), iNKT48 CD19 CAR / GFP (allogeneic) cells, and iNKT47mock (autologous) in response to CD1d+MV4:11-hfflucN target cells pulsed with different amounts of 7DW8-5 and αGalCer at 92 days of culture. CD19+ (K562CD19, Nalm6) and CD19- (K562) were used as positive and negative controls. [Figure 14A] Flow cytometry analysis plots showing the percentage of CD2 vs. CD7 and CD5 vs. CD7 positive cell populations in irradiated allogeneic PBMC and iNKT cells expanded with EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15 from a representative healthy blood donor (iNKT69). [Figure 14B]Graph showing statistical analysis of flow cytometry data of CD2, CD5 and CD7 positive cell populations in irradiated allogeneic PBMC and expanded iNKT cell products expanded with EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15 from 7-9 individual blood donors (iNKT45, 46, 47, 48, 50, 55, 56, 68, 69, except for iNKT47, 48 for CD2). Results were calculated as mean ± SD. ****p<0.0001. [Figure 15] 13 is a flow cytometry analysis plot showing CD7, CD1d and NGFR expression in bidirectional (humanized firefly luciferase and NGFR) lentivirally transduced human HSB2, Jurkat, and MOLT13 T-ALL cell lines, and KG1, Kasumi-1, and Kasumi-6 AML cell lines. [Figure 16] Graph showing T cell proliferation rate after lentiviral transduction of peripheral blood mononuclear cells (PBMCs) from donor 36 (PBL36) with CD19, ROR1 and CD7 CARs. A non-transduced mock was used as a control. [Figure 17] Graph showing iNKT cell proliferation rate of iNKT55 cells isolated from donor 55 after lentiviral transduction with CD7 CAR, CD19 CAR, or non-transduced mock control, and iNKT68 cells isolated from donor 68 after lentiviral transduction with CD7 CAR, or non-transduced mock control. iNKT55 and iNKT68 mock, or iNKT55 and iNKT68 transduced with CD19 CAR or CD7 CAR, were expanded with irradiated allogeneic PBMC and EBV-B cells, 7DW8-5, IL-2, and IL-15. [Figure 18]Representative cytotoxicity of CD7 CAR iNKT55 cells (mock iNKT55, CD19 CAR iNKT55, and CD7 CAR iNKT55 cells) derived from donor 55 against luciferase-expressing target cells including K562 (CML), CD19+ B-cell malignancies (Nalm6, Raji), CD7+ T-ALL (HSB2, Jurkat, MOLT13), CD7+ AML (KG1), CD7- AML (Molm13, MV4;11, U937), CD7- soft tissue sarcoma (Rh30, TC71, SaOS2), and CD7- neuroblastoma (BE(2)C, SKNFI) cell lines. Effector:target ratio. [Figure 19] Graph showing statistical analysis of five independent cytotoxicity assays of CD7 CAR iNKT cells (n=5) from donors 55, 56, 64, 68 and 69 against luciferase-expressing CD7+ T-ALL cell lines (HSB2, Jurkat, MOLT13), CD7+ AML (KG1), and CD19+ B-cell malignancies (Nalm6, Raji). Corresponding mock (n=5) and CD19 CAR (n=3) iNKT cells were used as baseline and CAR-T cell controls. Results were calculated as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. Significance of CD19 CAR iNKT vs. mock and CD7 CAR iNKT vs. mock is shown. [Figure 20]FIG. 20A is a graph showing the production of IFN-γ by enzyme-linked immunosorbent assay (ELISA) following recognition by representative CD7 CAR iNKT55 and mock iNKT55 or CD19 CAR iNKT55 controls of a panel of CD7+ T-ALL (HSB2, Jurkat, MOLT13), CD7+ AML (KG1, Kasumi-6), CD19+ B-cell malignancies (Daudi, Nalm6, Raji) and CD19+ EBV-transformed B cells (EBV-B) used as CD19 CAR iNKT55 controls, as well as CD7- CML (K562), AML (Kasumi-1, U937), multiple myeloma (RPMI8226), sarcoma (Rh30, TC71), and neuroblastoma (BE2(C), SKNFI) cell lines. FIG. 20B is a graph showing statistical analysis of five independent IFN-γ production assays for CD7 CAR iNKT cells (n=5) generated from donors 55, 56, 64, 68 and 69 in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13), and CD7+ AML (KG1, Kasumi-6), CD19+ B-cell malignancies (Daudi, Nalm6, Raji), and CD7- CML (K562), and AML (Kasumi-1, U937). Corresponding mock (n=5) and CD19 CAR (n=3) iNKT cells were used as baseline and CAR-T cell controls. Results were calculated as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. Statistical significance was shown between mock and CD7 CAR iNKT. [Figure 21] Flow cytometry analysis plots showing the levels of CD7 CAR expression following lentiviral transduction of iNKT cells derived from five blood donors (n=5). CD19 CAR iNKT (n=3) and mock iNKT (n=5) cells were used as specificity or background controls. [Figure 22A]Flow cytometry dot plots of CD2 vs. CD7 and CD5 vs. CD7 double staining showing exemplary loss of CD7+ cells in the final CD7 CAR iNKT55 product. Mock and CD19 CAR iNKT55 were used as controls. [Figure 22B] Graph showing statistical analysis of the percentage of remaining CD7+ cell population in the final CD7 CAR iNKT cell product (n=4) produced from donors 55, 56, 68 and 69 compared to mock (n=4) and unrelated CD19 CAR (iNKT55, iNKT56) or ROR1 CAR (iNKT69) iNKT cells (n=3). Results were calculated as mean ± SD. **p<0.01, ns, not significant. [Figure 23A] Flow cytometry analysis plots showing the percentage of CD3+iTCR(Vα24-Jα18)+ cell populations in CD7 CAR iNKT, mock iNKT, and CD19 CAR iNKT cells from one representative donor 55. Isotype control antibodies were used to set the background gate. [Figure 23B] Flow cytometry analysis plots showing the percentages of CD4+, CD8+, CD4-CD8-, and CD4+CD8+ cell populations in CD7 CAR iNKT, mock iNKT, and CD19 CAR iNKT cells from one representative donor 55. Isotype control antibodies were used to set the background gate. [Figure 23C] Flow cytometry histogram analysis showing PD1 expression levels in CD7 CAR iNKT, mock iNKT, and CD19 CAR iNKT cells from one representative donor55. [Figure 24A]Graph showing the experimental timeline of HSB2-hfflucN tumor cell injection, iNKT cell injection, and bioluminescence imaging (BLI) monitoring. 6-7 week old female NSG mice were injected iv with HSB2-hfflucN T-ALL cells on day -2. Tumor growth was determined by BLI on day 0. Mice were randomized into 3 groups (n=5 each) and treated with 3 iv injections of PBS, thawed mock iNKT55, or thawed CD7 CAR iNKT55 cells on days 0, 3, and 6. Mice were injected ip with PBS or IL-2 and IL-15 every 3 days for 2 weeks. Tumor growth was monitored by BLI on days 3, 6, 10, 17, and 24. [Figure 24B] Graph showing bioluminescence imaging (BLI) of tumor growth in mice treated with PBS, mock iNKT55 cells, or CD7 CAR iNKT55 cells (3 groups, n=5 each). Scale bar is shown as radiance (p / sec / cm2 / sr). All PBS or mock control mice died at day 23. One mouse (number 1) in the CD7 CAR iNKT treatment group died immediately after imaging at day 24, possibly due to the anesthesia method for imaging or injection of luciferin. [Figure 24C] FIG. 1 is a graph showing the overall kinetics of systemic tumor progression in mice. Each line represents an individual animal donation. [Figure 24D] Graph showing statistical analysis of tumor progression as determined by BLI imaging in each group of mice. P-values ​​<0.05 are considered significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. [Figure 24E] Graph showing Kaplan-Meier survival curves for mice treated with PBS, mock iNKT, or CD7 CAR iNKT cells. P=0.0009 by log-rank Mantel-Cox test. [Figure 25A]Graph showing the experimental timeline of KG1-hfflucN tumor cell injection, iNKT cell injection, and bioluminescence imaging (BLI) monitoring. 6-7 week old female NSG mice were injected iv with KG1-hfflucN T-ALL cells on day -2. Tumor growth was determined by BLI on day 0. Mice were randomized into 2 groups (n=5 each) and treated with 2 iv injections of thawed mock iNKT55, or thawed CD7 CAR iNKT55 cells on days 0 and 3. Mice were injected ip with PBS or IL-2 and IL-15 every 3 days for 2 weeks. Tumor growth was monitored by BLI on days 3, 6, 10, 17, 24, 31, 38, and 45. [Figure 25B] Graph showing bioluminescence imaging (BLI) of tumor growth in mice treated with mock iNKT55 cells or CD7 CAR iNKT55 cells (2 groups, n=5 each). Scale bar is shown as radiance (p / sec / cm2 / sr). The third mouse in the CD7 CAR iNKT treatment group showed a larger tumor burden than the other mice in the same group. This may be due to the ip injection of remaining T cells in the syringe during the second T cell iv infusion. One mouse (number 1) in the CD7 CAR iNKT treatment group died immediately after imaging at day 31, possibly due to the anesthesia method for imaging or injection of luciferin. [Figure 25C] FIG. 1 is a graph showing the overall kinetics of systemic tumor progression in mice. Each line represents an individual animal. [Figure 25D] Graph showing statistical analysis of tumor progression as determined by BLI imaging in each group of mice. P-values ​​<0.05 are considered significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. [Figure 25E]Graph showing Kaplan-Meier survival curves for mice treated with mock iNKT or CD7 CAR iNKT cells. P=0.0069 by log-rank Mantel-Cox test. One mouse (number 1) in the CD7 CAR iNKT treatment group died after imaging at day 31 and was not included in the survival curve. All control mice died on days 73-74. Two mice in the CD7 CAR iNKT treatment group survived for 368 days and were humanely euthanized. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure relates to type I NKT cells or invariant NKT (iNKT) cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + ) accounts for approximately 42.96% ± 17.21% of CD7 in iNKT cells. - Based on the recognition that CD7 CAR-modified CD7 expresses a subset of CD7-positive cells and can expand approximately 6,000-fold in culture after modification with lentivirus to express CD7 chimeric antigen receptor (CAR). - iNKT cells can avoid CD7 antigen-driven fratricide or suicide and can be expanded for therapeutic use. Importantly, CD7 CAR-modified iNKT cells can express anti-CD7 CARs in vitro and in animals. +It shows cancer activity. Furthermore, about 6,000-fold expansion of iNKT cells or iNKT cells modified with CD19 CAR can also be achieved using irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15. The present disclosure provides several advantages. First, CD7 CAR-modified iNKT cells are generated without genome editing, whereas most CD7 CAR-modified conventional T cells require CRISPR-mediated genome editing. Thus, CD7 CAR-modified iNKT cells may be safer than CD7 CAR-modified conventional T cells by avoiding genome editing-related off-target effects. Second, CD7 CAR-modified iNKT cells may survive in vivo for weeks to months, whereas most CD7 CAR-modified conventional T cells may persist for months to years, possibly resulting in more severe T cell deficiency and infection. Third, CD7 CAR-modified iNKT cells express CD7 CAR and invariant TCR, + CD1d + They may provide dual targeting for T-ALL and are likely more effective than CD7 CAR-modified conventional T cells in reducing relapse rates in these patients. Fourth, CD7 CAR-modified iNKT cells can be produced off-the-shelf from healthy donors without the need for genome editing, without inducing graft-versus-host disease (GVHD), whereas CD7 CAR-modified conventional T cells require CRISPR-mediated deletion of both CD7 and alloreactive TCR.

[0021] Thus, in certain aspects, the present disclosure provides a method for detecting CD7 + Methods and compositions (e.g., pharmaceutical compositions) using CD7 CAR-modified iNKT cells for treating or preventing cancer are provided. In some embodiments, the CD7 CAR-modified iNKT cells are + iTCR(Vα24-Jα18) + In some embodiments, the CD7 CAR-modified iNKT cells comprise or consist of CD3 + Vα24 +In some embodiments, the CD7 CAR-modified iNKT cells comprise or consist of CD3 + CD4 + In some embodiments, the CD7 CAR-modified iNKT cells comprise or consist of CD3 + CD4 - CD8 - In some embodiments, the CD7 CAR-modified iNKT cells comprise or consist of CD3 + CD8 + In some embodiments, the CD7 CAR-modified iNKT cells comprise or consist of CD3 + CD4 + cells, CD3 + CD4 - CD8 - , CD3 + CD8 + Cells and CD3 + CD4 + CD8 + In some embodiments, the CD7 CAR-modified iNKT cells are reactive to α-galactosylceramide (αGalCer) or 7DW8-5, or other glycolipid analogs presented by MHC class I-like CD1d molecules.

[0022] In some embodiments, the exemplary CD7 CAR-modified iNKT cells described herein are + In some embodiments, CD7 + The cancer is T-ALL or AML. + The cancer is refractory or relapsed T-ALL or AML. + The cancer is, for example, relapsed T-ALL or AML after hematopoietic stem cell transplant. +The cancer is one that expresses both CD7 and an additional antigen that is targeted by the iNKT cells and / or by a construct expressed by the iNKT cells (e.g., a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell receptor mimic antibody (TCRm), or a combination thereof). In some embodiments, CD7 + The cancer is one that is resistant or refractory to treatment in the absence of the cells. Exemplary such cancers are described and exemplified herein.

[0023] In some embodiments, the cancer is CD7 + Hematological malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes including early thymic precursor (ETP)-ALL (ETP-ALL), pro-T-ALL, pre-T-ALL, cortical T-ALL and mature T-ALL; subsets of peripheral T-cell lymphomas (PTCL) and PTCL subtypes including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ALCL), primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); CD7 + Other malignant tumors or CD7 + CD1d + Malignant tumors, possibly CD7 + The cancer is resistant or refractory to treatment in the absence of the cells.

[0024] In some embodiments, the present disclosure provides a method for treating or preventing CD7 + A method for treating or preventing cancer, comprising administering to a subject a CD7 CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18) + or CD3 +Vα24 + A therapeutically effective amount of a CD7 CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The present invention provides a method for treating a patient with a pulmonary artery disease comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of iNKT cells. In some embodiments, the pharmaceutical composition further comprises at least one pharma- ceutical acceptable carrier.

[0025] In some embodiments, the present disclosure provides a method for detecting CD7 + 1. A method of preparing a therapy for treating or preventing cancer, comprising: (a) administering to a patient a gene encoding one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + (b) isolating one or more iTCR(Vα24-Jα18) from a biological sample; + The methods include the steps of: (c) activating or activating iNKT cells using irradiated autologous PBMC negative fraction and / or total PBMC, α-galactosylceramide (αGalCer) or 7DW8-5, or other glycolipid analogs, IL-2 and IL-15, or IL-2, IL-7 and IL-15 in growth medium for expansion; (d) modifying the activated and expanding iNKT cells using a lentiviral vector to express a CAR (e.g., CD7); and (d) expanding one or more CAR (e.g., CD7) modified iNKT cells in growth medium using irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15, or IL-2, IL-7 and IL-15.

[0026] In some embodiments, the method further comprises modifying one or more cells to express the CAR, TCR or TCRm. In some embodiments, the modification comprises introducing one or more polynucleotides encoding the CAR, TCR or TCRm into the one or more cells. In some embodiments, the introduction of the one or more polynucleotides comprises electroporation, transduction, and / or transfection. In some embodiments, the one or more polynucleotides comprise mRNA and / or DNA. In some embodiments, the DNA comprises transposon DNA. In some embodiments, the one or more polynucleotides comprise one or more vectors. In some embodiments, the one or more vectors comprise one or more viral vectors. In some embodiments, the one or more vectors comprise one or more lentiviral vectors or gamma-retroviral vectors.

[0027] In some embodiments, the iNKT cells are isolated from a biological sample. In some embodiments, the biological sample is from a subject (e.g., a cancer patient). In some embodiments, the biological sample is from a donor (e.g., a healthy donor). In some embodiments, the biological sample comprises blood, bone marrow, lymph node tissue, spleen tissue, tumor tissue, one or more induced pluripotent stem cells, and / or one or more peripheral blood mononuclear cells. In some embodiments, the blood comprises peripheral blood and / or umbilical cord blood. In some embodiments, the iNKT cells are isolated from one or more peripheral blood mononuclear cells.

[0028] In some embodiments of the methods described herein, the iNKT cells are modified to express a chimeric antigen receptor (CAR) (e.g., a CD7 CAR). In some embodiments, the cells comprise one or more polynucleotides encoding a CAR. In some embodiments, the CAR comprises at least one antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0029] In some embodiments, the antigen binding domain of the CAR can bind to CD7, CD1a, CD1d, CD2, CD5, TRBC1, CD21, CCR9, CD30, CD123, CD33, CD38, CD138, CLL-1, LILRB4, Siglec-6, CD70, or PD-L1. In some embodiments, the antigen binding domain can bind to CD19 or ROR1.

[0030] In some embodiments, the antigen binding domain and / or CAR can bind to CD7. In some embodiments, the antigen binding domain and / or CAR can bind to CD1a. In some embodiments, the antigen binding domain and / or CAR can bind to CD1d. In some embodiments, the antigen binding domain and / or CAR can bind to CD2. In some embodiments, the antigen binding domain and / or CAR can bind to CD5. In some embodiments, the antigen binding domain and / or CAR can bind to TRBC1. In some embodiments, the antigen binding domain and / or CAR can bind to CD21. In some embodiments, the antigen binding domain and / or CAR can bind to CCR9. In some embodiments, the antigen binding domain and / or CAR can bind to CD30. In some embodiments, the antigen binding domain and / or CAR can bind to CD123. In some embodiments, the antigen binding domain and / or CAR can bind to CD33. In some embodiments, the antigen binding domain and / or CAR can bind to CD38. In some embodiments, the antigen binding domain and / or CAR can bind to CD138. In some embodiments, the antigen binding domain and / or CAR can bind to CLL-1. In some embodiments, the antigen binding domain and / or CAR can bind to LILRB4. In some embodiments, the antigen binding domain and / or CAR can bind to Siglec-6. In some embodiments, the antigen binding domain and / or CAR can bind to CD70. In some embodiments, the antigen binding domain and / or CAR can bind to PD-L1.

[0031] In some embodiments, the antigen binding domain and / or CAR can bind to CD7, CD1a, CD1d, CD2, CD5, TRBC1, TRBC2, CD21, CCR9, CD30, or CD70, and the cancer is T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma. In some embodiments, the antigen binding domain and / or CAR can bind to CD123, CD33, CD38, CD138, CLL-1, LILRB4, Siglec-6, or CD70, and the cancer is acute myeloid leukemia (AML). In some embodiments, the antigen binding domain and / or CAR can bind to CD70 and the cancer is T-cell leukemia and lymphoma, multiple myeloma, AML, diffuse large B-cell lymphoma-follicular lymphoma, Hodgkin's lymphoma, or various solid tumors (e.g., renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, glioma, lung cancer, breast cancer, cervical cancer, ovarian cancer, and mesothelioma). In some embodiments, the antigen binding domain and / or CAR can bind to PD-L1 and the cancer is T-cell leukemia and lymphoma, multiple myeloma, AML, or various solid tumors (e.g., glioma, lung cancer, breast cancer).

[0032] In some embodiments, the antigen binding domain and / or CAR can bind to CD19 and the cancer is a B cell malignancy (e.g., B cell precursor acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), or chronic lymphocytic leukemia (CLL)). In some embodiments, the antigen binding domain and / or CAR can bind to CD19 and the cancer is B-ALL, NHL, or CLL. In some embodiments, the antigen binding domain and / or CAR can bind to ROR1 and the cancer is Ewing's sarcoma, osteosarcoma, fibrosarcoma, rhabdomyosarcoma, chronic lymphocytic leukemia, mantle cell carcinoma, breast cancer, lung adenocarcinoma, melanoma, neuroblastoma, or ovarian cancer.

[0033] In some embodiments, the antigen-binding domain of the CAR comprises an antibody, or an antigen-binding fragment thereof, or a non-antibody protein scaffold. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, or a single domain antibody (known as a nanobody). In some embodiments, the antigen-binding fragment comprises a single chain variable fragment (scFv).

[0034] In some embodiments, the intracellular signaling domain of the CAR comprises a functional signaling domain of at least one stimulatory molecule. In some embodiments, the at least one stimulatory molecule comprises a zeta chain associated with the T cell receptor complex. In some embodiments, the at least one stimulatory molecule comprises a CD3 zeta chain. In some embodiments, the intracellular signaling domain further comprises a functional signaling domain of at least one costimulatory molecule. In some embodiments, the at least one costimulatory molecule comprises 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, or DAP12.

[0035] In some embodiments of the methods described herein, the CD7 CAR-modified iNKT cells are further modified to express a T cell receptor (TCR). In some embodiments, the cells comprise one or more polynucleotides encoding a TCR. In some embodiments, the TCR comprises at least one alpha chain and a beta chain. In some embodiments, the alpha chain and / or the beta chain can bind to an antigen. In some embodiments, the antigen is an intracellular antigen. In some embodiments, the antigen is Wilms' tumor 1 (WT1), a melanoma preferentially expressed antigen (PRAME), a minor histocompatibility antigen (MiHA, e.g., HA-1), or a mutant nucleophosmin 1 (ΔNPM1).

[0036] In some embodiments, the alpha and / or beta chain of the TCR can bind to WT1, PRAME, HA-1, or ΔNPM1. In some embodiments, the alpha chain, beta chain, and / or TCR can bind to WT1. In some embodiments, the alpha chain, beta chain, and / or TCR can bind to PRAME. In some embodiments, the alpha chain, beta chain, and / or TCR can bind to HA-1. In some embodiments, the alpha chain, beta chain, and / or TCR can bind to ΔNPM1. In some embodiments, the antigen is WT1, PRAME, HA-1, or ΔNPM1 and the cancer is AML.

[0037] In some embodiments of the methods described herein, the CD7 CAR-modified iNKT cells are further modified to express a T cell receptor mimic antibody (TCRm). In some embodiments, the cells comprise one or more polynucleotides encoding the TCRm. In some embodiments, the TCRm comprises at least one antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0038] In some embodiments, the antigen binding domain of the TCRm is capable of binding to a composite antigen, hi some embodiments, the composite antigen comprises a peptide and a human leukocyte antigen (HLA) molecule.

[0039] In some embodiments, the HLA molecule is an HLA class I molecule. In some embodiments, the HLA molecule is an HLA class II molecule.

[0040] In some embodiments, the peptide comprises a WT1 peptide. In some embodiments, the WT1 peptide comprises an amino acid sequence of RMFPNAPYL. In some embodiments, the composite antigen comprises a WT1 peptide and an HLA-A2 molecule. In some embodiments, the cancer is AML.

[0041] In some embodiments, the peptide comprises a Preferentially Expressed Melanoma Antigen (PRAME) peptide. In some embodiments, the PRAME peptide comprises an amino acid sequence of VLDGLDVLL. In some embodiments, the PRAME peptide comprises an amino acid sequence of ALYVDSLFFL. In some embodiments, the PRAME peptide comprises an amino acid sequence of SLYSFPEPEA. In some embodiments, the PRAME peptide comprises an amino acid sequence of SLLQHLIGL. In some embodiments, the PRAME peptide comprises an amino acid sequence of LYVDSLFFLC. In some embodiments, the composite antigen comprises a PRAME peptide and an HLA-A * In some embodiments, the composite antigen comprises a PRAME peptide and an HLA-A * 2402 molecule. In some embodiments, the cancer is AML, multiple myeloma, T-cell lymphoma, B-ALL, neuroblastoma, sarcoma, melanoma, non-small cell lung cancer, colon adenocarcinoma, or breast adenocarcinoma.

[0042] In some embodiments, the peptide comprises an HA-1 peptide. In some embodiments, the HA-1 peptide comprises the amino acid sequence of VLHDDLLEA. In some embodiments, the composite antigen comprises an HA-1 peptide and an HLA-A2 molecule. In some embodiments, the cancer is AML and multiple myeloma.

[0043] In some embodiments, the peptide comprises a ΔNPM1 peptide. In some embodiments, the ΔNPM1 peptide comprises the amino acid sequence of CLAVEEVSL. In some embodiments, the composite antigen comprises a ΔNPM1 peptide and an HLA-A2 molecule. In some embodiments, the cancer is AML.

[0044] In some embodiments, the antigen-binding domain of the TCRm comprises an antibody, or an antigen-binding fragment thereof, or a non-antibody protein scaffold. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, or a single domain antibody (nanobody). In some embodiments, the antigen-binding fragment comprises a single chain variable fragment (scFv).

[0045] In some embodiments, the intracellular signaling domain of the TCRm comprises a functional signaling domain of at least one stimulatory molecule. In some embodiments, the at least one stimulatory molecule comprises a zeta chain associated with the T cell receptor complex. In some embodiments, the at least one stimulatory molecule comprises a CD3 zeta chain. In some embodiments, the intracellular signaling domain further comprises a functional signaling domain of at least one costimulatory molecule. In some embodiments, the at least one costimulatory molecule comprises 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, or DAP12.

[0046] In some embodiments of the methods described herein, the CD7 CAR modified iNKT cells are further modified to include an exogenous cytokine, growth factor, antibody or antigen-binding fragment, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment comprises a bispecific T cell engager (BiTE).

[0047] Further provided herein in some embodiments are CD7 CAR-modified iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the present disclosure provides a pharmaceutical composition comprising CD7 iNKT cells in a subject in need of treatment or prevention. + and administering to a patient a CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the pharmaceutical composition includes a CD7 CAR-modified iNKT cell and at least one pharma- ceutical acceptable carrier. In some embodiments, the CD7 CAR-modified iNKT cell in the pharmaceutical composition is a CD3 + iTCR(Vα24-Jα18) + CD7 CAR + iNKT cells (e.g., CD3 + CD4 +cells, CD3 + CD4 - CD8 - , CD3 + CD8 + cells, CD3 + CD4 + CD8 + In some embodiments, the CD7 CAR-modified iNKT cells in the pharmaceutical composition are CD3 + CD4 + In some embodiments, the CD7 CAR-modified iNKT cells in the pharmaceutical composition are CD3 + CD4 - CD8 - In some embodiments, the CD7 CAR-modified iNKT cells in the pharmaceutical composition are CD3 + CD8 + In some embodiments, the CD7 CAR-modified iNKT cells in the pharmaceutical composition are CD3 + CD4 + CD8 + In some embodiments, the CD7 CAR-modified iNKT cells in the pharmaceutical composition are CD3 + CD4 + cells, CD3 + CD4 - CD8 - cells, CD3 + CD8 + Cells, and CD3 + CD4 + CD8 + It is a cell.

[0048] Also provided herein in some embodiments are CD7 CAR-modified iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The present invention relates to a therapeutic use of a human iNKT cell, or a pharmaceutical composition comprising the same.

[0049] Specific Illustrative Embodiments In order to make this disclosure more readily understandable, certain terms are defined throughout the detailed description. Unless otherwise defined herein, all scientific and technical terms used in connection with this disclosure have the same meaning as commonly understood by one of ordinary skill in the art.

[0050] All references cited herein are also incorporated by reference in their entirety. To the extent that a cited reference conflicts with the disclosure of this specification, the specification shall control.

[0051] As used herein, the singular forms of words include the plural forms unless the context clearly dictates otherwise; for example, the terms "a," "an," and "the" are understood to be either singular or plural. By way of example, "an element" means one or more elements. The term "or" is intended to mean "and / or" unless the specific context dictates otherwise. All ranges include their endpoints and all intermediate points unless the context dictates otherwise.

[0052] The term "about" or "approximately" as used herein in the context of numerical values ​​and numerical ranges refers to a value or range that is close to or proximate to the recited value or range such that the embodiment can function as intended to be clear to one of ordinary skill in the art from the teachings contained herein. This is due, at least in part, to different characteristics of nucleic acid composition, age, race, sex, anatomical and physiological variations, and imprecision of biological systems. Thus, these terms include values ​​beyond those resulting from systematic error. In some embodiments, "about" or "approximately" means plus or minus (±) 10% of the amount expressed as a number, sometimes preferably ±5%, and sometimes more preferably ±2%.

[0053] NKT cells In certain aspects, the present disclosure provides methods for treating CD7 CAR-modified type I NKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 +The present invention provides methods and compositions for using the CD7 CAR-modified iNKT cells described herein.

[0054] As used herein, the term "natural killer T cells" or "NKT cells" refers to a T cell or population of T cells that exhibit characteristics of both conventional T cells and natural killer (NK) cells. For example, in some embodiments, NKT cells are mature lymphocytes that possess both T cell receptors and NK cell receptors. In some embodiments, NKT cells are CD4 T cells that have undergone T cell receptor (TCR) gene rearrangement in the thymus. + CD8 + They arise from cortical thymocytes.

[0055] Two classifications of NKT cells exist in the literature: the classical classification of NKT cells classifies NKT cells into a subpopulation of unconventional T cells that recognize lipid antigens presented by MHC class I-like CD1d molecules and divides NKT cells into type I and type II NKT cells (Godfrey et al., Nat Immunol. 2010;11(3):197-206; Dhodapkar and Kumar. J Immunol. 2017;198(3):1015-21; Godfrey et al., Immunity. 2018;48(3):453-73). The other classification of NKT cells includes type I, type II, and type III NKT (NKT-like) cells (Godfrey et al., Nat Rev Immunol. 2004;4(3):231-237; Farr et al., Proc Natl Acad Sci USA. 2014;111(35):12841-6).

[0056] As used herein, the term "type I NKT cells" or "invariant NKT cells" or "iNKT cells" refers to NKT cells or populations of NKT cells that express an invariant or semi-invariant TCR repertoire and bind to the glycosphingolipid α-galactosylceramide (α-GalCer) associated with MHC class I-like CD1d molecules. In some embodiments, type I NKT cells express an invariant TCR α chain and a limited number of non-invariant TCR β chains. In some embodiments, type I NKT cells express a semi-invariant Vα chain (e.g., Vα14-Jα18 TCR in mice, and Vα24-Jα18 in humans) paired with a limited repertoire of Vβ chains (e.g., Vβ8.2, Vβ7, and Vβ2 in mice, and Vβ11 in humans). In some embodiments, type I NKT cells recognize the glycosphingolipid α-galactosylceramide (α-GalCer) or its synthetic analogs (e.g., 7DW8-5) when presented by MHC class I-like CD1d molecules. In some embodiments, the terms "type I NKT cells" or "invariant NKT cells" or "iNKT cells" may be used interchangeably.

[0057] As used herein, the term "iTCR" or "invariant TCR" refers to the invariant TCR expressed on iNKT cells, including, but not limited to, the invariant TCR α chain (e.g., Vα24-Jα18, Vα24-JαQ, Vα24) paired with a limited repertoire of Vβ chains (e.g., Vβ11 in humans) and a limited number of non-invariant TCR β chains.

[0058] As used herein, the term "genome editing" or "CRISPR-mediated genome editing" or "gene editing" or "genome engineering" refers to a type of genetic engineering in which DNA is deleted, inserted, modified, or replaced within a genome. Unlike lentiviral transduction, which randomly inserts genetic material into the host genome for expression of a CAR, genome editing targets insertions or deletions at site-specific locations. Current nuclease-based genome editing tools include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR / Cas9.

[0059] In some embodiments, the NKT cells (e.g., iNKT cells) are single cells. In some embodiments, the NKT cells (e.g., iNKT cells) are a homogenous cell population. In some embodiments, the NKT cells (e.g., iNKT cells) are a heterogeneous cell population. In some embodiments, the NKT cells (e.g., iNKT cells) cause, stimulate, and / or help the production of at least one cytokine (e.g., IL-4 and / or IFN-γ). In some embodiments, the NKT cells (e.g., iNKT cells) are cytotoxic. In some embodiments, the NKT cells (e.g., iNKT cells) can exert cytotoxicity against a variety of cells, including cancer cells or cell lines (e.g., T-ALL cells or cell lines), as described and exemplified herein.

[0060] In some embodiments, the iNKT cells of the present disclosure may express any number or combination of cell surface markers. For example, in some embodiments, the iNKT cells may express CD3 and iTCR (Vα24-Jα18), or CD3 and Vα24, on the cell surface. In some embodiments, the CD3 and iTCR (Vα24-Jα18) cell surface markers may be expressed alone or in combination with one or more additional cell surface markers (e.g., CD4 + , CD8 + , CD4 - CD8 - , CD4 + CD8 +This may occur when used in combination with other drugs.

[0061] In some embodiments, the iNKT cells of the present disclosure (e.g., CD3 + iTCR(Vα24-Jα18) + iNKT cells) can be obtained or isolated from a biological sample. In some embodiments, iNKT cells of the present disclosure can be obtained or isolated from one or more peripheral blood mononuclear cells.

[0062] In some embodiments, the biological sample is from a human (e.g., fetus, newborn, child, or adult). In some embodiments, the biological sample is from a non-human animal. Non-human animals include all vertebrates (e.g., mammals or non-mammals), such as mice, rats, rabbits, dogs, monkeys, and pigs. In some embodiments, the biological sample is from a subject in need of treatment (e.g., a cancer patient, e.g., a T-ALL patient). In some embodiments, the biological sample is from a donor (e.g., a healthy donor). In some embodiments, the biological sample comprises blood (e.g., peripheral blood and / or umbilical cord blood), bone marrow, lymph node tissue, spleen tissue, tumor tissue, one or more induced pluripotent stem cells, and / or one or more peripheral blood mononuclear cells. In some embodiments, the biological sample and / or blood comprises peripheral blood and / or umbilical cord blood. In some embodiments, the biological sample and / or blood is collected (e.g., from a subject or donor) by apheresis and / or leukapheresis.

[0063] In some embodiments, the iNKT cells of the present disclosure (e.g., CD3 + iTCR(Vα24-Jα18) + The iNKT cells can be isolated, for example, from a human biological sample. In some embodiments, the iNKT cells are isolated iNKT cells.

[0064] As used herein, the term "isolated" refers to material that has been removed from its source environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide, polypeptide, or cell present in an organism is not isolated, but the same polynucleotide, polypeptide, or cell separated from some or all of the coexisting materials in an organism is isolated.

[0065] As used herein, an "isolated cell" refers to a cell or cell population (e.g., a type I NKT cell or cell population) that has been identified and separated from one or more (e.g., the majority) of the components of its source environment (e.g., from the components of a cell culture or biological sample). In some embodiments, the separation is performed to sufficiently remove components that may otherwise interfere with the suitability of the cell for a desired application (e.g., for therapeutic use of the type I NKT cell or cell population). In some embodiments, the separation is performed to sufficiently separate cells expressing a particular marker or set of markers (e.g., iTCR(Vα24-Jα18)) from cells expressing alternative markers or sets of markers. Methods of isolating cells are known in the art and include, but are not limited to, for example, separation using antibody-conjugated microbeads, flow cytometry using a cocktail of monoclonal antibodies against cell surface markers, by positive and / or negative selection techniques, or by cell sorting, etc. Exemplary isolation and separation techniques are described and exemplified herein.

[0066] In some embodiments, the iNKT cells of the present disclosure (e.g., CD3 + iTCR(Vα24-Jα18) +iNKT cells (e.g., iTCR(Vα24-Jα18)) may be isolated or separated by affinity-based separation methods. Exemplary techniques for affinity separation may, in some embodiments, include magnetic separation (e.g., using antibody-coated magnetic beads), affinity chromatography, cytotoxic agents (e.g., complement and cytotoxins) linked to or used in conjunction with monoclonal antibodies, and "panning" with antibodies attached to a solid matrix (e.g., a plate), or other suitable techniques. In some embodiments, separation techniques may also include the use of fluorescence-activated cell sorters, which may have various degrees of sophistication, e.g., multiple color channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, etc. iNKT cells (e.g., iTCR(Vα24-Jα18)) + It is understood that any technique that allows for the isolation or separation of iNKT cells may be utilized.

[0067] In some embodiments, the affinity reagents utilized in the various isolation or separation methods may be specific receptors or ligands for cell surface markers on iNKT cells. In some embodiments, the antibodies may be conjugated to a label, which may be used in some embodiments for isolation or separation. Labels may include, in some embodiments, magnetic beads (e.g., which may allow for direct separation), biotin (e.g., which may be removed by avidin or streptavidin bound to a support; e.g., biotin-conjugated anti-TCRVα24-Jα18 (6B11 clone) or anti-Vα24 antibody in conjunction with streptavidin microbeads), fluorescent dyes (e.g., phycoerythrin, fluorescein, Texas Red, or combinations thereof, which may be used by a fluorescence-activated cell sorter), and the like.

[0068] In some embodiments, cell separation using antibodies may include, for example, adding antibodies to a suspension of cells for a period of time sufficient for available cell surface markers to bind. Incubation may be for a variety of times. For example, in some embodiments, incubation may be for about 2 minutes, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes or more. Any length of time that results in specific labeling by the antibody and minimal non-specific binding may be considered as contemplated for this aspect of the disclosure.

[0069] In some embodiments, the staining intensity of iNKT cells can be monitored by flow cytometry, e.g., a laser detects the quantitative level of fluorochrome, which can be proportional to the amount of cell surface antigen bound by the antibody. Flow cytometry or FACS can also be used in some embodiments to separate cell populations based on the intensity of antibody staining, as well as other parameters, e.g., cell size and light scatter.

[0070] In some embodiments, iNKT cells are separated based on the expression of at least one cell surface marker. The separated cells may be collected in any suitable medium that maintains cell viability. In some embodiments, the culture containing the cells may contain serum, cytokines, or growth factors to which the cells are responsive. In some embodiments, the cytokines or growth factors may promote cell survival, proliferation, function, or a combination thereof. The cytokines and growth factors may include polypeptide and non-polypeptide factors in some embodiments.

[0071] The term "activation" as used herein refers to the process of rendering isolated iNKT cells or conventional T cells active (e.g., proliferative, cytokine releasing, or cytolytic). In some embodiments, isolated iNKT cells or conventional T cells are activated by co-culture with a T cell receptor (TCR) engaging reagent (e.g., microbeads coated with antibodies against CD2, CD3, and CD28, or soluble anti-CD3 antibodies). In some embodiments, isolated iNKT cells are activated by co-culture with irradiated autologous PBMC negative fraction, αGalCer or 7DW8-5, IL-2, IL-15, and / or IL-7 in human T cell medium.

[0072] In some embodiments, the iNKT cells of the present disclosure (e.g., CD3 + iTCR(Vα24-Jα18) + In some embodiments, the CD7 CAR-modified iNKT cells of the present disclosure are modified to express a CD7 CAR construct capable of binding to a CD7 target antigen. + They have been further engineered to express a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor mimic antibody (TCRm), or any combination thereof, to target additional antigens on the cancer.

[0073] In some embodiments, the iNKT cells are modified to express a chimeric antigen receptor (CAR). In some embodiments, the CAR can be engineered using an antigen binding domain such that the CAR and / or the cell binds to a target antigen (e.g., CD7 or other exemplary antigens described herein) when the CAR is expressed on the cell (e.g., iNKT cell). In some embodiments, the CAR sequence is cloned into a cell or cell population (e.g., iNKT cell or iNKT cell population) and expanded using our own and / or currently available protocols for irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15, or IL-2, IL-7 and IL-15). In some embodiments, the cell or cell population comprises one or more polynucleotides encoding a CAR. In some embodiments, the cell or cell population is from a donor or patient (e.g., a patient having or suspected of having cancer, e.g., T-ALL or other exemplary cancers described herein). In some embodiments, when used as a therapeutic and when the cells or cell population are from a patient, the CAR modified cells or cell population can be administered to the same patient and / or other patients in need of such treatment. In some embodiments, when used as a therapeutic and when the cells or cell population are from a donor, the CAR modified cells or cell population can be administered to any patient in need of such treatment.

[0074] As used herein, the terms "CAR-expressing" and "CAR-modified," when used to describe a cell or cell population, refer to a cell or cell population that has been artificially engineered to contain one or more polynucleotides encoding the sequence of a CAR peptide, which can be transcribed, translated, and expressed on the cell surface. In some embodiments, a CAR-expressing cell or cell population comprises an iNKT cell or cell population. In some embodiments, a CAR-expressing cell or cell population comprises an iTCR(Vα24-Jα18) + In some embodiments, the CAR-expressing cell or cell population comprises a CD3 +iTCR(Vα24-Jα18) + In some embodiments, the CAR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 + In some embodiments, the CAR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - In some embodiments, the CAR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD8 + In some embodiments, the CAR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + In some embodiments, the CAR-expressing cell or cell population comprises a mixture of cells, e.g., CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD8 + Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD4 + A mixture of CD8+ cells or CD3 + iTCR(Vα24-Jα18) + CD4 + , CD3 + iTCR(Vα24-Jα18)+ CD8 + , CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - , and CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + In some embodiments, when used as a therapeutic agent, the CAR-expressing cell or cell population administered to a subject may comprise a CAR-modified NKT cell, or a population of CAR-modified NKT cells from a subject. In some embodiments, when used as a therapeutic agent, the CAR-expressing cell or cell population administered to a subject may comprise a CAR-modified NKT cell, or a population of CAR-modified NKT cells from a donor.

[0075] In some embodiments, the CAR modified cells or cell populations can engage and kill cells (e.g., malignant cancer cells) expressing a target antigen (e.g., CD7). Methods and compositions for constructing and administering the disclosed CAR-based immunotherapies are provided herein. Exemplary methods for constructing CAR-based immunotherapies are also disclosed, for example, in US publications U.S. Publication No. 2014 / 0271635 and U.S. Publication No. 2016 / 0310532, both of which are incorporated herein by reference with respect to such methods.

[0076] As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a polypeptide or set of polypeptides that, when expressed by a cell, provides the cell with specificity for a target antigen-expressing cell (e.g., a malignant cancer cell) and with the generation of an intracellular signal. In some embodiments, a CAR comprises at least one extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. The domains may be present in a single polypeptide or a set of polypeptides. In some embodiments, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In some embodiments, the costimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, and / or DAP12.

[0077] In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain comprising (i) a functional signaling domain derived from a stimulatory molecule; (ii) a functional signaling domain derived from a stimulatory molecule and a functional signaling domain derived from a costimulatory molecule; or (iii) a functional signaling domain derived from a stimulatory molecule and at least two functional signaling domains derived from one or more costimulatory molecules. In some embodiments, the CAR optionally comprises a leader sequence at the N-terminus of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, which is optionally cleaved from the antigen binding domain during cellular processing and localization of the CAR to the cell membrane.

[0078] In some embodiments, the antigen binding domain of the CAR comprises an antibody, or an antigen binding fragment thereof. In some embodiments, the antigen binding domain and / or antibody comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, or a single domain antibody (also known as a nanobody). In some embodiments, the antigen binding domain and / or antigen binding fragment comprises a single chain variable fragment (scFv) or a Fab fragment. In some embodiments, the antigen binding domain and / or antigen binding fragment comprises an scFv.

[0079] The term "antibody" as used herein refers to any functional immunoglobulin molecule that recognizes and binds, e.g., specifically binds, a target, e.g., a protein, a polypeptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" includes antibodies having sequences from any species of origin, e.g., mouse, rabbit, goat, llama, alpaca, non-human primate, and human. The term further includes human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, so long as it exhibits the desired binding and / or biological activity. In some embodiments, the antibody is capable of binding, e.g., specifically binding, to a target antigen (e.g., CD7) expressed on a cancer cell. Antibodies can be generated using any suitable technique, e.g., recombinant expression, hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries, or any combination thereof. The term "antibody" includes full-length antibodies, as well as antigen-binding domains and antigen-binding fragments thereof. In some embodiments, the antibodies used in the CAR and / or other constructs described herein are full length or intact antibodies. In some embodiments, the antibodies used in the CAR and / or other constructs described herein are monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, or single domain antibodies. In some embodiments, the antibodies used in the CAR and / or other constructs described herein are antigen-binding domains or antigen-binding fragments of antibodies.

[0080] A "full-length" or "intact" antibody typically comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Recognized classes of immunoglobulin genes encoding antibody chains include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. In some embodiments, an antibody comprises a kappa light chain. In some embodiments, an antibody comprises a lambda light chain. A kappa or lambda light chain can be selected from any kappa or lambda light chain sequence from any species. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. The four subclasses of IgG (IgG1, IgG2, IgG3 and IgG4) differ in their constant regions and exhibit different effector functions.

[0081] Antibodies that may be used in the CAR and / or other constructs described herein further include antigen-binding fragments. As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of a full-length antibody that retain the ability to bind, e.g., specifically bind, to a target antigen (e.g., CD7) and / or provide a function of the full-length antibody (e.g., the ability to specifically bind to CD7). The antigen-binding function of an antibody may be performed by a fragment of the full-length antibody. Fragments may also be present in larger macromolecules, e.g., bispecific antibodies. Examples of such antibody fragments include a Fab fragment, which is a monovalent fragment containing at least one VL, CL, VH and CH1 domain; an F(ab)2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of a VH domain and a CH1 domain; an Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; a single domain antibody (SdAb) fragment consisting of a VH domain or a VL domain; isolated complementarity determining regions (CDRs); and a half-body which contains only one heavy chain and one light chain rather than the typical pairing of two heavy chains and two light chains on separate arms. Furthermore, the two domains of the Fv fragment, VL and VH, although encoded by separate genes, can be linked using recombinant methods, for example by an artificial peptide linker, which generates the VL and VH as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single chain variable fragment (scFv)) (see, e.g., Bird et al., Science 1988;242(4877):423-6; Huston et al., PNAS 1988;85(16):5879-83). Such single chain antibodies include one or more antigen-binding fragments or portions of an antibody. Other examples of antigen-binding fragments include bispecific T cell engagers (BiTEs) consisting of two scFvs of different antibodies or amino acid sequences from four different genes on a single peptide chain. In some embodiments, the antigen-binding fragment is a Fab fragment or a scFv. In some embodiments, the antigen-binding fragment is a scFv.

[0082] In some embodiments, the antigen binding domain of the CAR comprises a cell binding agent. In some embodiments, the antigen binding domain of the CAR and / or the cell binding agent comprises a DARPin, a duobody, a bicyclic peptide, a nanobody, a sentinelin, MSH (melanocyte stimulating hormone), a receptor-Fc fusion molecule, a T cell receptor structure, a natural ligand (e.g., a receptor expressed in mature non-malignant and / or malignant B cells, including plasma cells, exemplary ligands for B cell maturation antigen (BCMA) include, but are not limited to, B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL)), a steroid hormone (e.g., androgen or estrogen), a growth factor, a colony stimulating factor (e.g., EGF), or other non-antibody scaffold. In some embodiments, non-antibody scaffolds can be broadly categorized into two structural classes: domain size compounds (approximately 6-20 kDa) and constrained peptides (approximately 2-4 kDa). Exemplary domain size scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (e.g., adnectins and sentinels), finomers, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusion proteins, and exemplary constrained peptides include avimers, bicyclic peptides, and Cys-knots. In some embodiments, the antigen binding domain of the CAR and / or cell binding agent comprises an affibody, affilin, anticalins, atrimers, DARPins, FN3 scaffolds, e.g., adnectins or sentinels, finomers, Kunitz domains, pronectins, O-bodies, receptor-Fc fusion proteins, avimers, bicyclic peptides, and / or Cys-knots. Non-antibody scaffolds are reviewed, for example, in Vazquez-Lombardi et al., Drug Dis Today 2015;20(10):1271-83.

[0083] In some embodiments, the antigen binding domain of the CAR can bind to CD7, CD1a, CD1d, CD5, TRBC1, TRBC2, CD21, CCR9, CD30, CD123, CD33, CD38, CD138, CLL-1, LILRB4, Siglec-6, CD70, or PD-L1. In some embodiments, the antigen binding domain can bind to CD19 or ROR1.

[0084] The term "CD7, CD1a, CD1d, CD5, TRBC1, CD21, CCR9, CD123, CD33, CD38, CD138, CLL-1, LILRB4, CD30, Siglec-6, CD70, PD-L1, CD19, or ROR1" refers to any native form of an antigen, such as CD7. The term may include full length CD7 (e.g., UniProt Reference Sequence: P09564; SEQ ID NO: 1), CD1a (e.g., UniProt Reference Sequence: P06126; SEQ ID NO: 2), CD1d (e.g., UniProt Reference Sequence: P15813; SEQ ID NO: 3), CD2 (e.g., UniProt Reference Sequence: P06729; SEQ ID NO: 4), CD5 (e.g., UniProt Reference Sequence: P06127; SEQ ID NO: 5), TRBC1 (e.g., UniProt Reference Sequence: P06128; SEQ ID NO: 6), or any of the following: 01850; SEQ ID NO: 6), TRBC2 (e.g., UniProt Reference Sequence: A0A5B9; SEQ ID NO: 7), CD21 (e.g., UniProt Reference Sequence: P20023; SEQ ID NO: 8), CCR9 (e.g., UniProt Reference Sequence: P51686; SEQ ID NO: 9), CD30 (e.g., UniProt Reference Sequence: P28908; SEQ ID NO: 10), CD123 (e.g., UniProt Reference Sequence: P26951; SEQ ID NO: 11), CD33 (e.g., UniProt Reference Sequence: P27024; SEQ ID NO: 12), iProt Reference Sequence: P20138; SEQ ID NO: 12), CD38 (e.g., UniProt Reference Sequence: P28907; SEQ ID NO: 13), CD138 (e.g., UniProt Reference Sequence: P18827; SEQ ID NO: 14), CLL-1 (e.g., UniProt Reference Sequence: Q5QGZ9; SEQ ID NO: 15), LILRB4 (e.g., UniProt Reference Sequence: Q8NHJ6; SEQ ID NO: 16), Siglec-6 (e.g., UniProt Reference Sequence: O4369 9; SEQ ID NO:17), CD70 (e.g., UniProt Reference Sequence: P32970; SEQ ID NO:18), PD-L1 (e.g., UniProt Reference Sequence: Q9NZQ7; SEQ ID NO:19), CD19 (e.g., UniProt Reference Sequence: P15391; SEQ ID NO:20), ROR1 (e.g., UniProt Reference Sequence: Q01973; SEQ ID NO:21), or any antigen, as well as any form of an antigen (e.g., CD7) that may result from cellular processing.The term also includes functional variants or fragments of the antigen, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biological functions of the antigen (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein).Antigens (e.g., CD7) can be isolated from humans or produced recombinantly or by synthetic methods.

[0085] In some embodiments, the CD7 CAR-modified iNKT cells are modified to express a T cell receptor (TCR). In some embodiments, the TCR can be engineered using antigen-binding alpha and / or beta chains such that the TCR and / or cell binds to a target antigen (e.g., WT1 or other exemplary antigens described herein) when the TCR is expressed on the cell (e.g., CD7 CAR-modified iNKT cell). In some embodiments, the TCR sequence is cloned into a cell or cell population (e.g., CD7 CAR-modified iNKT cell or iNKT cell population) and expanded using our own and / or currently available protocols for irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15, or IL-2, IL-7 and IL-15). In some embodiments, the cell or cell population comprises one or more polynucleotides encoding the TCR. In some embodiments, the cells or cell populations are from a donor or patient (e.g., a patient having or suspected of having cancer, e.g., T-ALL or other exemplary cancers described herein). In some embodiments, when used as a therapeutic and when the cells or cell populations are from a patient, the TCR modified cells or cell populations may be administered to the same patient and / or other patients in need of such treatment. In some embodiments, when used as a therapeutic and when the cells or cell populations are from a donor, the TCR modified cells or cell populations may be administered to any patient in need of such treatment.

[0086] As used herein, the terms "TCR-expressing" and "TCR-modified," when used to describe a cell or cell population, refer to a cell or cell population that has been artificially engineered to contain one or more polynucleotides encoding the sequence of a TCR peptide, which can be transcribed, translated, and expressed on the cell surface. In some embodiments, a TCR-expressing cell or cell population comprises a CD7 CAR-modified iNKT cell or cell population. In some embodiments, a TCR-expressing cell or cell population comprises a CD3 CAR-modified iNKT cell or cell population. + iTCR(Vα24-Jα18) + CD4 + In some embodiments, the TCR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - In some embodiments, the TCR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD8 + In some embodiments, the TCR-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + In some embodiments, the TCR-expressing cell or cell population comprises a mixture of cells, e.g., a CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD8 + Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3+ iTCR(Vα24-Jα18) + CD4 + CD8 + A mixture of cells or CD3 + iTCR(Vα24-Jα18) + CD4 + , CD3 + iTCR(Vα24-Jα18) + CD8 + , CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - , and CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + In some embodiments, when used as a therapeutic agent, the TCR-expressing cell or cell population administered to a subject may comprise a TCR-modified NKT cell or a population of TCR-modified NKT cells from a subject. In some embodiments, when used as a therapeutic agent, the TCR-expressing cell or cell population administered to a subject may comprise a TCR-modified NKT cell or a population of TCR-modified NKT cells from a donor.

[0087] In some embodiments, the TCR modified cell or cell population can engage and kill cells (e.g., malignant cancer cells) expressing a target antigen (e.g., WT1). Methods and compositions for constructing and administering the disclosed TCR-based immunotherapies are provided herein. Exemplary methods for constructing TCR-based immunotherapies are also disclosed, for example, in U.S. Patent No. 9,115,372, which is incorporated herein by reference with respect to such methods.

[0088] As used herein, the terms "T cell receptor" and "TCR" refer to a polypeptide or set of polypeptides that, when expressed by a cell, provides the cell with specificity for a target antigen-expressing cell (e.g., a malignant cancer cell) and with the generation of an intracellular signal. In some embodiments, a TCR comprises at least an alpha chain and a beta chain. The chains may be present in a single polypeptide or set of polypeptides. In some embodiments, the alpha chain and / or the beta chain are capable of binding to an antigen.

[0089] In some embodiments, the TCR comprises an alpha chain and a beta chain. In some embodiments, both the alpha and beta chains comprise a constant region (c) and a variable region (v). In some embodiments, the variable region determines the antigen specificity. In some embodiments, the variable region recognizes a target antigen, e.g., an antigen ligand comprising a short contiguous amino acid sequence of a protein, presented on a target cell by a major histocompatibility complex (MHC) molecule (also known as a human leukocyte antigen (HLA) molecule). In some embodiments, accessory adhesion molecules expressed by T cells are also involved, e.g., CD4 for MHC class II and CD8 for MHC class I. In some embodiments, TCR signaling is via the associated invariant CD3 complex. In some embodiments, the CD3 complex comprises different CD3 proteins, which form two heterodimers (CD3δε and CD3γε) and one homodimer (CD3ζζ).

[0090] In some embodiments, the alpha and / or beta chains of the TCR are capable of binding to an antigen. In some embodiments, the alpha and / or beta chains are capable of binding to WT1, PRAME, HA-1, or ΔNPM1.

[0091] The term "WT1, PRAME, HA-1, and ΔNPM1" as used herein refers to any of the native forms of human WT1, PRAME, HA-1, and ΔNPM1. This term includes full-length WT1 (e.g., UniProt Reference Sequence: P19544; SEQ ID NO: 22), PRAME (e.g., UniProt Reference Sequence: P78395; SEQ ID NO: 23), HA-1 (e.g., UniProt Reference Sequence: Q92619; SEQ ID NO: 24), and ΔNPM1 (e.g., UniProt Reference Sequence: P06748; SEQ ID NO: 25), as well as any of the forms of human WT1, PRAME, HA-1, and ΔNPM1 that may result from cellular processing. The term also includes functional variants or fragments of WT1, PRAME, HA-1, and ΔNPM1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biological functions of human WT1, PRAME, HA-1, and ΔNPM1 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein).WT1, PRAME, HA-1, and ΔNPM1 can be isolated from humans or produced recombinantly or by synthetic methods.

[0092] In some embodiments, the CD7 CAR-expressing iNKT cells are further modified to express a T cell receptor mimic antibody (TCRm). In some embodiments, the TCRm can be engineered using an antigen-binding domain such that when the TCRm is expressed on a cell (e.g., an iNKT cell), the TCRm and / or the cell binds to a target antigen (e.g., a composite antigen, e.g., a composite antigen comprising a peptide and a human leukocyte antigen (HLA) molecule, as described herein). In some embodiments, the TCRm sequence is cloned into a cell or cell population (e.g., an iNKT cell or an iNKT cell population) and expanded using our own and / or currently available protocols for irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15, and / or IL-7). In some embodiments, the cell or cell population comprises one or more polynucleotides encoding the TCRm. In some embodiments, the cells or cell populations are from a donor or patient (e.g., a patient having or suspected of having cancer, e.g., T-ALL or other exemplary cancers described herein). In some embodiments, when used as a therapeutic and when the cells or cell populations are from a patient, the TCRm modified cells or cell populations may be administered to the same patient and / or other patients in need of such treatment. In some embodiments, when used as a therapeutic and when the cells or cell populations are from a donor, the TCRm modified cells or cell populations may be administered to any patient in need of such treatment.

[0093] As used herein, the terms "TCRm-expressing" and "TCRm-modified," when used to describe a cell or cell population, refer to a cell or cell population that has been artificially engineered to contain one or more polynucleotides encoding the sequence of a TCRm peptide, and which are capable of transcribing, translating, and expressing the TCRm peptide on the cell surface. In some embodiments, a TCRm-expressing cell or cell population comprises a CD7 CAR-modified iNKT cell or cell population. In some embodiments, a TCRm-expressing cell or cell population comprises a CD3 CAR-modified iNKT cell or cell population. + iTCR(Vα24-Jα18)+ CD4 + In some embodiments, the TCRm-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - In some embodiments, the TCRm-expressing cell or cell population comprises a CD3 + iTCR(Vα24-Jα18) + CD8 + In some embodiments, the TCRm-expressing cell or cell population comprises a mixture of cells, e.g., a CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD8 + Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - Mixture of cells, CD3 + iTCR(Vα24-Jα18) + CD4 + and CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + A mixture of cells or CD3 + iTCR(Vα24-Jα18) + CD4 + , CD3 + iTCR(Vα24-Jα18) + CD8 + , CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - , and CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 +In some embodiments, when used as a therapeutic agent, the TCRm-expressing cell or cell population administered to a subject may comprise a TCRm-modified NKT cell or a population of TCRm-modified NKT cells from a subject. In some embodiments, when used as a therapeutic agent, the TCRm-expressing cell or cell population administered to a subject may comprise a TCRm-modified NKT cell or a population of TCRm-modified NKT cells from a donor.

[0094] In some embodiments, the TCRm modified cell or cell population can engage and kill a cell (e.g., a malignant cancer cell) expressing a target antigen (e.g., a composite antigen, e.g., a composite antigen comprising a peptide and a human leukocyte antigen (HLA) molecule, as described herein). Methods and compositions for constructing and administering the disclosed TCRm-based immunotherapies are provided herein. Exemplary methods for constructing TCRm-based immunotherapies are also disclosed, for example, in U.S. Publication No. 2019 / 092876, which is incorporated herein by reference, with respect to such methods.

[0095] As used herein, the terms "T cell receptor mimic antibody" and "TCRm" refer to a polypeptide or set of polypeptides that, when expressed by a cell, provides the cell with specificity for a target antigen-expressing cell (e.g., a malignant cancer cell) and with the generation of an intracellular signal. In some embodiments, the TCRm comprises at least one extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. The domains may be present in a single polypeptide or a set of polypeptides. In some embodiments, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In some embodiments, the costimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, and / or DAP12.

[0096] In some embodiments, the antigen binding domain of the TCRm comprises an antibody, or an antigen binding fragment thereof. In some embodiments, the antigen binding domain and / or antibody comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, or a single domain antibody. In some embodiments, the antigen binding domain and / or antigen binding fragment comprises a single chain variable fragment (scFv) or a Fab fragment. In some embodiments, the antigen binding domain and / or antigen binding fragment comprises an scFv.

[0097] In some embodiments, the antigen binding domain of the TCRm comprises a cell binding agent. In some embodiments, the antigen binding domain of the TCRm and / or the cell binding agent comprises a DARPin, a duobody, a bicyclic peptide, a nanobody, centilin, MSH (melanocyte stimulating hormone), a receptor-Fc fusion molecule, a T cell receptor structure, a natural ligand (e.g., a receptor expressed on mature non-malignant and / or malignant B cells, including plasma cells, exemplary ligands for B cell maturation antigen (BCMA) include, but are not limited to, B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL)), a steroid hormone (e.g., androgen or estrogen), a growth factor, a colony stimulating factor (e.g., EGF), or other non-antibody scaffold. In some embodiments, the antigen binding domain of the TCRm and / or the cell binding agent comprises an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN3 scaffold, e.g., an adnectin or sentinelin, a finomer, a Kunitz domain, a pronectin, an O-body, a receptor-Fc fusion protein, an avimer, a bicyclic peptide, and / or a Cys-knot.

[0098] In some embodiments, the antigen binding domain of the TCRm is capable of binding to a complex antigen comprising a peptide and a human leukocyte antigen (HLA) molecule.

[0099] In some embodiments, the HLA molecule is an HLA class I molecule. In some embodiments, the HLA molecule is an HLA class I binding peptide. In some embodiments, the HLA class I binding peptide is about 9 or 10 amino acids in length. Exemplary HLA class I binding peptides include WT1-derived HLA-A * 0201 binding peptide RMFPNAPYL (SEQ ID NO: 26); WT1 derived HLA-A * 2402 binding peptide CMTWNQMNL (SEQ ID NO: 27); PRAME derived HLA-A * 0201 binding peptide VLDGLDVLL (SEQ ID NO: 28); PRAME derived HLA-A * 0201 binding peptide ALYVDSLEFL (SEQ ID NO: 29); PRAME derived HLA-A * 0201 binding peptide SLYSFPEPEA (SEQ ID NO: 30; PRAME derived HLA-A * 0201 binding peptide SLLQHLIGL (SEQ ID NO: 31); PRAME derived HLA-A * 2402 binding peptide LYVDSLFFLC (SEQ ID NO: 32); HA-1 derived HLA-A * 0201 binding peptide VLHDDLLEA (SEQ ID NO: 33); ΔNPM1 derived HLA-A * Examples include, but are not limited to, the 0201 binding peptide CLAVEEVSL (SEQ ID NO:34).

[0100] In some embodiments, the antigen binding domain of TCRm can bind to a complex antigen comprising an HLA molecule having the amino acid sequence of RMFPNAPYL (sequence number 26); CMTWNQMNL (sequence number 27); VLDGLDVLL (sequence number 28); ALYVDSLEFL (sequence number 29); SLYSFPEPEA (sequence number 30); SLLQHLIGL (sequence number 31); LYVDSLFFLc (sequence number 32); VLHDDLLEA (sequence number 33); and / or CLAVEEVSL (sequence number 34).

[0101] In some embodiments, the HLA molecule is an HLA class II molecule. In some embodiments, the HLA molecule is an HLA class II binding peptide. In some embodiments, the HLA class II binding peptide is about 13 to 25 amino acids in length. Exemplary HLA class II binding peptides include HLA-DRB1 derived from WT1. * 0405, -DRB1 * 1501, -DRB1 * 1502, -DPB1 * 0501 and -DPB1 * Examples include, but are not limited to, the 0901 binding peptide KRYFKLSHLQMHSRKH (SEQ ID NO:35).

[0102] In some embodiments, the antigen binding domain of the TCRm is capable of binding to a complex antigen comprising an HLA molecule having the amino acid sequence of KRYFKLSHLQMHSRKH (SEQ ID NO: 35).

[0103] In some embodiments, the iNKT cells of the present disclosure (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The iNKT cells) are further modified to include an exogenous cytokine, growth factor, antibody or antigen-binding fragment, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment comprises a bispecific T cell engager (BiTE).

[0104] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0105] Therapeutic Methods, Uses and Compositions The CD7 CAR-modified iNKT cells described herein (e.g., CD3 + iTCR(Vα24-Jα18) + , or CD3 + Vα24 + iNKT cells, also known as type I NKT cells, can be utilized in a variety of therapeutic and prophylactic applications. For example, in some embodiments, the CD7 CAR-modified iNKT cells described herein are + The CD7 CAR modified iNKT cells described herein may be administered per se or in any suitable pharmaceutical composition, which may be useful in the treatment or prevention of cancer.

[0106] Thus, in certain aspects of the disclosure, CD7 CAR-modified iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In a subject in need of treatment or prevention, a therapeutically effective amount of iNKT cells is administered to the subject to inhibit CD7 + In certain aspects, the present disclosure provides a method for treating or preventing cancer. + 1. A method of preparing a therapy for treating or preventing cancer, comprising: (a) administering to a patient a gene encoding one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 +(b) activating or activating one or more iNKT cells using, but not limited to, irradiated autologous PBMC negative fraction and / or total PBMC, α-galactosylceramide (αGalCer) or 7DW8-5, IL-2, IL-15 and / or IL-7 in growth medium for expansion; (c) engineering the activated and expanding iNKT cells using a lentiviral vector to express a CAR (e.g., CD7); and (d) expanding the one or more CAR (e.g., CD7) engineered iNKT cells in growth medium using our own protocol for irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15 and / or IL-7). In certain aspects, the disclosure further provides a method for the production of expanded cell populations (e.g., CD7 CAR-modified iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + and administering to the subject a therapeutically effective amount of an expanded cell population comprising iNKT cells (iNKT cells) to the subject, thereby inhibiting the proliferation and proliferation of CD7 + Methods for treating or preventing cancer are provided. + Use of the disclosed CD7 CAR-modified iNKT cells in the treatment or prevention of cancer is also provided. + iTCR(Vα24-Jα18) + or CD3 + Vα24 + Pharmaceutical compositions comprising a therapeutically effective amount of iNKT cells are also disclosed and are useful in the therapeutic methods and uses provided herein.

[0107] One exemplary embodiment is a method for treating or preventing CD7 + A method for treating or preventing cancer, comprising administering to a subject a CD7 CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18)+ or CD3 + Vα24 + A therapeutically effective amount of a CD7 CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The method includes administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of iNKT cells and at least one pharma- ceutical acceptable carrier.

[0108] Another exemplary embodiment is a method for treating or preventing CD7 + 1. A method of preparing a therapy for treating or preventing cancer, comprising: (a) administering to a patient a gene encoding one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + (b) isolating one or more iTCRs (iNKT cells) from a biological sample; + (c) activating or activating the iNKT cells using, but not limited to, irradiated autologous PBMC negative fraction and / or total PBMC, α-galactosylceramide (αGalCer) or 7DW8-5, IL-2, IL-15, and / or IL-7 in growth medium for expansion; (d) engineering the activated and expanding iNKT cells using a lentiviral vector to express a CAR (e.g., CD7); and (d) expanding one or more CAR (e.g., CD7) engineered iNKT cells in growth medium using our own protocol of irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15, and / or IL-7).

[0109] Another exemplary embodiment is a method for treating or preventing CD7 + A method of treating or preventing cancer, comprising administering to a patient a population of expanded cells (e.g., CD7 CAR-modified iNKT cells (e.g., CD3+ iTCR(Vα24-Jα18) + or CD3 + Vα24 + a therapeutically effective amount of an expanded cell population (e.g., CD7 CAR-modified iNKT cells (e.g., CD3 iNKT cells)) or an expanded cell population (e.g., as described herein and / or prepared by the methods described herein) + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The method includes administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of an expanded cell population comprising iNKT cells and at least one pharma- ceutical acceptable carrier.

[0110] Another exemplary embodiment is a method for treating or preventing CD7 + Isolated iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the use comprises administering to a subject a therapeutically effective amount of the cells, or a pharmaceutical composition comprising a therapeutically effective amount of the cells and at least one pharma- ceutical acceptable carrier.

[0111] Another exemplary embodiment is a method for treating or preventing CD7 + Isolated iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the use comprises administering to a subject a therapeutically effective amount of the cells, or a pharmaceutical composition comprising a therapeutically effective amount of the cells and at least one pharma- ceutical acceptable carrier.

[0112] Another exemplary embodiment is a method for treating or preventing CD7 +In the manufacture of a medicament for treating or preventing cancer, isolated iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the medicament comprises a therapeutically effective amount of the cells, or a pharmaceutical composition comprising a therapeutically effective amount of the cells and at least one pharma- ceutical acceptable carrier.

[0113] The term "treat" and its cognates as used herein refer to the amelioration of a disease, disorder, or condition (e.g., cancer), or at least one discernible symptom thereof. The term "treat" includes, but is not limited to, completing treatment or completing amelioration of one or more symptoms of cancer. In some embodiments, "treat" refers to at least a partial amelioration of at least one measurable physical parameter, not necessarily discerned by the subject. In some embodiments, "treat" refers to physical inhibition (e.g., stabilization of discernible symptoms), physiological inhibition (e.g., stabilization of physical parameters), or both, of the progression of a disease, disorder, or condition. In some embodiments, "treat" refers to slowing down or reversing the progression of a disease, disorder, or condition. As used herein, "treat" and its cognates further include delaying the onset of, or reducing the risk of contracting, a given disease, disorder, or condition. In some embodiments, "treating" refers to administering to a subject suspected of having a disease, disorder, or condition (e.g., cancer or a precancerous condition) a CD7 CAR-modified iNKT cell, cell population, or composition disclosed herein. + A subject suspected of having cancer and / or a precancerous condition, and / or a sample from a subject, may contain one or more cells that are abnormal, malignant, and / or premalignant.

[0114] The terms "subject" and "patient" are used interchangeably herein to refer to any human or non-human animal in need of treatment. Non-human animals include all vertebrates (e.g., mammals and non-mammals). Non-limiting examples of mammals include humans, mice, rats, rabbits, dogs, monkeys, and pigs. In some embodiments, the subject is a human.

[0115] As used herein, the term "donor" refers to any human or non-human animal that provides a biological sample (e.g., a blood sample) for use in a subject in need of treatment and / or for use in the preparation of a therapy (e.g., a CD7 CAR modified iNKT cell therapy disclosed herein) for a subject in need of treatment. In some embodiments, the donor is a human.

[0116] The term "cancer" as used herein refers to the presence of cells with characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and / or specific morphological characteristics. Cancer cells may be in the form of a tumor or mass, but such cells may exist alone in a subject or circulate in the bloodstream as independent cells, e.g., leukemia or lymphoma cells. The term "cancer" includes all types of cancer and cancer metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumor cancers.

[0117] As used herein, the term "CD7 +"Cancer" refers to cancer cells that express the CD7 antigen on the cell surface, including T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), pro-T-ALL, pre-T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) subtypes, including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ACL) lymphoma (ALCL), primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); CD7 + Other malignant tumors or CD7 + CD1d + In some embodiments, the CD7+ cancer is T-ALL or AML. + The cancer is a refractory or recurrent cancer (e.g., refractory or recurrent T-ALL or AML). In some embodiments, the cancer is refractory or recurrent T-ALL or AML. In some embodiments, the cancer expresses a target antigen.

[0118] The term "target antigen" as used herein refers to any antigen targeted by CD7 CAR modified iNKT cells and / or by constructs (e.g., CAR, TCR or TCRm) expressed by iNKT cells. The term "antigen" as used herein is synonymous with "antigenic determinant" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids, or a conformational configuration consisting of distinct regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding moiety (e.g., an antigen-binding moiety of a CAR, TCR or TCRm) binds and forms an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, inside or on the surface of cancer cells, inside or on the surface of virus-infected cells, inside or on the surface of other diseased cells, free in serum, and / or in the extracellular matrix (ECM).

[0119] Exemplary target antigens are disclosed herein and include, but are not limited to, CD7, CD1a, CD1d, CD2, CD5, TRBC1, CD21, CCR9, CD30, CD123, CD33, CD38, CD138, CLL-1, LILRB4, Siglec-6, CD70, PD-L1, CD19, ROR1, WT1, PRAME, HA-1, and ΔNPM1. Target antigens can include full-length antigens (e.g., any of the exemplary antigens disclosed herein), as well as any type of antigen that may result from cellular processing. Target antigens also include functional variants or fragments of an antigen (e.g., any of the exemplary antigens disclosed herein), including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of the antigen (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type antigen). In some embodiments, the target antigen is a functional fragment of a full-length antigen.

[0120] In some embodiments of the methods and uses described herein, the CD7 CAR-modified iNKT cells are formulated and / or used as a pharmaceutical composition. Thus, in certain aspects, the present disclosure provides a pharmaceutical composition comprising CD7 CAR-modified iNKT cells. One exemplary embodiment is directed to, for example, administering to a subject in need of treatment or prevention of CD7 CAR-modified iNKT cells. + and administering to a patient a CAR-modified iNKT cell (e.g., a CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the pharmaceutical composition further comprises at least one pharma- ceutical carrier. The pharmaceutical composition may also include one or more additional therapeutic agents suitable for treating or preventing cancer (e.g., anti-cancer agents, standard of care agents for the particular cancer being treated, etc.). The pharmaceutical composition may also include one or more inert carriers, excipients, and / or stabilizer components, etc. Methods for formulating pharmaceutical compositions and suitable formulations (e.g., for intravenous, systemic, or other modes of administration) are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.). Suitable formulations may depend on the route of administration.

[0121] As used herein, a "pharmaceutical composition" refers to a composition that contains an NKT cell or cell population (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The pharmaceutical composition provided herein is in a form that allows for administration and subsequently provides the intended biological activity of the active ingredient and / or achieves a therapeutic effect. The pharmaceutical composition provided herein does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation will be administered.

[0122] As used herein, the expressions "pharmacologically acceptable carrier" and "physiologically acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the subject and does not abolish the biological activity and properties of the administered iNKT cells or cell population, or any additional therapeutic agent in the composition. A pharmaceutically acceptable carrier may enhance or stabilize the composition and / or may be used to facilitate preparation of the composition. A pharmaceutically acceptable carrier may include physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like. A carrier may be selected to minimize adverse side effects in the subject and / or to minimize degradation of the active ingredient. Adjuvants may also be included in any of these formulations.

[0123] The term "excipient" as used herein refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Formulations for parenteral administration may contain, for example, excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated naphthalenes. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants, including, for example, polysorbate 20. Certain components included in the pharmaceutical compositions of the present disclosure may be considered as pharma-ceutically acceptable carriers or excipients.

[0124] The pharmaceutical compositions of the present disclosure may be administered by various methods known in the art. The route and / or mode of administration may vary depending on the desired results. In some embodiments, administration is intratumoral, intraventricular, intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, spinal, or epidermal. In some embodiments, the pharmaceutically acceptable carrier is suitable for intratumoral, intraventricular, intravenous, intramuscular, intraperitoneal, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion).

[0125] CD7 CAR-modified iNKT cells or cell populations (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The iNKT cells or cell populations) may be administered alone or in combination with at least one additional therapeutic agent (e.g., an anti-cancer agent, a standard of care agent for the particular cancer being treated, etc.) and may be administered in any acceptable formulation, dose, or dosing regimen. When administered in combination with an additional therapeutic agent, the additional therapeutic agent may be administered according to its standard dose and / or dosing regimen. Alternatively, the additional therapeutic agent may be administered in a greater or lesser amount, and / or more or less frequently than its standard dose and / or dosing regimen. In some embodiments, the additional therapeutic agent is administered in a smaller amount and / or less frequently.

[0126] As used herein, the term "agent" refers to a compound, a mixture of compounds, a biological polymer, or an extract from biological material. The term "therapeutic agent" refers to an agent that can regulate a biological process and / or has biological activity. The CD7 CAR-modified iNKT cells and cell populations described herein are exemplary therapeutic agents. Additional therapeutic agents (e.g., those that can be administered in combination with the CD7 CAR-modified iNKT cells or cell populations described herein) can include any active ingredient appropriate for the particular indication (e.g., cancer) being treated, e.g., that has complementary activities and does not adversely affect each other.

[0127] Typically, a therapeutically effective dose of the CD7 CAR-modified iNKT cells or cell populations is utilized in the pharmaceutical compositions of the present disclosure. The CD7 CAR-modified iNKT cells or cell populations can be formulated into a pharma- ceutically acceptable dosage form by conventional methods known in the art.

[0128] The dosing regimen of the CD7 CAR modified iNKT cells or cell population alone or in combination with at least one additional therapeutic agent can be adjusted to provide the desired optimal response (e.g., therapeutic response). For example, a single bolus of one or both agents can be administered at once, several divided doses can be administered over a period of time, or the dose of one or both agents can be proportionally lowered or increased as dictated by the requirements of the therapeutic situation. For any particular subject, a specific dosing regimen can be adjusted over time according to the individual need and the professional judgment of the treating clinician. Parenteral compositions can be formulated in unitary dosage form for ease of administration and uniformity of dosage. Unitary dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect together with the required pharma-ceutically acceptable carrier.

[0129] The dose value of the composition comprising the CD7 CAR modified iNKT cells or cell population and / or any additional therapeutic agent may be selected based on the unique characteristics of the active agent and the particular therapeutic effect to be achieved. A physician or veterinarian may start the dose of the CD7 CAR modified iNKT cells or cell population utilized in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, the effective dose of the composition of the present disclosure for the treatment of cancer may vary depending on many different factors, including the means of administration, the target site, the physiological condition of the subject, whether the subject is human or animal, other medications, and whether the treatment is prophylactic or therapeutic. The dose level selected may also depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure utilized, the route of administration, the time of administration, the excretion rate of the particular compound utilized, the duration of treatment, other drugs, compounds and / or substances utilized in combination with the particular composition utilized, the age, sex, weight, condition, general health and medical history of the subject to be treated, and similar factors. Treatment doses may be titrated to optimize safety and efficacy.

[0130] As used herein, the terms "therapeutically effective dose" and "therapeutically effective amount" are used to refer to an amount sufficient to measurably reduce at least one symptom or measurable parameter associated with a medical condition or infirmity, to normalize bodily function in a disease or disorder resulting in impairment of a particular bodily function, or to cause improvement or slow the progression of one or more parameters of a disease as measured clinically. A therapeutically effective amount may be sufficient, for example, to treat, prevent, reduce the severity, delay the onset, and / or reduce the risk of development of one or more symptoms of cancer. The therapeutically effective amount, as well as the therapeutically effective frequency of administration, may be determined by methods known in the art and discussed herein. In some embodiments of the compositions and methods described herein, the CD7 CAR-modified iNKT cells or cell populations are administered in an amount that is therapeutically effective when administered as a single agent. In some other embodiments, the CD7 CAR-modified iNKT cells or cell populations and the at least one additional therapeutic agent are each administered in an amount that is therapeutically effective when the agents are administered in combination. In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is the amount required to kill a cancer cell population or a portion thereof in a subject. In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is the amount required to reduce or slow the growth of a cancer cell population in a subject. In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is the amount required to reduce or slow the growth of a tumor in a subject.

[0131] In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is about 1×10 7 From 5 to 10 9 In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is about 1×10 7 pieces, approximately 2×10 7 pieces, about 3 x 10 7 pieces, about 4×10 7 pieces, about 5×10 7 pieces, about 6×107 pieces, about 7×10 7 pieces, about 8×10 7 Pieces, or about 9 x 10 7 In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is about 1×10 8 pieces, approximately 2×10 8 pieces, about 3 x 10 8 pieces, about 4×10 8 pieces, about 5×10 8 pieces, about 6×10 8 pieces, about 7×10 8 pieces, about 8×10 8 Pieces, or about 9 x 10 8 In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is about 1×10 9 pieces, approximately 2×10 9 pieces, about 3 x 10 9 pieces, about 4×10 9 Pieces, or about 5 x 10 9 In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is about 1×10 7 In some embodiments, a therapeutically effective amount of a CD7 CAR-modified iNKT cell or cell population is less than about 5×10 9 In some embodiments, the cells, cell populations, or pharmaceutical compositions are administered to the subject one time only. In some embodiments, the cells, cell populations, or pharmaceutical compositions are administered to the subject multiple times (e.g., hourly, daily, weekly, biweekly, monthly, or yearly).

[0132] A therapeutically effective dose of the CD7 CAR-modified iNKT cells or cell populations described herein generally provides therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the CD7 CAR-modified iNKT cells or cell populations can be determined by standard pharmaceutical methods, e.g., in cell culture or animal models. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some embodiments, the CD7 CAR-modified iNKT cells or cell populations exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. In some embodiments, the dosage falls within a range of circulating concentrations that includes the ED50 with minimal or no toxicity. The dosage can vary within this range depending on various factors, such as the dosage form utilized, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration, and dosage can be chosen by the attending physician in view of the subject's condition.

[0133] In some embodiments, the CD7 CAR modified iNKT cells, cell populations, or pharmaceutical compositions are administered one time only. In some embodiments, the CD7 CAR modified iNKT cells, cell populations, or pharmaceutical compositions are administered multiple times. The interval between single doses can be, for example, hourly, daily, weekly, biweekly, monthly, or yearly. To maintain a relatively consistent concentration of the agent, the interval can also be irregular, based on the measured level of the administered agent (e.g., CD7 CAR modified iNKT cells or cell populations) in the subject. The dose and frequency of administration of the CD7 CAR modified iNKT cells or cell populations can also vary depending on whether the treatment is preventative or therapeutic. In preventative applications, relatively low doses can be administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the disease is reduced or terminated and / or the subject shows partial or complete improvement of one or more symptoms of the disease. Thereafter, the subject may be administered a lower, e.g., prophylactic, dosage regimen.

[0134] In some embodiments, kits and articles of manufacture for use in the therapeutic and prophylactic applications described herein are also provided. In some embodiments, the present disclosure provides kits or articles of manufacture comprising CD7 CAR-modified iNKT cells or cell populations. In some embodiments, the kits or articles of manufacture further comprise one or more additional components, including, but not limited to, instructions for use; other reagents, such as therapeutic agents (e.g., anti-cancer agents); devices, containers, or other materials for preparing the CD7 CAR-modified iNKT cells or cell populations for administration; pharma-ceutically acceptable carriers; and devices, containers, or other materials for administering the CD7 CAR-modified iNKT cells or cell populations to a subject. The instructions for use may include, for example, guidance for therapeutic applications, including suggested doses and / or modes of administration in subjects having or suspected of having cancer. In some embodiments, the kits comprise CD7 CAR-modified iNKT cells or cell populations, and instructions for using the CD7 CAR-modified iNKT cells or cell populations in the treatment and / or prevention of cancer.

[0135] [Example] The following examples provide illustrative embodiments of the present disclosure. The examples provided are not intended to limit the present disclosure in any way.

[0136] Materials and Methods Cell culture: K562 (chronic myeloid leukemia, CML), Daudi (B-cell Burkitt lymphoma), Raji (B-cell Burkitt lymphoma), Nalm-6 (B-cell precursor leukemia), EBV-B (Epstein-Barr virus transformed B-cell line), HSB2 (T-cell acute lymphoblastoid leukemia, T-ALL), Jurkat (T-ALL), MOLT13 (T-ALL), KG1 (acute myeloid leukemia, AML), Kasumi-1 (AML), Kasumi-6 (AML), Molm13 (AML), M V4:11 (AML), U937 (AML), SaOS2 (osteosarcoma), Rh30 (alveolar rhabdomyosarcoma), and TC71 (Ewing's sarcoma) cell lines and their luciferase-expressing counterparts were maintained in RPMI 1640 medium (Corning, Catalog No. 10-040-CM) supplemented with 10% heat inactivated fetal bovine serum (FBS, Corning, Catalog No. 35-015-CV), 2 mM L-glutamine (Corning, Catalog No. 25-005-CI), 50 U / mL penicillin, and 50 μg / mL streptomycin (Corning, Catalog No. 30-002-CI). Kasumi-1 (AML) and Kasumi-6 (AML) were maintained in RPMI 1640 medium supplemented with 20% heat-inactivated FBS, 2 mM L-glutamine, 50 U / mL penicillin, 50 μg / mL streptomycin, and 2–20 ng / mL human granulocyte-macrophage colony-stimulating factor (GM-CSF, Leukine, Partner Therapeutics). BE(2)C (neuroblastoma) and SKNFI (neuroblastoma) were maintained in a 1:1 mixture of Eagle's Minimum Essential Medium (ATCC, Catalog No. 30-2003) and F12 medium (ATCC, Catalog No. 30-2006) supplemented with 10% FBS (Corning, Catalog No. 35-015-CV) and Dulbecco's Modified Eagle's Medium (DMEM) (Corning, Catalog No. 10-013-CM) supplemented with 10% FBS, 0.1 mM non-essential amino acids (Corning, Catalog No. 45000-700).

[0137] K562, Daudi, Raji, Nalm-6, BE(2)C, and SKNFI were purchased from ATCC. EBV-transformed B (EBV-B) cell lines were provided by Ludwig Cancer Research Branch (Belgium). HSB2, Jurkat, MOLT13, KG-1, Kasumi-1, Kasumi-6, Molm-13, MV4:11, U937, Rh30, and SaOs2 were provided by the University of Minnesota. TC71 was provided by the University of Utah. Validation of HSB2, Jurkat, MOLT13, and KG1 expressing luciferase and NGFR (hfflucN) for 10 short tandem repeat (STR) loci plus the sex-determining locus amelogenin was performed by the Oregon Health Science University Integrated Genomics Laboratory.

[0138] Peripheral blood mononuclear cell isolation, CAR-T production, and culture: Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from whole blood units purchased from the New York Blood Center using Ficoll Paque Plus (Cytiva, Cat. No. GE17-1440-03). PBMCs were thawed and cultured at 1 × 10 per well in a 24-well plate. 6Cells were cultured in human T cell medium consisting of RPMI 1640 (Corning, Cat. No. 10-040-CM), 10% FBS (Corning, Cat. No. 35-015-CV), 2 mM L-glutamine (Corning, Cat. No. 25-005-CI), 50 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich, Cat. No. 60-24-2), 100 IU / mL penicillin, and 100 μg / mL streptomycin (Corning, Cat. No. 30-002-CI) with the addition of Dynabeads human T-Activator CD3 / CD28 (Invitrogen, Cat. No. 11131D). On day 3, cultures were transduced with CD19, ROR1, CD7 CAR lentivirus (multiplicity of infection (MOI) = 9–20) on RetroNectin (Takara Bio, Cat. No. T100B)-coated 24-well plates by centrifugation at 1,902 g (3000 rpm) at 32 °C for 2 h and incubated at 37 °C for 48 h. After removal of CD3 / CD28 beads, transduced or non-transduced mock T cells were cultured in human T cell medium supplemented with human IL-2 (50 IU / mL, Proleukin, Novartis Pharmaceuticals), IL-7 (10 ng / mL, Peprotech, Cat. No. 200-07), and IL-15 (10 ng / mL, Peprotech, Cat. No. 200-15). On days 11 and 16, cell numbers were determined on a hemocytometer by trypan blue staining.

[0139] Isolation, CAR transduction, and expansion of iNKT cells: Use anti-iNKT microbeads (Miltenyi Biotec, catalog no. 130-094-842) to positively select human cells expressing TCR Vα24-Jα18 to culture iNKT cells (1–2 × 10 per well). 5 ) were isolated from fresh PBMCs and then transfected with irradiated (40 Gy) autologous PBMC negative fraction (3 × 10 per well) in 48-well plates after iNKT isolation. 6iNKT cell cultures were cultured in 1 mL of human T cell medium supplemented with α-GalCer (200 ng / mL, DiagnoCine, Cat. No. KRN7000; FlycoFineChem, Cat. No. FC-070) or 7DW8-5 glycolipid analog (200 ng / mL, kindly provided by Dr. Moriya Tsuji, Columbia University; DiagnoCine, Cat. No. 7DW8-5), 250 IU / mL IL-2 (Proleukin, Novartis Pharmaceuticals), and 10 ng / mL IL-15 (Peprotech, Cat. No. 200-15) together with 1,000 μg of NKT cells. On day 3 or 4, iNKT cell cultures were transduced with CAR lentivirus (MOI=20-200) on RetroNectin (Takara Bio, Cat. No. T100B)-coated 24-well plates by centrifugation at 1,902 g (3000 rpm) at 32°C for 2 h. Transduced iNKT cells were transferred to 48-well plates after 48 hours, supplemented with human T cell medium supplemented with IL-2 and IL-15, and split into new wells when confluent. At 2–3 weeks, iNKT cells were expanded initially in 24-well plates and then transferred to T75 flasks. Briefly, iNKT cells (5 × 10 per well) were cultured in 24-well plates and then split into new wells when confluent. 5 PBMCs (3.5 × 10 per well) were mixed with irradiated autologous PBMC negative fraction and / or total PBMCs (referred to as autologous), or mixed PBMCs from 3–5 allogeneic donors (3.5 × 10 per well). 6 cells, 40 Gy) and irradiated EBV-B cells (5 × 10 per well 5The mice were co-cultured with 100- and 80-Gy (referred to as allogeneic) in human T cell medium supplemented with α-GalCer or 7DW8-5 (100–200 ng / mL), IL-2 (250 IU / mL), and IL-15 (10 ng / mL) at 2 mL per well of a 24-well plate. iNKT cells were expanded in T25 flasks with 5 × 10 iNKT cells per flask in 25 mL of human T cell medium supplemented with either α-GalCer or 7DW8-5 (100–200 ng / mL), IL-2 (250 IU / mL), and IL-15 (10 ng / mL), or with anti-human CD3 monoclonal antibody (OKT3, 30 ng / mL, Miltenyi Biotec, Cat. No. 130-093-387), IL-2 (50 IU / mL), IL-7 (10 ng / mL), and IL-15 (10 ng / mL). 5 ), irradiated mixed PBMCs from 3–5 allogeneic donors (3 × 10 per flask) 7 cells, 40 Gy), and irradiated EBV-B cells (3 × 10 per flask 6 The expanded iNKT cells were cultured at 8 × 10 per vial. 7 Cells were stored frozen in 1 mL of 90% FBS and 10% dimethyl sulfoxide (DMSO, MPBio, SKU# 021960559). αGalCer or 7DW8-5 were dissolved in DMSO at 1 mg / mL or 4 mg / mL, respectively, and stored at -20°C after heating in a beaker of water at 60-80°C for 10 min, vortexing for 30 s, and aliquoting at 10 μl or 2 μl per tube. For use, αGalCer (10 μl per tube) or 7DW8-5 (2 μl per tube) were heated in a beaker of water at 60-80°C for 10 min, vortexed for 30 s, and diluted to 100 μg / mL in T cell medium.

[0140] Lentiviral production and tumor cell transduction: An HIV-1-based bidirectional vector expressing humanized firefly luciferase and NGFR (hfflucN) was constructed as previously described (Huang et al., Mol Ther. 2008;16(3):580-9; Huang et al., PLoS ONE. 2015;10(7):e0133152). Lentiviral vectors expressing CD7 CAR, CD19 CAR, and ROR1, containing the single chain variable regions (scFv) of anti-CD7 (SEQ ID NO: 36), anti-CD19 (SEQ ID NO: 37), and anti-ROR1 (SEQ ID NO: 38) antibodies, the CD8α hinge and transmembrane regions, and the intracellular domains of 4-1BB and the CD3 zeta chain, were constructed based on previously described lentiviral vectors (Milone et al., Mol Ther. 2009;17(8):1453-64; Tammana et al., Hum Gene Ther. 2010;21:75-86; Baskar et al., mAbs. 2012;4(3):349-361; Huang et al. PLoS ONE. 2015;10(7):e0133152). CD7 CARs were generated using a commercially available gene synthesis of the anti-CD7 single chain variable fragment (scFv) sequence found in patent WO 2003051926 (Cooper et al., Leuk. 2018;32(9):1970-1983). Lentiviral supernatants were harvested 48h and 96h after transfection into the Lenti-X 293T cell line (Takara Bio, Catalog No. 632180) with four plasmids (pLVCARsin, pMDLg / pRRE, PRSV-REV, pMD2.G) and Lipofectamine 2000 (ThermoFisher, Catalog No. 11668019) and concentrated using a Lenti-X Concentrator (Takara Bio, Catalog No. 631231). Viral titers were determined in the Lenti-X 293T cell line and were 2.3×10 7 ~2×10 8The results ranged from 1000 to 10000 transducing units / mL. Leukemia cell lines were spin transduced with hfflucN lentivirus in the presence of polybrene (8 μg / mL) at 1170 g and 32°C for 1 h and then sorted by FACS or enriched with biotin anti-human CD271 (NGFR, BD Biosciences, Cat. No. 557195) and anti-biotin microbeads (Miltenyi Biotec, Cat. No. 130-090-485) or CD271 microbeads for NGFR expression (Miltenyi Biotec, Cat. No. 130-099-023). All transduced cell lines were verified for hffluc bioluminescence activity by flow cytometric analysis of NGFR expression using a Synergy 2 microplate reader (BioTek).

[0141] Luciferase-based killing assay: target cells (2 × 10 5 αGalCer- or DMSO-pulsed target cells were prepared by incubating 100 cells / mL with αGalCer (200 ng / mL) or DMSO (1:500 of 10% stock) overnight at 37°C, 5% CO2, and used for the cytotoxicity assay after spinning to remove the medium containing αGalCer or DMSO. Luciferase-expressing target cells (1 × 10 cells / mL in 50 μL per well) were plated in 96-well flat-bottom white polystyrene microplates (Corning, Cat. No. 3912). 4) were incubated in quadruplicates at different effector T cell:target cell (E / T) ratios with 50 μL of iNKT cells per well. Spontaneous or maximal killing was set up by adding 50 μL of culture medium or 1% Triton X-100 per well, respectively, in place of iNKT cells. After standard incubation at 37°C for 4 h or 16 h (as indicated), 10 μL of D-luciferin (1:10 of 30 mg / mL stock) or 50 μL (1:50 of 30 mg / mL) (Caliper Life Sciences, Cat. No. XR-1001) was added to each well. Luciferase activity was measured using a Synergy 2 microplate reader (BioTek). Percent specific lysis was calculated as follows: Specific lysis (%) = (Spontaneous mortality RLU - Sample RLU) / (Spontaneous mortality RLU - Maximum mortality RLU) x 100. RLU = Relative Luminescence Units

[0142] Antibodies and flow cytometry: FITC anti-human TCR Vα24-Jα18 (clone 6B11, catalog number 342906, recognizing the invariant CDR3 region of iNKT cells, TCR Vα24-Jα18 and TCR Vα24-JQ), PE anti-human CD1d (clone 51.1, catalog number 350306), FITC anti-human PD1 (clone EH12.2H7, catalog number 329904), APC anti-human CD3 (clone UCHT1, catalog number 300439), PE anti-His tag (clone J095G46, catalog number 362603), PE streptavidin (clone 100610 ... Putavidin (cat. no. 405204), PE mouse IgG1, kappa isotype control (clone MOPC-21, cat. no. 981804), APC mouse IgG1, kappa isotype control (clone MOPC-21, cat. no. 981806), PE mouse IgG2a, kappa isotype control (clone MOPC-173, cat. no. 400246), and PE mouse IgG2b, kappa isotype control (clone MPC-11, cat. no. 400314) were purchased from BioLegend.PE anti-human CD2 (clone RPA-2.10, catalog number 555327), APC anti-human CD3 (clone UCHT1, catalog number 555335), PE anti-human CD5 (clone UCHT2, catalog number 555353), PE anti-human CD4 (clone PRA-T4, catalog number 555347), APC anti-human CD4 (clone PRA-T4, catalog number 555349), PE anti-human CD8 (clone HIT8a, catalog number 555635), APC anti-human CD8 (clone RPA-T8, catalog number 555369), V450 anti-human CD Antibody IgG1, Kappa isotype control (clone MOPC-21, catalog no. 555749), APC mouse IgG1, Kappa isotype control (clone MOPC-21, catalog no. 555751), and V450 mouse IgG1, Kappa isotype control (clone MOPC-21, catalog no. 560373) were purchased from BD Biosciences. Biotinylated human CD19 protein (Cat. No. 11880-H08H-B), biotinylated human ROR1 protein (Cat. No. 13968-HCCH1-B), and human CD7 protein (His tag, Cat. No. 11028-H08H) were purchased from SinoBiological Co., Ltd. Surface CAR expression was detected by biotinylated human CD19 protein or biotinylated human ROR1 and PE streptavidin, or human CD7 protein His tag and PE anti-His tag, or Alexa Fluor 647 (AF-647)-conjugated F(ab')2 fragment of goat anti-mouse IgG F(ab')2 (anti-CAR, Jackson ImmunoResearch, Cat. No. 115-606-006) and Alexa Fluor 647-conjugated F(ab')2 fragment of ChromPure goat IgG isotype control (Cat. No. 015-600-006). Flow cytometric analysis was performed using a BD FACSCelesta or BD Accuri C6 cytometer.Data was analyzed with FlowJo software 7.2.2 or V10.

[0143] Cytokine release assay: Cytokine release assays were performed in duplicate in 96-well flat-bottom plates, with 1–2 × 10 cells per well. 5 2 x 10 T cells 4 After 24 h, supernatants were assayed using the LEGEND MAX Human IFN-γ ELISA Kit (BioLegend, Cat. No. 430107).

[0144] In vivo antitumor assay: NOD.Cg-Prkdc scid IL2rg tm1WjI / SzJ NSG (stock no. 005557) mice were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free facility at New York Medical College. To establish xenograft models of T-ALL and AML, 6- to 7-week-old NSG mice were inoculated with 5 × 10 5 HSB2-hfflucN or KG1-hfflucN cells were inoculated intravenously (iv) on day -2. Mice were examined for human leukemia engraftment by bioluminescence imaging (BLI) using a Perkin Xenogen IVIS Spectral Imaging System on day 0. Mice bearing T-ALL or AML were randomly divided into 3 or 2 groups (n=5 per group) based on imaging intensity. On days 0, 3, and 6, mice bearing HSB2-hfflucN T-ALL were inoculated with PBS, thawed mock iNKT55 cells, and thawed CD7 CAR-iNKT55 cells (5×10 per mouse). 6Mice bearing KG1-hfflucN AML were injected iv with thawed mock iNKT55 and CD7 CAR-iNKT55 on days 0 and 3. Mock iNKT55 and CD7 CAR-iNKT55 cells were thawed from a liquid nitrogen dewar, washed twice with human T cell medium and PBS, and resuspended in PBS. Mice were also injected intraperitoneally (ip) with a cytokine mixture (2,000 IU / mouse IL-2, 2 μg / mouse IL-15) or PBS every 3 days for 2 weeks (Tian et al., J Clin Invest. 2016;126(6):2341-55; Xu et al., Clin Cancer Res. 2019;25(23):7126-38). Tumor BLI was performed on days 3, 6, 10, 17, and 24 for mice with T-ALL, or on days 0, 3, 6, 10, 17, 24, 31, 38, and 45 for mice with AML after the first T cell infusion. BLI was performed under isoflurane anesthesia after intraperitoneal injection of D-luciferin (Caliper Life Sciences, Cat. No. XR-1001). Images were collected and analyzed using an IVIS imaging system and Living Image 4.7.3 software (PerkinElmer). Regions of interest (ROIs) were drawn over the entire mouse body, and the intensity of the signal, measured as total photon flux, was normalized to exposure time and surface area and expressed as photons / sec / cm. 2 / steradian (p / sec / cm 2 The results were expressed in units of sr. The survival of the mice was then monitored without any imaging procedures.

[0145] Statistical analysis: Data were calculated as mean ± standard deviation (SD) and analyzed using one-way ANOVA followed by Tukey's Honestly Significant Difference (Tukey's HSD) post-hoc test for pairwise comparisons. Statistical significance in Kaplan-Meier survival curves was assessed by the Mantel-Cox log-rank test. All p values ​​were calculated using Prism 7 software (GraphPad).

[0146] result Figure 1 shows that CD1d was expressed in most AML cell lines, including HL60, KG1, Molm13, MV4:11, THP-1, and U937, but not in Mo7e AML cell line. CD1d expression in KG1 was weakly positive. CD1d expression in B cell tumor cell lines (Daudi, Raji, Nalm6) and CML cell lines (K562, K562CD19) was negative. All luciferase-transduced tumor cell lines expressed high levels (>90%) of NGFR, indicating that all could express luciferase.

[0147] Figure 2A shows that iNKT1 (autologous) cells grown with irradiated autologous PBMC negative fraction and / or whole PBMC and αGalCer, which is a common method for iNKT cell expansion, show that iNKT1 (autologous) cells grown with irradiated allogeneic PBMC and EBV-B cells and αGalCer antigen upregulate αGalCer-sensitized CD1d + (Molm13, THP-1, U937) but not CD1d - (Daudi, Nalm6) target cells were not. Control DMSO-sensitized CD1d + and CD1d - Target cells were not lysed by iNKT1 (autologous) and iNKT1 (allogeneic).

[0148] Figure 2B shows that iNKT2 (autologous) cells grown with irradiated autologous PBMC negative fraction and / or total PBMC and αGalCer suppressed the expression of αGalCer-sensitized CD1d + (KG1, Molm13, MV4:11, THP-1) were specifically lysed, whereas CD1d - (Daudi, Nalm6) target cells were not. These results based on Figure 2A and Figure 2B suggest that iNKT cells grown with irradiated allogeneic PBMCs and EBV-B cells and αGalCer antigen remain αGalCer-CD1d specific compared to iNKT cells grown with irradiated autologous PBMC negative fraction and / or total PBMCs and αGalCer.

[0149] FIG. 3A shows that irradiated allogeneic PBMCs and EBV-B cells, and iNKT1 (OKT3) cells grown with a soluble anti-CD3 monoclonal antibody (OKT3), surprisingly, upregulated CD1d αGalCer-independently. + As controls, irradiated allogeneic PBMC and EBV-B cells, and iNKT1(αGalCer) cells grown with αGalCer antigen recognized αGalCer-treated CD1d + Target cells were recognized but not DMSO-treated CD1d + Both iNKT1(OKT3) and iNKT1(αGalCer) expressed CD1d IgG, regardless of αGalCer or DMSO treatment. - It did not recognize K562 cells.

[0150] FIG. 3B shows that irradiated allogeneic PBMCs and EBV-B cells, as well as iNKT2 (OKT3) cells grown with a soluble anti-CD3 monoclonal antibody (OKT3), upregulated CD1d IL-16 expression in a manner independent of αGalCer. +As controls, irradiated allogeneic PBMC and EBV-B cells, and iNKT2(αGalCer) cells grown with αGalCer antigen, recognized αGalCer-treated CD1d + Target cells were recognized but not DMSO-treated CD1d + Both iNKT2(OKT3) and iNKT2(αGalCer) expressed CD1d IgG1-dependent NKT3 ... - (K562, Daudi) cells. The slight recognition of DMSO-treated KG1 cells by iNKT2 (OKT3) may possibly be due to the low level expression of CD1d in KG1 cells (FIG. 1).

[0151] FIG. 3C shows that irradiated allogeneic PBMCs and EBV-B cells, as well as iNKT12 (OKT3) cells grown with a soluble anti-CD3 monoclonal antibody (OKT3), upregulated CD1d IL-16 expression independent of αGalCer. + As controls, irradiated allogeneic PBMC and EBV-B cells, and iNKT12 (αGalCer), iNKT2 (αGalCer), and iNKT1 (αGalCer) cells grown with αGalCer antigen recognized αGalCer-treated CD1d + Target cells were recognized but not DMSO-treated CD1d + iNKT12(OKT3), but not iNKT12(αGalCer) and iNKT2(αGalCer), did not recognize CD1d cells, regardless of αGalCer or DMSO treatment. - (Daudi, Raji) also demonstrated low background recognition of cells.

[0152] FIG. 4A shows that iNKT cells from four donors (iNKT1, iNKT2, iNKT11, iNKT12) expanded with irradiated allogeneic PBMCs and EBV-B cells, αGalCer, and mixed cytokines (IL-2, IL-7, and IL-15) upregulated αGalCer-treated CD1d +(THP1, U937) target cells, whereas DMSO treatment does not.

[0153] Figure 4B shows that iNKT1 and iNKT2 cells expanded with OKT3 or αGalCer were >92% CD3 + iTCR + and + iTCR + All these results, based on iNKT cells from three donors (iNKT1, iNKT2, iNKT12) expanded with OKT3 and iNKT cells from four donors (iNKT1, iNKT2, iNKT11, iNKT12) expanded with αGalCer, suggest that OKT3 expansion may alter the antigen specificity of iNKT cells. αGalCer stimulation of iNKT cells is crucial for maintaining their antigen specificity.

[0154] Next, we examined the function, phenotype, and proliferation rate of iNKT cells grown with irradiated allogeneic PBMCs and EBV-B cells, and αGalCer antigen, supplemented with IL-2 and IL-15 in both cases (referred to as iNKT(allogeneic)), and compared them to iNKT cells grown with irradiated autologous PBMC negative fraction and / or total PBMCs, and αGalCer (referred to as iNKT(autologous)). Figure 5A shows that iNKT45(autologous), iNKT45(allogeneic), iNKT46(autologous), and iNKT46(allogeneic), isolated from healthy blood donors 45 and 46 and generated using autologous or allogeneic expansion protocols, increased the function, phenotype, and proliferation rate of iNKT cells grown with αGalCer-treated CD1d + (MV4:11, U937) target cells, but not to DMSO treatment. Furthermore, these iNKT45 and iNKT46 cells were not CD1d-specific, regardless of DMSO or αGalCer. - (Nalm6) target cells were recognized indiscriminately.

[0155] FIG. 5B shows that after primary stimulation with irradiated autologous PBMC negative fraction and αGalCer, iNKT45 and iNKT46 cells expressed more than 96% CD3 + iTCR+ As shown in Figure 5C, after secondary stimulation or expansion with irradiated autologous PBMC negative fraction and / or total PBMC and αGalCer, or irradiated allogeneic PBMC and EBV-B cells and αGalCer, over 94% of the CD3 + iTCR + Homogeneous populations of cells were demonstrated in iNKT45 and iNKT46 cells grown in autologous or allogeneic conditions.

[0156] To demonstrate that irradiated allogeneic PBMCs and EBV-B cells, and iNKT cells expanded with αGalCer can be used to produce CAR-modified iNKT cells, iNKT47 and iNKT48 cells isolated from donors 47 and 48 were transduced via lentivirus to express CD19 CAR or CD19 CAR / GFP and expanded in autologous or allogeneic conditions. Figure 6 shows that CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47 and iNKT48 cells expanded in autologous or allogeneic conditions express CD19 CAR in an E / T ratio-dependent manner. + Specifically killed B-cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji), but not CD19 - CML (K562) cells did not. As expected, mock iNKT47 and iNKT48 cells expressed CD19 + Furthermore, CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47 and iNKT48 cells grown in autologous or allogeneic conditions were unable to kill B-cell leukemia and lymphoma cell lines. + (Moml13, U937) target cells were reactive to DMSO treatment but not to DMSO treatment. CD1d treated with DMSO or αGalCer - Mo7e cells were not recognized by either iNKT47 or iNKT48.

[0157] FIG. 7 shows that CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47 and iNKT48 cells grown in autologous or allogeneic conditions express CD19- It does not respond to CML (K562) and AML (Mo7e) cells, but it responds to CD19 + We show that αGalCer-treated CD1d specifically produced higher amounts of IFN-γ than their mock counterparts in various B cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji). + THP1 cells significantly enhanced IFN-γ production by all iNKT47 and iNKT48 cells compared to DMSO-treated THP1 cells, and CD19 CAR-modified iNKT cells expanded in allogeneic conditions were cytotoxic and CD19 + We show that IFN-γ is produced in response to tumor cells while remaining specific for αGalCer.

[0158] FIG. 8 shows that over 93% of CD19 CAR-modified iNKT47 and iNKT48 cells expanded in autologous or allogeneic conditions expressed CD3 + iTCR + We show that iNKT cells express a population of iNKT cells and that iNKT cells grown in allogeneic conditions have some of these phenotypes.

[0159] FIG. 9 shows that CD 19 CAR or CD19 CAR / GFP expression was confirmed in iNKT47 and iNKT48 cells grown in autologous or allogeneic conditions, indicating that autologous or allogeneic culture conditions did not affect the levels of CAR expression.

[0160] FIG. 10 shows that CD19 CAR-modified or CD19 CAR / GFP-modified iNKT50 cells isolated from donor 50 and grown in autologous or allogeneic conditions express CD19 CAR-modified iNKT50 cells in an E / T ratio-dependent manner. + Specifically killed B-cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji), as well as CD19-transfected K562 (K562CD19), but not CD19 - CML (K562) and AML (HL60, KG1, Molm13, THP1) cells did not. As expected, mock cells expressed CD19 +Furthermore, CD19 CAR-modified or CD19 CAR / GFP-modified iNKT50 cells grown in autologous or allogeneic conditions were unable to kill B-cell leukemia and lymphoma cell lines, as well as K562CD19. + (MV4:11, U937) target cells were reactive to DMSO-treated CD1d cells, but not to DMSO-treated CD1d cells. - Mo7e cells were not recognized by any of the iNKT50 cells.

[0161] FIG. 11A shows that over 87% of CD19 CAR-modified iNKT50 cells expanded in autologous or allogeneic conditions expressed CD3 + iTCR + These results indicate that iNKT cells expanded in allogeneic conditions remain phenotypically intact.

[0162] Figure 11B shows that CD 19 CAR or CD19 CAR / GFP expression was confirmed in transduced iNKT50 cells, but not in mock cells grown in autologous or allogeneic culture conditions, indicating that autologous or allogeneic culture conditions did not affect the levels of CAR expression.

[0163] Figure 12 shows the proliferation rates of non-transduced iNKT45 and iNKT46, and CD19 CAR-modified iNKT47, iNKT48, and iNKT50 grown in autologous or allogeneic culture conditions. Approximately 245-fold and 76-fold proliferation was achieved in iNKT45 and iNKT46, respectively, 13 days after primary stimulation with irradiated autologous PBMC negative fraction and αGalCer. At day 26, 10,800-fold and 2,800-fold proliferation was obtained in iNKT45 and iNKT46 grown in allogeneic conditions, compared with 6,080-fold and 2,106-fold proliferation in iNKT45 and iNKT46 cultured in autologous conditions. Allogeneic culture conditions appear to be as effective as autologous culture conditions in iNKT cell proliferation.

[0164] Next, CD19 CAR-modified iNKT cell expansion in iNKT47, iNKT48, and iNKT50 was examined using autologous and allogeneic expansion conditions. As shown in Figure 12, at the end of the culture at 35 days, iNKT47 CD19 CAR (allogeneic) and iNKT47 CD19 CAR / GFP (allogeneic) cells expanded in allogeneic conditions demonstrated 4,160-fold and 4,065-fold expansion, respectively, compared to 2,250-fold and 2,680-fold expansion in iNKT47 CD19 CAR (autologous) and iNKT47 CD19 CAR / GFP (autologous) expanded in autologous conditions. With regard to iNKT48 expansion, at the end of the culture on day 35, 4,417-fold and 3,650-fold expansion was recorded for iNKT48 CD19 CAR (allogeneic) and iNKT48 CD19 CAR / GFP (allogeneic) cells expanded in allogeneic conditions, respectively, compared to 441-fold and 985-fold expansion for iNKT48 CD19 CAR (autologous) and iNKT48 CD19 CAR / GFP (autologous) expanded in autologous conditions.

[0165] Similarly, CD19 CAR-modified iNKT cell expansion was performed with iNKT50 cells. As shown in Figure 12, at 35 days, 18,500-fold, 8,400-fold, and 10,680-fold expansion was recorded for iNKT50mock (autologous), iNKT50 CD19 CAR (autologous), and iNKT50 CD19 CAR / GFP (autologous) cells expanded in allogeneic conditions, respectively, compared to 14,666-fold, 5,200-fold, and 15,353-fold expansion for iNKT50mock (autologous), iNKT50 CD19 CAR (autologous), and iNKT50 CD19 CAR / GFP (autologous) cells expanded in autologous conditions. At the end of the culture, 4.4 x 10 8 iNKT50mock (self), 5.5 x 10 8 iNKT50mock (same type), 3.9 x 10 8 iNKT50 CD19 CAR (autologous), 6.3 × 10 8 iNKT50 CD19 CAR (allogeneic), 1.1 × 10 9 iNKT50 CD19 CAR / GFP (autologous), and 8.0 × 10 8iNKT50 CD19 CAR / GFP (allogeneic) were compared to their starting value of 0.3 × 10 5 pieces (mock) and 0.75×10 5 The CD19 CAR-modified and CD19 CAR / GFP-modified iNKT cells were generated from isolated iNKT50 cells (CAR). Expansion rates of 5,863 (5,863±2,858, n=6)-fold and 4,484 (4,484±5,576, n=6)-fold, respectively, of CD19 CAR-modified and CD19 CAR / GFP-modified iNKT cells in allogeneic or autologous conditions were demonstrated. Allogeneic conditions may be advantageous over autologous conditions for iNKT expansion. For example, allogeneic feeder cells can be pre-produced, banked, and used for any iNKT expansion, whereas autologous feeders are individually produced, potentially simplifying the regulatory process and reducing manufacturing costs and product variability.

[0166] FIG. 13A shows that thawed and cultured iNKT47 CD19 CAR (allogeneic) and iNKT48 CD19 CAR (allogeneic) were able to upregulate CD19 CARs in a 16 h assay, even at a low E / T ratio of 2.2:1. + It can significantly kill B-cell leukemia and lymphoma cells (Daudi, Raji, Nalm6), as well as CD19-transfected K562 (K562CD19), but - CML (K562) cells show that this is not the case, indicating that the freezing and thawing conditions have minimal effect on the cytotoxicity of CAR-modified iNKT cells. Furthermore, Figure 13B shows that thawed and cultured iNKT47 CD19 CAR cells (allogeneic) inhibited the cytotoxicity of 7DW8-5 or αGalCer-treated CD1d + 7DW8-5 killed Molm13 and THP1 target cells in a dose-dependent manner. +7DW8-5 appeared to be at least 16-fold and 64-fold more potent than αGalCer in sensitizing Molm13 and THP1 target cells. Furthermore, FIG. 13C shows that 7DW8-5 was more potent than αGalCer in stimulating 3-month cultured iNKT47 CD19 CAR (allogeneic) and iNKT48 CD19 CAR / GFP (allogeneic) to produce IFN-γ, confirming that 7DW8-5 exhibits a superior effect on iNKT cell activity than αGalCer (Li et al., Proc Natl Acad Sci USA. 2010;107(29):13010-13015). As a control, CD19 CAR-modified iNKT47 cells (allogeneic) and CD19 CAR / GFP-modified iNKT48 cells (allogeneic) did not stimulate CD19 + IFN-γ was produced in response to target cells (K562CD19, Nalm6), but not mock iNKT47 (autologous).

[0167] FIG. 14A shows that iNKT cells from a representative blood donor were >95% monoclonal CD2+ after primary stimulation with irradiated autologous PBMC negative fraction and αGalCer or 7DW8-5, and secondary expansion with irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, IL-2, IL-15, and / or IL-7. + or CD5 + Compared to the population, CD7 + (approximately 32%) and CD7 - We show that the two populations of CD7 expressing T cells (approximately 66%) were unexpectedly found to represent a subset of CD7-positive T cells (approximately 66%). This finding was surprising because it is well documented that the majority of conventional peripheral blood T and NK cells (median 90% and 97%) express CD7 (Gomes-Silva et al., Mol Ther. 2019;27(1):272-280; Kim et al., JCI Insight. 2021;6(16);e149819).

[0168] FIG. 14B shows that after primary stimulation with irradiated autologous PBMC negative fraction and αGalCer or 7DW8-5, and secondary expansion with irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, IL-2, IL-15, and / or IL-7, approximately 56±17.96% and 42.96±17.21% of iNKT cells from nine healthy blood donors (n=9) were CD7 + and CD7 - Again, the majority (>96%) of iNKT cells were CD2 + (n=7) and CD5+ (n=9). Thus, the CD7 CAR-expressing iNKT cells - A subset may be able to partially avoid fratricide without the use of nuclease (e.g., CRISPR)-mediated genome editing to knock out CD7, and may be able to partially avoid fratricide without the use of CD7 + We hypothesized that they could be expanded in our allogeneic culture conditions for adoptive cell therapy of cancer.

[0169] Figure 15 shows that expression of CD7, CD1d and NGFR in lentivirally engineered human T-ALL (HSB2, Jurkat and MOLT13) and AML (KG1) cell lines expressing luciferase and NGFR was confirmed by flow cytometry analysis. Two additional AML cell lines, Kasumi-1 and Kasumi-6, expressed CD7 - and CD7 + , and CD1d - It was.

[0170] Figure 16 shows that CD7 CAR-transduced peripheral blood T cells failed to induce substantial fratricide and proliferation. To confirm that CD7 CAR-transduced T cells induce fratricide and prevent CAR-T cell proliferation, activated human T cells from a blood donor (PBL36) were transduced with lentiviral CD7 CAR constructs. CAR-T targeting CD19 or ROR1, or non-transduced mock T cells were used as controls. At days 11 and 16 after T cell transduction, there were approximately 19-30 and 41-59 fold fewer CD7 CAR-T than CD19 CAR-T or ROR1 CAR-T or mock T cells, respectively, confirming that CD7 antigen-driven fratricide prevents CD7 CAR-T expansion (Gomes-Silva et al., Blood 2017;130(3):285-296; Cooper et al., Leuk. 2018;32(9):1970-1983).

[0171] Figure 17 shows that CD7 CAR-modified iNKT cells can be expanded in culture. On days 22 and 41, CD7 CAR-modified iNKT55 cells (iNKT55 CD7 CAR) gave expansion rates of 46-fold and 6,407-fold, whereas CD19 CAR-modified iNKT55 (iNKT55 CD19 CAR) and iNKT55mock cells recorded expansion rates of 334-732-fold and 25,661-50,492-fold. Using our homogeneous expansion protocol, at the end of culture, 6.5 x 10 9 pcs iNKT55mock, 3.30×10 9 iNKT55 CD19 CAR, and 8.3 x 10 8 iNKT55 CD7 CAR cells were cultured at their starting value of 1.3 × 10 5 iNKT68 cells were generated from 10 iNKT cells. Additionally, CD7 CAR-modified iNKT68 cells from donor 68 could also be expanded in culture. On days 24 and 39, CD7 CAR-modified iNKT68 cells (iNKT68 CD7 CAR) yielded 176-fold and 7,470-fold expansion rates, whereas iNKT68 mock cells recorded 600-fold and 27,120-fold expansion rates. At the end of the culture, 5.4 × 109 pcs iNKT68 mock and 1.5×10 9 iNKT68 CD7 CARs were cultured at their starting value of 2 × 10 5 iNKT cells were generated from 10 iNKT cells. After two rounds of stimulation, it appears that an approximately 6,000-fold expansion of CD7 or CD19 CAR-modified iNKT cells can be achieved. On average, 2.526 ± 1.495 × 10 iNKT cells were generated using anti-iNKT microbeads. 5 iNKT cells (n=27 donors) were 8 Isolated from 10 PBMCs. 8 PBMC or one Leukopack (approximately 5-8 × 10 9 PBMCs), approximately 1.5 × 10 9 pcs or 50 x 10 9 70-90% of CAR + It is anticipated that CD7 CAR-modified iNKT cells with the following characteristics can be generated: 6 CARs + T cells (up to 2 × 10 for patients with large B-cell lymphoma) 8 Based on 175 infusions of CD7 CAR-modified iNKT cells, approximately 175 infusions of CD7 CAR-modified iNKT cells can be achieved from one leukopack.

[0172] FIG. 18 shows a representative cytotoxicity plot, in which CD7 CAR-modified iNKT55 inhibited luciferase-expressing CD7 + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + The AML (KG1) cell line was killed in an E / T ratio-dependent manner, but CD7 -Tumor cell lines did not. CD7 CAR-modified iNKT55 showed low background cytotoxicity against B cell leukemia and lymphoma cell lines (Nalm6, Raji) compared to mock cells. As a control, CD19 CAR-modified iNKT55 showed low background cytotoxicity against CD19 + Specifically lysed malignant B cells (Nalm6, Raji) but not CD19 - T-ALL, AML, sarcoma, and neuroblastoma did not. Non-transduced mock iNKT cells were CD1d + It did not kill any of the target cells tested, including AML (KG1, Molm13, MV4:11, U937), but it did kill CD1d + Jurkat cells were killed, and many human leukemic T cells expressed CD1d and Va24 + This confirms previous work showing that killing can be effected directly by NKT cells (Takahashi et al., Br J Haematol. 2003;122(2):231-239).

[0173] FIG. 19 shows a composite cytotoxicity plot with statistical analysis, showing that CD7 CAR-modified iNKT cells (n=5 donors) inhibited luciferase-expressing CD7 CAR-modified iNKT cells at all E / T ratios (20:1, 6.7:1, and 2.2:1) compared to mock and CD19 CAR-modified iNKT cells. + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + CD7 CAR iNKT cells specifically killed AML (KG1) (p<0.0001). CD7 CAR iNKT cells were significantly more potent than mock iNKT cells (n=5 donors). - They appeared to display low background cytotoxicity against B-cell leukemia and lymphoma cells (Nalm6, Raji) and U937 (at E / T ratios of 6.7:1 and 2.2:1, but not 20:1) (p<0.05). As a control, CD19 CAR-modified iNKT cells (n=3 donors) were significantly more potent than CD19 + Specific killing of B-cell tumor cells, but CD19 - T-ALL and AML cells were not (p<0.0001).+ It showed minimal cytotoxicity against all target cells except Jurkat T-ALL cells.

[0174] CD7 CAR-modified iNKT cells further + To demonstrate IFN-γ secretion in response to tumor cell stimulation, co-culture supernatants were tested for IFN-γ levels by ELISA. Figure 20A shows that CD7 CAR-modified iNKT55 cells from one representative donor secreted IFN-γ in response to tumor cell stimulation. + T-ALL (HSB2, MOLT13) and CD7 + In co-culture with AML (KG1, Kasumi-6), CD7 CAR-modified iNKT55 cells secreted at least 4,000-fold more IFN-γ than CD19 CAR iNKT and mock iNKT cells against Jurkat T-ALL. As expected, CD19 CAR-modified iNKT cells secreted 1,000-fold more IFN-γ than CD19 CAR iNKT and mock iNKT cells against Jurkat T-ALL. + In co-culture with leukemia and lymphoma cells (Daudi, Nalm6, Raji) and EBV-transformed B cells (EBV-B), CD7 CAR iNKT and CD19 CAR iNKT cells produced at least 2,500-fold more IFN-γ than CD7 - They produced marginal amounts of IFN-γ against AML (Kasumi-1, U937), sarcoma (Rh30, TC71), neuroblastoma (BE(2)C, SKNFI), and CML (K562). Non-transduced mock iNKT cells produced negligible amounts of IFN-γ against any tumor cell line tested. We note that CD7 CAR iNKT or mock cells grown with irradiated allogeneic PBMC and EBV-B, αGalCer or 7DW8-5, IL2, and IL-15 did not recognize the EBV-B cells used for iNKT expansion, suggesting that CD7 CAR iNKT cells grown in allogeneic conditions are CD7 specific and may not be alloreactive.

[0175] To demonstrate that CD7 CAR-modified iNKT cells reproducibly produce IFN-γ in an antigen-specific manner, IFN-γ production was examined in CD7 CAR iNKT cells from multiple donors (n=5). As shown in Figure 20B, CD7 CAR-modified iNKT cells from all five donors (n=5) produced IFN-γ in a CD7 + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + In response to AML (KG1, Kasumi-6), mock iNKT (n=5, p<0.001) and CD19 CAR iNKT cells (p<0.05 except Jurkat, p<0.01) produced significant amounts of IFN-γ. Control CD19 CAR-modified iNKT cells (n=3) produced significantly less IFN-γ than CD19 + Against B-cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji), mock iNKT and CD7 CAR iNKT cells produced significantly more IFN-γ than CD7 CAR iNKT cells (p<0.0001). - or CD19 - Mock iNKT did not produce significant amounts of IFN-γ in recognition of K562 (CML), Kasumi-1 (AML), and U937 (AML). Mock iNKT showed minimal IFN-γ production against any of the target cells tested, and showed no significant cytotoxicity-sensitive CD1d + Even against Jurkat (T-ALL) cells (Figures 18 and 19, Takahashi et al., Br J Haematol. 2003;122(2):231-239).

[0176] FIG. 21 shows that CD7 CAR expression in transduced iNKT cells (n=5) ranged from 64.6% to 98.8%, whereas CD19 CAR or ROR1 CAR expression varied from 5.37% to 43.9%. CD7 antigen-mediated fratricide was associated with increased expression of CD7 CAR + Because the iNKT cell population can be enriched, the CD7 CAR transduction efficiency in iNKT cells appears to be higher than that of CD19 or ROR1 CARs.

[0177] Fratricide is a CD7 CAR-modified iNKT cell-mediated + To confirm complete depletion of iNKT cells, flow cytometry dot plot analysis of CD2 vs. CD7 and CD5 vs. CD7 expression was performed. Figure 22A shows data from one representative donor,55 in which 45-50% of CD7 in mock iNKT and CD19 CAR iNKT cells were depleted. + Figure 22B shows that CD7 CAR iNKT cells reduced nearly all CD7 cells compared to mock iNKT (mean ± SD, 61.6% ± 24.4, n = 4) and CD19 or ROR1 CAR iNKT cells (69.9% ± 24.4, n = 3). + The results demonstrate a significant loss of cells (0.4%±0.1, n=4) (p<0.0001).

[0178] As shown in Figure 23A, phenotypically, CD3 in CD7 CAR iNKT cells + iTCR + Expression of CD7 CAR iNKT cells remained unchanged compared to mock iNKT and CD19 CAR iNKT cells. Similar to mock iNKT and CD19 CAR iNKT cells, CD7 CAR iNKT cells expressed CD4 + , CD4 - CD8 - , CD8 + , and CD4 + CD8 + The CD7 CAR iNKT cells were composed of CD8 + CD4 rather than cells + These iNKT cells differentiated into cells that appeared to express less cell surface PD-1 inhibitory molecules than mock or CD19 CAR iNKT cells (Figure 23B,C).

[0179] To evaluate the anti-leukemia effect of CD7 CAR modified iNKT cells, we used time-dynamic bioluminescence imaging (BLI) technology in live mice. For the xenograft T-ALL model, NSG mice were injected iv with aggressive HSB2-hfflucN T-ALL cells and randomly divided into three groups (n=5 mice per group) based on BLI readings (p=0.942). Mice were then injected iv with thawed CD7 CAR iNKT cells, mock iNKT cells, or PBS (Figure 24A). Administration of three infusions of thawed CD7 CAR iNKT cells to leukemia-bearing mice significantly suppressed T-ALL growth at days 3 (p<0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024), and 17 (p<0.0001) compared to PBS and mock controls (Figure 24B, Figure 24C, Figure 24D). A significant survival advantage was observed after CD7 CAR iNKT cell therapy compared to PBS and mock control groups (p=0.0009, Figure 24E).

[0180] In addition, xenograft CD7 +The anti-leukemia effect of CD7 CAR modified iNKT cells in an AML model was evaluated. Figure 25A shows the experimental schedule of tumor cell injection, CD7 CAR iNKT cell infusion, and BLI monitoring. NSG mice were iv injected with KG1-hfflucN AML cells, and then iv injected with thawed CD7 CAR iNKT cells and mock iNKT cells (n=5 mice per group) (Figure 25A). Before treatment, no significant difference in tumor burden was found between the two groups (p=0.3787). Two injections of thawed CD7 CAR iNKT cells into pre-established AML mice significantly suppressed AML growth at 3 days (p=0.0096), 6 days (p<0.0001), 10 days (p=0.0026), 17 days (p<0.0001), 24 days (p<0.0001), 31 days (p<0.0001), 38 days (p<0.0001), and 45 days (p=0.0003) compared to mock controls (Figure 25B, Figure 25C, Figure 25D). CD7 CAR iNKT cell therapy also significantly extended animal survival for more than one year in 50% of treated mice, while mock control mice died around day 74 (p=0.0069, Figure 25E). Overall, these results demonstrate that CD7 CAR-modified iNKT cells grown in our allogeneic culture conditions without genome editing can upregulate anti-CD7 + It has been demonstrated that it is possible to initiate a tumor response.

[0181] The above embodiments and examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Based on this disclosure, many variations to the above are possible and may be apparent to those skilled in the art. Such modifications and variations are within the spirit and scope of the present invention. All cited patents or non-patent documents are incorporated herein by reference in their entirety without any admission as prior art.

Claims

1. Isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells that have been modified to express a CD7-specific chimeric antigen receptor (CAR) without the use of genome editing.

2. CD3 isolated from biological samples and activated in the presence of α-galactosylceramide (αGalCer) or 7DW8-5 or other glycolipid analogs + iTCR(Vα24-Jα18) + or CD3 + Vα24 + 2. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of claim 1, wherein the iNKT cells or type I natural killer T (NKT) cells are modified with a lentiviral vector to express a CD7 chimeric antigen receptor (CAR), and expanded in culture using irradiated allogeneic peripheral blood mononuclear cells and Epstein-Barr virus-transformed B cells, αGalCer or 7DW8-5, IL-2, IL-15, and / or IL-7.

3. CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells express CD3 + iTCR + CD4 + cells, CD3 + iTCR + CD8 + cells, CD3 + iTCR + CD4 - CD8 - cells, or CD3 + iTCR + CD4 + CD8 + 2. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of claim 1, having a phenotype of a type I invariant natural killer T (iNKT) cell, a type I invariant natural killer T (NKT) cell, or a mixture thereof.

4. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of claim 2, wherein the biological sample is selected from blood, bone marrow, lymph node tissue, spleen tissue, tumor tissue, induced pluripotent stem cells, and peripheral blood mononuclear cells, and combinations thereof, and optionally the blood is peripheral blood and / or umbilical cord blood.

5. 2. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell of claim 1, isolated from one or more peripheral blood mononuclear cells and comprising one or more polynucleotides encoding a CD7-specific CAR.

6. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell of claim 1, wherein the CD7-specific CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, and the antigen-binding domain is capable of binding to CD7.

7. 7. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell of claim 6, wherein the antigen-binding domain comprises an antibody, or an antigen-binding fragment thereof, or a non-antibody protein scaffold, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, or a single-domain antibody (or nanobody), wherein the antigen-binding fragment comprises a single-chain variable fragment (scFv), and wherein the intracellular signaling domain comprises a functional signaling domain of at least one stimulatory molecule.

8. 8. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of claim 7, wherein the at least one stimulatory molecule comprises a zeta chain or a CD3 zeta chain associated with the T cell receptor complex, and the intracellular signaling domain further comprises a functional signaling domain of at least one costimulatory molecule, optionally wherein the at least one costimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, or DAP12.

9. 2. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of claim 1, further modified to include an exogenous cytokine, growth factor, antibody or antigen-binding fragment, or any combination thereof, wherein the antibody or antigen-binding fragment optionally comprises a bispecific T cell engager (BiTE).

10. A pharmaceutical composition comprising the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. 10. A method for treating a pulmonary artery disease comprising administering to a subject the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of any one of claims 1 to 9 a CD7 + A pharmaceutical composition for use in the treatment of cancer.

12. Cancer, CD7 + Hematological malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), pro-T-ALL, pre-T-ALL, cortical T-ALL, and mature T-ALL; subsets of peripheral T-cell lymphomas (PTCL) and PTCL subtypes, including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ALCL), primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); and CD7 + Other malignant tumors or CD7 + CD1d + 12. The pharmaceutical composition for use according to claim 11, selected from malignant tumors, optionally wherein the cancer is resistant or refractory to treatment in the absence of said cells.

13. Isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells are isolated from a biological sample of a subject or donor and expressed as CD3 + iTCR + CD4 + cells, CD3 + iTCR + CD8 + cells, CD3 + iTCR + CD4 - CD8 - cells, or CD3 + iTCR + CD4 + CD8 + CD3 activated and engineered with synthetic receptors that have the phenotype of cells, or a mixture thereof + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The pharmaceutical composition for use according to claim 11, which is an iNKT cell.

14. 10. Use of the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells of any one of claims 1 to 9 in the manufacture of a medicament for treating or preventing cancer in a subject in need thereof, comprising: Cancer, CD7 + Hematological malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), pro-T-ALL, pre-T-ALL, cortical T-ALL, and mature T-ALL; subsets of peripheral T-cell lymphomas (PTCL) and PTCL subtypes, including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ALCL), primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); CD7 + Other malignant tumors or CD7 + CD1d + The use of any one of claims 1 to 4, wherein the cancer is selected from malignant tumors, and optionally the cancer is resistant or refractory to treatment in the absence of said cells.

15. CD7 in a subject in need of treatment or prevention + 1. A method of preparing a therapy for treating or preventing cancer, comprising: a. isolating one or more invariant natural killer T cells from a biological sample based on expression of the TCR alpha chain Vα24-Jα18 (iTCR); b. One or more iTCRs + activating or activating invariant natural killer T cells in growth medium for expansion using, but not limited to, irradiated autologous peripheral blood mononuclear cell negative fraction and / or whole peripheral blood mononuclear cells, α-galactosylceramide (αGalCer) or 7DW8-5, or other glycolipid analogs, IL-2 and IL-15 and / or IL-7; c. lentivirally engineering activated invariant natural killer T cells to express a CAR (e.g., CD7); d. Expanding one or more CAR (e.g., CD7) engineered invariant natural killer T cells in a growth medium using irradiated allogeneic peripheral blood mononuclear cells and Epstein-Barr virus transformed B cells, αGalCer or 7DW8-5, IL-2, and IL-15 and / or IL-7. A method comprising:

16. 16. The method of claim 15, further comprising modifying one or more invariant natural killer T cells to express a CAR or TCR or TCRm, wherein the modification comprises introducing one or more polynucleotides encoding a CAR into the one or more cells, wherein the introduction of the one or more polynucleotides optionally comprises electroporation, transduction, and / or transfection, wherein optionally the one or more polynucleotides comprise mRNA and / or DNA, and optionally the DNA comprises transposon DNA.

17. 17. The method of claim 16, wherein the one or more polynucleotides comprise one or more vectors, and the one or more vectors comprise one or more viral vectors, lentiviral vectors, or gamma-retroviral vectors.

18. Cancer, CD7 + Hematological malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), pro-T-ALL, pre-T-ALL, cortical T-ALL, and mature T-ALL; subsets of peripheral T-cell lymphomas (PTCL) and PTCL subtypes, including PTCL not otherwise specified (PTCL-NOS); anaplastic large cell lymphoma (ALCL), primary cutaneous ALCL, angioimmunoblastic T-cell lymphoma (AITL), nasal NK / T-cell lymphoma, adult T-cell acute lymphoblastic lymphoma or leukemia (ATLL) associated with human T-cell leukemia virus 1 (HTLV-1) infection, enteropathy-associated lymphoma, hepatosplenic lymphoma, subcutaneous panniculitis-like lymphoma, precursor T-cell acute lymphoblastic lymphoma or leukemia, blastic NK-cell lymphoma, and cutaneous T-cell lymphoma (CTCL); CD7 + Acute myeloid leukemia (AML); CD7 + Other malignant tumors or CD7 + CD1d + 18. The method of any one of claims 15 to 17, wherein the cancer is selected from malignant tumors, and optionally the cancer is resistant or refractory to treatment in the absence of said cells.

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