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

Abstract

The present disclosure relates to modified invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells (e.g., CD3 + iTCRV [alpha] 24-J [alpha] 18 + or CD3 + V [alpha] 24 +) for expressing a CD7 chimeric antigen receptor (CAR) as therapeutic agents for the treatment of CD7 + cancer, and pharmaceutical compositions, methods of preparation, and therapeutic uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 313,186, filed on February 23, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to modified invariant natural killer T (NKT) cells (e.g., iNKT cells, type I NKT cells) expressing CD7 chimeric antigen receptors, pharmaceutical compositions of the cells, methods of preparing the cells, and use of the cells or pharmaceutical compositions thereof in treating or preventing CD7. + Therapeutic uses for cancer. Background Art

[0004] Natural killer T (NKT) cells are a subset of T cells that exhibit properties of both conventional T cells and natural killer (NK) cells. NKT cells are usually produced in the thymus by CD4 + CD8 + Cortical thymocytes produce. NKT cells have traditionally been defined as CD1d-restricted lipid antigen-reactive T cells and have been classified into 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 dependence, 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).

[0005] Type I or invariant NKT (iNKT) cells express invariant TCR α chains (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 and 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 well-defined TCR repertoire and recognize non-αGalCer molecules (such as sulfatides) presented by CDld molecules. Type III NKT or NKT-like cells have a diverse TCR repertoire and recognize CDld-independent molecules.

[0006] Single-chain bispecific antibodies using iNKT cells co-expressing chimeric antigen receptors (CAR-iNKT) with interleukin-15 (IL-15) or allogeneic hematopoietic stem cell-engineered iNKT (HSC-iNKT) or stabilized invariant T cell receptor (iTCR)-CD1d complexes have recently produced promising antitumor results in patients with neuroblastoma and in mice transplanted 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. 2019 ... Med. 2021; 2(11): 100449; Lameris et al., Nat Cancer. 2020; 1(11): 1054-1065). The advantages of CAR-iNKT over conventional CAR-modified T (CAR-T) cells include: 1) CAR-iNKT can more effectively infiltrate tumors and suppress tumor-associated macrophages (TAMs), and may be more effective in solid tumors; 2) iNKT cells have metabolic properties that can confer differential functional characteristics and adaptability and lifespan in the nutrient-poor tumor microenvironment (TME), and 3) they do not cause graft-versus-host disease (GVHD) and can be used as allogeneic off-the-shelf drugs (S 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).

[0007] Since human iNKT cells in peripheral blood account for about 0.01-1%, it is a challenge to expand iNKT cells for treatment. The most common method is to stimulate the separated iNKT cells with the negative part of irradiated PBMC supplemented with αGalCer, IL-2, IL-7 and IL-15, and expand them with αGalCer pulsed autologous dendritic cells (DC) or autologous PBMC (Poels et al., Int J Mol Sci. 2021; 22: 1096; Tian et al., J Clin Invest. 2016; 126 (6): 2341-2355). The separated iNKT cells stimulated with OKT3 anti-CD3 monoclonal antibody, irradiated autologous PBMC and IL-2 are used in clinical trials to treat advanced melanoma (Exley et al., Clin Cancer Res. 2017; 23 (14): 3510-3519). INKT cells isolated and modified to express GD2 CAR were stimulated with αGalCer pulsed autologous PBMC, IL-2 and IL-21 and were used in clinical trials for the treatment of neuroblastoma (Heczey et al., Nat Med. 2020; 26(11): 1686-1690). Alternatively, isolated iNKT cells were stimulated with autologous PBMC, anti-CD3 / CD28 beads and IL-15 (Rotolo et al., Cancer Cell. 2018; 34(10): 596-610).

[0008] Adoptive transfer of tumor antigens by CAR-T cells has emerged as a powerful strategy for treating 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, due to the shared expression of many targetable antigens (e.g., CD1a, CD2, CD5 and CD7) between CAR-T cells and malignant T cells, the main obstacle in developing CAR-T therapy for T cell malignancies is the fratricide caused by suicide injury by CAR-T cells, resulting in insufficient number 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).

[0009] CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome disruption expressed by CD7 and / or TCRα chain (TRAC) provides an alternative method to enable expansion of CD7 CAR-transduced T cells from allogeneic healthy donors without extensive autoantigen-driven fratricide. Edited CD7 CAR-T cells produce powerful cytotoxicity against malignant T cell lines and primary tumors, and are protective in a mouse xenograft model 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 significance of CRISPR-based gene editing remain. 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 breakage), and large structural variants at on-target and off-target sites in vivo, which are passed on to the next generation (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). In addition, recent studies have documented a high frequency of chromosome loss and truncation in genetically engineered T cells after CRISPR-Cas9 transfection (Nahmad et al., Nat Biotechnol. 2022; 40(12): 1807-1813).

[0010] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood disorder. T-ALL subtypes include ETP (early thymic precursor), early precursor T, precursor T, cortex and mature T-ALL (Bayon-Calderon et al., Int J Mol Sci. 2020; 21 (20): 7685). T-ALL represents about 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 more than 85%. However, adult T-ALL still presents poor results, with significantly lower survival rates compared to pediatric T-ALL. Children and adolescents with ETPALL have the worst response to initial treatment (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, new therapies are urgently needed for patients with refractory or relapsed and high-risk T-ALL. Summary of the invention

[0011] The present disclosure relates to invariant natural killer T (iNKT) or type I NKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + ), which are modified to express a CD7 chimeric antigen receptor (CAR) for the treatment of CD7 without the use of genome editing (e.g., CRISPR / Cas). + Cancer. In some embodiments, the iNKT cells modified with CD7 CAR disclosed herein are CD3 + 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 mixtures thereof).

[0012] The present disclosure also relates to methods and compositions (e.g., pharmaceutical compositions) using the iNKT cells modified with CD7 CAR disclosed herein. In some embodiments, the iNKT cells modified with CD7 CAR disclosed herein can be used, for example, in the treatment or prevention of CD7 + Therapeutic agents in cancer (e.g., T cell leukemia and lymphoma or acute myeloid leukemia). In some embodiments, the iNKT cells disclosed herein can be isolated (e.g., from a biological sample, such as from a patient or donor), cultured, modified to express CD7 CAR and expanded into a cell population. In some embodiments, the iNKT cells modified by CD7 CAR disclosed herein are present in and / or used in a pharmaceutical composition.

[0013] Also provided are pharmaceutical compositions comprising the iNKT cells disclosed herein. In one aspect, the present disclosure relates to a pharmaceutical composition comprising the isolated CD3 T cells according to any embodiment disclosed herein. + iTCR(Vα24-Jα18) + iNKT cells) and a pharmaceutically acceptable carrier. In some embodiments, the cells are 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 + mixture.

[0014] In one embodiment, the present disclosure relates to a method for treating or preventing CD7 in a subject in need thereof. + A method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of iNKT cells modified with CD7 CAR according to any embodiment disclosed herein, or a pharmaceutical composition comprising a therapeutically effective amount of iNKT cells modified with CD7 CAR and a pharmaceutically acceptable carrier.

[0015] In another embodiment, the present disclosure relates to a method for treating or preventing CD7 in a subject in need thereof. + A method of treating cancer, the method comprising:

[0016] a) isolating one or more CD3 + iTCR(Vα24-Jα18) + iNKT cells; and

[0017] b) culturing one or more CD3 + iTCR(Vα24-Jα18) + iNKT cells were expressed with CD7CAR and generated into expanded cell populations.

[0018] In some embodiments, the method further comprises: + iTCR(Vα24-Jα18) + iNKT cells are modified to express CD7 CAR. In some embodiments, the modification comprises introducing one or more polynucleotides encoding the CD7 CAR into the one or more cells.

[0019] In some embodiments, the CD7 CAR-modified iNKT cells are further modified to comprise a CAR, a T cell receptor (TCR), a TCR mimetic antibody (TCRm), an exogenous cytokine, a growth factor, an antibody or an antigen-binding fragment, or any combination thereof, wherein the antibody or antigen-binding fragment optionally comprises a bispecific T cell engager (BiTE).

[0020] In any aspect, the isolated iNKT cells are modified to express CD7 CAR and can be used to treat or prevent cancers including but not limited to CD7 + Cancer or CD7 +malignancies; for example, T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL, precursor T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and subsets of PTCL subtypes, including PTCL, not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma (ALCL), primary cutaneous 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 malignancies or CD7 + CD1d + Malignant tumors (e.g., T-ALL).

[0021] Other aspects or advantages of the present disclosure may be better understood through the following description of the drawings, detailed description of the present disclosure, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flow cytometry diagrams showing CD1d expression and NGFR expression in luciferase-expressing tumor cell lines used in the present disclosure. Tumor cells were transduced with a bidirectional lentiviral vector expressing humanized firefly luciferase and a cytoplasmic domain-deleted nerve growth factor receptor (NGFR) (hffluc N). K562, chronic myeloid leukemia (CML); K562CD19, K562 stably transfected with CD19; Daudi and Raji, B cell lymphoma; Nalm6, B cell precursor leukemia; HL60, KG1, Mo7e, Molm13, MV4:11, THP1 and U937, acute myeloid leukemia (AML). The data are partially used in PCT / US20 / 47253.

[0023] Figure 2A Figure 2 shows the effect of iNKT1(auto) and iNKT1(allo) cells on luciferase-expressing CD1d cells pre-incubated overnight with DMSO or αGalCer at different effector / target (E / T) ratios. - (Daudi, Nalm6) and CD1d +Figure 3 shows luciferase-based cytotoxicity of target cells (Molm13, THP1, U937). iNKT1(auto) and iNKT1(allo) were generated by co-culturing iNKT1 cells with irradiated autologous peripheral blood mononuclear cell (PBMC) negative fraction, αGalCer, IL-2 and IL-15 in 24-well plates or co-culturing iNKT1 cells with irradiated allogeneic PBMC and Epstein-Barr virus-transformed B (EBV-B) cells, αGalCer, IL-2, IL-7 and IL-15 in T25 flasks after secondary stimulation or expansion. In the 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.

[0024] Figure 2B Figure 2 shows the expression of luciferase in iNKT2(auto) and iNKT2(allo) cells at different E / T ratios to CD1d expressing luciferase pre-incubated with DMSO or αGalCer. - (Daudi, Nalm6) and CD1d + Figure 2 shows the cytotoxicity of target cells (KG1, Molm13, MV4:11, THP1). iNKT2(auto) and iNKT2(allo) were generated by co-culturing iNKT2 cells with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15 in 24-well plates or co-culturing iNKT2 cells with irradiated allogeneic PBMC and EBV-B cells, αGalCer, IL-2, IL-7 and IL-15 in T25 flasks after secondary stimulation or expansion. In the 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.

[0025] Figure 3A Figure 2 shows CD1d cells pre-incubated with DMSO or αGalCer. - (K562) and CD1d + (U937) Graph of specific lysis of target cells by iNKT1 cells expanded in T25 flasks with αGalCer or anti-CD3 antibody (OKT3) plus irradiated allogeneic PBMCs and EBV-B cells, IL-2, IL-7, and IL-15.

[0026] Figure 3B Figure 2 shows CD1d cells pre-incubated with DMSO or αGalCer. - (K562, Daudi) and CD1d+ Figure 3. Specific lysis of (HL60, KG1, Molm13, MV4:11, U937) target cells by iNKT2 cells expanded in T25 flasks with αGalCer or anti-CD3 antibody (OKT3) plus irradiated allogeneic PBMCs and EBV-B cells, IL-2, IL-7 and IL-15.

[0027] Figure 3C Figure 2 shows CD1d cells pre-incubated with DMSO or αGalCer. - (Daudi, Raji) and CD1d + Figure 3 shows specific lysis of (MV4:11, U937) target cells by iNKT12(αGalCer) and iNKT12(OKT3) cells expanded in T25 flasks with αGalCer or anti-CD3 antibody (OKT3) plus irradiated allogeneic PBMC and EBV-B cells, IL-2, IL-7 and IL-15. In the primary stimulation, isolated iNKT12 cells from blood donor 12 were co-cultured with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15 in 48-well plates. iNKT2 cells and iNKT1 expanded in T25 flasks with αGalCer, irradiated allogeneic PBMC 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.

[0028] Figure 4A Figure 2 shows CD1d cells pre-incubated with DMSO or αGalCer. + Figure 3. Specific lysis of (THP1, U937) target cells at different E / T ratios by iNKT1, iNKT2, iNKT11 and iNKT12 expanded in T25 flasks with αGalCer, irradiated allogeneic PBMCs and EBV-B cells, IL2, IL-7 and IL-15.

[0029] Figure 4B Figure 2 shows that CD3 + iTCR + Flow cytometry analysis of the percentage of cells expressing iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to establish gates.

[0030] Figure 5AFigure 2 shows the responses of iNKT45(auto), iNKT45(allo), iNKT46(auto), and iNKT46(allo) cells to luciferase-expressing CD1d3 cells pre-incubated with DMSO or GalCer. - (Nalm6) and CD1d + Figure 2 shows the cytotoxicity of target cells (MV4:11, U937). iNKT45 (auto) or iNKT46 (auto) and iNKT45 (allo) or iNKT46 (allo) refer to iNKT cell expansion in 24-well plates using irradiated autologous PBMC negative fraction or irradiated allogeneic PBMC and EBV-B cells (all supplemented with αGalCer, IL-2 and IL-15), respectively. The iNKT cells were then transferred to T75 flasks for further expansion. The isolated iNKT45 and iNKT46 cells from blood donors 45 and 46 were initially stimulated in 48-well plates with irradiated autologous PBMC negative fraction, αGalCer, IL-2 and IL-15.

[0031] Figure 5B Figure 2 shows CD3 expression in iNKT45 and iNKT46 cells after initial stimulation. + iTCR + Flow cytometry analysis of the percentage of cells expressing iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to establish gates.

[0032] Figure 5C Figure 2 shows CD3 in iNKT45(auto), iNKT45(allo), iNKT46(auto) and iNKT46(allo) cells after secondary stimulation (i.e., expansion). + iTCR + Flow cytometry analysis of the percentage of cells expressing iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to establish gates.

[0033] Figure 6 Figure 2 shows the expression of CD19 CAR modified iNKT47 and iNKT48 cells in response to luciferase-expressing CD19 - (K562) and CD19 + (Daudi, Nalm6, Raji) and CD1d-(Mo7e) and CD1d treated with DMSO or αGalCer +Figure 2 shows the cytotoxicity of target cells (Molm13, U937). Isolated iNKT47 and iNKT48 were modified with CD19CAR or CD19CAR / GFP and expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells (all supplemented with αGalCer, IL2 and IL-15).

[0034] Figure 7 It was shown that in the enzyme-linked immunosorbent assay (ELISA), it can recognize CD19 + B cell malignancies (Daudi, Nalm6, Raji) and CD1d treated with DMSO or αGalCer - (Mo7e) and CD1d + Figure 3. IFN-γ production by CD19 CAR-modified iNKT47 and iNKT48 after expansion with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15, following (THP1) target cells.

[0035] Figure 8 Figure 2 shows the expression of CD3 in iNKT47 and iNKT48 cells modified with CD19 CAR. + iTCR + Flow cytometric analysis of the percentage of cells that CD19 CAR-modified iNKT47 and iNKT48 cells expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2, and IL-15. iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to establish gates.

[0036] Fig. 9 Figure 1 shows the expression of CAR in iNKT47 and iNKT48 cells modified with CD19 CAR or CD19 CAR / GFP. + or CAR + GFP + Figure 3 Flow cytometric analysis of the percentage of cells that were expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15.

[0037] Fig.10 Figure 2 shows the expression of luciferase-expressing CD19 in iNKT50 cells modified with CD19 CAR or CD19 CAR / GFP. - (K562, HL60, KG1, Molm13, THP1) and CD19 + (K562CD19, Daudi, Nalm6, Raji) and CD1d treated with DMSO or αGalCer - (Mo7e) and CD1d + Figure 4. (MR4:11, U937) Target cell cytotoxicity. Isolated iNKT50 cells were modified with lentiviral CD19 CAR or CD19CAR / GFP and expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15.

[0038] Fig.11A Figure 2 shows the expression of CD3 in iNKT50 or mock cells modified with CD19 CAR. + iTCR + Flow cytometric analysis of the percentage of cells that CD19 CAR-modified iNKT50 or mock cells were expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2, and IL-15. iTCR, Vα24-Jα18. Isotype antibodies were used as background controls to establish gates.

[0039] Fig. 11B Figure 2 shows the expression of CAR in iNKT50 cells modified with CD19 CAR or modified with CD19 CAR / GFP or in mock cells. + or CAR + GFP + Flow cytometric analysis of the percentage of cells, wherein the CD19CAR-modified or CD19 CAR / GFP-modified iNKT50 or mock cells were expanded with irradiated autologous PBMC negative fraction and whole PBMC or irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL-2 and IL-15.

[0040] Fig.12is a graph showing the expansion rate of CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47, iNKT48 and iNKT50 or unmodified iNKT45 and iNKT46 cells, which were expanded with irradiated autologous PBMC negative fraction and whole PBMCs or irradiated allogeneic PBMCs and EBV-B cells in the presence of αGalCer, IL-2 and IL-15.

[0041] Fig.13A Figure 3 shows the effect of thawed CD19 CAR-modified iNKT47 and iNKT48 on CD19 - (K562) and CD19 + Figure 1 shows overnight (16 hours) cytotoxicity of (K562CD19, Daudi, Nalm6, Raji) target cells with CD19 CAR-modified iNKT47 and iNKT48 expanded with irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2 and IL15. CD19 CAR-modified iNKT47 (allo) and iNKT48 (allo) were thawed and cultured in 24-well plates for 5 days in human T cell culture medium supplemented with IL-2 and IL-15.

[0042] Fig. 13B It is a graph showing the overnight (16 hours) cytotoxicity of thawed CD19 CAR-modified iNKT47 cells to Molm13 and THP1 cells expressing luciferase in the presence of 7DW8-5 or αGalCer at a concentration range of 200 ng / mL to 3.12 ng / mL, wherein the CD19 CAR-modified iNKT47 cells are expanded with irradiated allogeneic PBMC and EBV-B cells in the presence of αGalCer, IL2 and IL-15. iNKT47 (allo) modified by CD19 CAR is thawed and cultured in 24-well plates for 14 days in human T cell culture medium supplemented with IL-2 and IL-15. E / T ratio, 10 / 1.

[0043] Fig. 13C Figure 2 shows the responses of iNKT47 CD19 CAR (allo), iNKT48 CD19CAR / GFP (allo) cells and iNKT47 mimic (auto) cultured on day 92 to CD1d cells pulsed with different amounts of 7DW8-5 and αGalCer. +Figure 3. IFN-γ production by MV4:11-hfflucN target cells. + (K562CD19, Nalm6) and CD19-(K562) were used as positive and negative controls.

[0044] Fig.14A Graphs showing flow cytometric analysis of iNKT cells (from a representative healthy blood donor (iNKT69)) expanded in the presence of irradiated allogeneic PBMCs and EBV-B cells, GalCer or 7DW8-5, IL-2 and IL-15, the percentage of CD2 to CD7 and CD5 to CD7 positive cell populations.

[0045] Fig. 14B Is a graph showing statistical analysis of flow cytometry data of CD2, CD5 and CD7 positive cell populations in expanded iNKT cell products from 7-9 individual blood donors (iNKT45, 46, 47, 48, 50, 55, 56, 68, 69 (except iNKT47, 48) in the case of CD2) expanded with irradiated allogeneic PBMC and EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15. Results are calculated as mean ± SD. ****p < 0.0001.

[0046] Fig.15 Graphs showing flow cytometry analysis of CD7, CDld and NGFR expression in bidirectional (humanized firefly luciferase and NGFR) lentivirally transduced human HSB2, Jurkat and MOLT13T-ALL cell lines and KG1, Kasumi-1 and Kasumi-6 AML cell lines.

[0047] Fig.16 is a graph showing T cell expansion rate after lentiviral transduction of peripheral blood mononuclear cells (PBMCs) from donor 36 (PBL36) with CD19, ROR1 and CD7 CARs. Untransduced mock was used as a control.

[0048] Fig.17 Graph showing the expansion rate of iNKT cells after lentiviral transduction of iNKT55 cells isolated from donor 55 with CD7 CAR, CD19 CAR or untransduced mock control, and after lentiviral transduction of iNKT68 cells isolated from donor 68 with CD7 CAR or untransduced mock control. Transduced iNKT55 and iNKT68 mocks or CD19 CAR or CD7 CAR were expanded with irradiated allogeneic PBMCs and EBV-B cells, 7DW8-5, IL-2 and IL-15.

[0049] Fig.18 is a graph showing representative cytotoxicity of CD7 CAR iNKT55 cells (mock iNKT55, CD19CAR iNKT55, and CD7 CAR iNKT55 cells) derived from donor 55 against target cells expressing luciferase, 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 sarcomas (Rh30, TC71, SaOS2) and CD7 - Neuroblastoma (BE(2)C, SKNFI) cell lines. E:T (effector:target) ratio.

[0050] Fig.19 Figure 2 shows the expression of CD7 CAR iNKT cells (n=5) from donors 55, 56, 64, 68, and 69 to CD7 expressing luciferase. + T-ALL cell lines (HSB2, Jurkat, MOLT13), CD7 + AML (KG1) and CD19 + Figure of statistical analysis of five independent cytotoxicity assays for B cell malignancies (Nalm6, Raji). The corresponding mimics (n=5) and CD19CAR (n=3) iNKT cells were used as baseline and CAR-T cell controls. The results were calculated as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant. It shows that there is significance between CD19 CAR iNKT relative to mimics and CD7 CARiNKT relative to mimics.

[0051] Fig. 20 A is a graph showing recognition of CD7 in an enzyme-linked immunosorbent assay (ELISA) + 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) (for CD19 CAR iNKT55 control) and CD7- Figure 3. IFN-γ production by a panel of CML (K562), AML (Kasumi-1, U937), multiple myeloma (RPMI8226), sarcoma (Rh30, TC71), and neuroblastoma (BE2(C), SKNFI) cell lines followed by representative CD7 CAR iNKT55 and either mock iNKT55 or CD19 CAR iNKT55 controls.

[0052] Fig. 20 B is a graph showing the CD7 CAR iNKT cells prepared from donors 55, 56, 64, 68 and 69 (n=5) in response to CD7 + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + AML (KG1, Kasumi-6), CD19 + B-cell malignancies (Daudi, Nalm6, Raji) and CD7 - Figure of statistical analysis of five independent IFN-γ production assays for CML (K562) and AML (Kasumi-1, U937). Corresponding mimics (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. It shows that there is statistical significance between mimics and CD7 CAR iNKT.

[0053] Fig.21 is a flow cytometric analysis showing the level of CD7 CAR expression after lentiviral transduction in iNKT cells derived from 5 blood donors (n=5). CD19 CAR iNKT (n=3) and mock iNKT (n=5) cells were used as specificity or background controls.

[0054] Fig.22A The final CD7 CAR iNKT55 product shows CD7 + Flow cytometry dot plots of exemplary depleted CD2 vs. CD7 and CD5 vs. CD7 dual staining of cells. Mock and CD19CAR iNKT55 were used as controls.

[0055] Fig. 22B Figure 2 shows the residual CD7 in the final CD7 CAR iNKT cell products (n=4) prepared from donors 55, 56, 68, and 69 compared to mock (n=4) and irrelevant CD19 CAR (iNKT55, iNKT56) or ROR1 CAR (iNKT69) iNKT cells (n=3).+ Graph of statistical analysis of percentage of cell population. Results are calculated as mean ± SD. **p < 0.01, ns, not significant.

[0056] Fig.23A Figure 5 shows CD3 in CD7 CAR iNKT, mock iNKT, and CD19 CARiNKT cells from a representative donor 55. + iTCR(Vα24-Jα18) + Flow cytometry analysis of percentage of cell population. Isotype control antibody was used to establish background gate.

[0057] Fig. 23B Figure 5 shows CD4 in CD7 CAR iNKT, mock and CD19 CAR iNKT cells from a representative donor 55. + 、CD8 + 、CD4-CD8 - and CD4 + CD8 + Flow cytometry analysis of percentage of cell population. Isotype control antibody was used to establish background gate.

[0058] Fig.23C is a flow cytometry histogram analysis showing PD1 expression levels in CD7 CAR iNKT, mock, and CD19 CAR iNKT cells from one representative donor 55.

[0059] Fig.24A It is a diagram showing the experimental scheme of HSB2-hfflucN tumor cell injection, iNKT cell infusion and bioluminescent imaging (BLI) monitoring. HSB2-hfflucN T-ALL cells were intravenously injected into 6-7 week-old female NSG mice on the 2nd day. Tumor growth was determined by BLI on the 0th day. Mice were randomly divided into three groups (n=5 per group) and treated with three intravenous infusions of PBS, thawed mimics iNKT55 or thawed CD7 CAR iNKT55 cells on the 0th, 3rd and 6th days. PBS or IL-2 and IL-15 were injected intraperitoneally into mice every three days for two weeks. Tumor growth was monitored by BLI on the 3rd, 6th, 10th, 17th and 24th days.

[0060] Fig. 24B Graph showing bioluminescent imaging (BLI) of tumor growth in mice treated with PBS, mock iNKT55 cells, or CD7 CAR iNKT55 cells (three groups, n=5 each). Scale bars represent radiance (p / sec / cm 2All PBS or mock control mice died on day 23. One mouse (the first one) in the CD7 CARiNKT-treated group died immediately on day 24 after imaging, possibly due to the anesthesia procedure used for imaging or the injection of luciferin.

[0061] Fig.24C is a graph showing the overall kinetics of systemic tumor progression in mice. Each line indicates an individual animal.

[0062] Fig.24D is a graph showing statistical analysis of tumor progression in each group of mice as determined by BLI imaging. P-values ​​< 0.05 were considered significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

[0063] Fig.24E is a 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.

[0064] Fig.25A It is a diagram showing the experimental scheme of KG1-hfflucN tumor cell injection, iNKT cell infusion and bioluminescent imaging (BLI) monitoring. KG1-hfflucN cells were injected intravenously into 6-7 week-old female NSG mice on the 2nd day. Tumor growth was determined by BLI on day 0. Mice were randomly divided into two groups (n=5 per group) and treated with two intravenous infusions of thawed mimics iNKT55 or thawed CD7 CAR iNKT55 cells on days 0 and 3. PBS or IL-2 and IL-15 were injected intraperitoneally into mice every three days for two weeks. Tumor growth was monitored by BLI on days 3, 6, 10, 17, 24, 31, 38 and 45.

[0065] Fig.25B Graph showing bioluminescent imaging (BLI) of tumor growth in mice treated with mock iNKT55 cells or CD7 CAR iNKT55 cells (two groups, n=5 each). Scale bars represent radiance (p / sec / cm 2 / sr). The third mouse in the CD7CAR iNKT treatment group showed a larger tumor mass than the other mice in the same group. This may be due to the intraperitoneal injection of the remaining T cells in the syringe during the second intravenous T cell infusion. One mouse (the first one) in the CD7CAR iNKT treatment group died immediately on day 31 after imaging, probably due to the anesthesia procedure used for imaging or injection of luciferin.

[0066] Fig.25C is a graph showing the overall kinetics of systemic tumor progression in mice. Each line indicates an individual animal.

[0067] Fig.25D is a graph showing statistical analysis of tumor progression in each group of mice as determined by BLI imaging. P-values ​​< 0.05 were considered significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

[0068] Fig.25E is a graph showing the Kaplan-Meier survival curves of mice treated with mock iNKT or CD7 CAR iNKT cells. P=0.0069 by log-rank Mantel-Cox test. One mouse (the first one) in the CD7 CARiNKT treatment group died on day 31 after imaging and was not counted in the survival curve. All control mice died on days 73-74. Two mice in the CD7 CAR iNKT treatment group survived 368 days and were humanely euthanized. DETAILED DESCRIPTION

[0069] The present disclosure is based on the recognition that type I NKT cells or invariant NKT (iNKT) cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + ) expressed approximately 42.96% ± 17.21% CD7 in iNKT cells - subgroups and can be expanded approximately 6,000-fold in culture after being modified with a lentivirus to express a CD7 chimeric antigen receptor (CAR). - iNKT cells can avoid CD7 antigen-driven fratricide or suicide and can be expanded for therapeutic purposes. Importantly, iNKT cells modified with CD7 CAR showed anti-CD7 in vitro and in animals. +Cancer activity. In addition, irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15 can also be used to achieve about 6,000-fold expansion of iNKT cells or iNKT cells modified with CD19 CAR. The present disclosure provides multiple advantages. First, iNKT cells modified with CD7 CAR are generated without the use of genome editing, while most conventional T cells modified with CD7 CAR require CRISPR-mediated genome editing. Therefore, by avoiding off-target effects associated with genome editing, iNKT cells modified with CD7 CAR can be safer than conventional T cells modified with CD7 CAR. Second, iNKT cells modified with CD7 CAR can survive in vivo for weeks to months, while most conventional T cells modified with CD7 CAR can last for months to years and may cause more severe T cell deficiency and infection. Third, iNKT cells modified with CD7 CAR can provide CD7 by CD7 CAR and invariant TCR. + CD1d + Dual targeting of T-ALL and may be more effective than conventional T cells modified with CD7CAR in reducing the relapse rate of these patients. Fourth, iNKT cells modified with CD7CAR can be prepared off-the-shelf from healthy donors without causing graft-versus-host disease (GVHD) and the need for genome editing, while conventional T cells modified with CD7CAR require CRISPR-mediated deletion of both CD7 and alloreactive TCRs.

[0070] Therefore, in certain aspects, the present disclosure provides methods for treating or preventing CD7 in a subject in need thereof using iNKT cells modified with CD7 CAR. + In some embodiments, the iNKT cells modified with CD7CAR comprise CD3 + iTCR(Vα24-Jα18) + iNKT cells or CD3 + iTCR(Vα24-Jα18) + iNKT cell composition. In some embodiments, the iNKT cells modified with CD7 CAR comprise CD3 + Vα24 + CD3 + Vα24 + Cell composition. In some embodiments, the iNKT cells modified with CD7 CAR comprise CD3 + CD4 + CD3 + CD4 +Cell composition. In some embodiments, the iNKT cells modified with CD7 CAR comprise CD3 + CD4 - CD8 - CD3 + CD4 - CD8 - Cell composition. In some embodiments, the iNKT cells modified with CD7 CAR comprise CD3 + CD8 + CD3 + CD8 + Cell composition. In some embodiments, the iNKT cells modified with CD7 CAR comprise CD3 + CD4 + Cells, CD3 + CD4-CD8- cells, CD3 + CD8 + Cells and CD3 + CD4 + CD8 + CD3 + CD4 + Cells, CD3 + CD4 - CD8 - Cells, CD3 + CD8 + Cells and CD3 + CD4 + CD8 + In some embodiments, the iNKT cells modified with CD7 CAR are reactive to γ-galactosylceramide (αGalCer) or 7DW8-5 or other glycolipid analogs presented by MHC class I-like CD1d molecules.

[0071] In some embodiments, the exemplary CD7 CAR-modified iNKT cells described herein can be used to treat and / or prevent CD7 + Cancer (e.g., T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), e.g., refractory or relapsed T-ALL or AML). In some embodiments, CD7 + The cancer is T-ALL or AML. In some embodiments, CD7 + The cancer is refractory or relapsed T-ALL or AML. In some embodiments, CD7 + The cancer is, for example, relapsed T-ALL or AML after hematopoietic stem cell transplantation. In some embodiments, CD7 +The cancer is a cancer that expresses CD7 and an additional antigen targeted by the iNKT cells and / or a construct (e.g., a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a T cell receptor mimetic antibody (TCRm), or a combination thereof) expressed by the iNKT cells. In some embodiments, CD7 + A cancer is one that is resistant or refractory to treatment in the absence of cells. Such exemplary cancers are described and exemplified herein.

[0072] In some embodiments, the cancer is CD7 + Hematologic malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL, precursor T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and subsets of 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 malignancies or CD7 + CD1d + malignant tumor, and wherein optionally, CD7 + Cancer becomes resistant or refractory to treatment in the absence of the cells.

[0073] In some embodiments, the present disclosure provides methods for treating or preventing CD7 in a subject in need thereof. + A method for treating cancer, comprising administering to a subject a therapeutically effective amount of iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells), or a therapeutically effective amount of iNKT cells modified with CD7 CAR (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 +In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier.

[0074] In some embodiments, the present disclosure provides a method for treating or preventing CD7 in a subject in need thereof. + A method for treating cancer, the method comprising: (a) isolating one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + (b) using irradiated autologous PBMC negative fractions and / or whole PBMCs, α-galactosylceramide (αGalCer) or 7DW8-5 or other glycolipid analogs, IL-2 and IL-15 or IL-2, IL-7 and IL-15 in the growth medium, for one or more iTCRs (Vα24-Jα18) + iNKT cells are enabled or activated to proliferate; (c) the activated or proliferated iNKT cells are modified to express a CAR (e.g., CD7) using a lentiviral vector; and (d) one or more iNKT cells modified with a CAR (e.g., CD7) are expanded using irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5, IL-2 and IL-15 or IL-2, IL-7 and IL-15 in growth medium.

[0075] In some embodiments, the method further comprises modifying the one or more cells to express CAR, TCR or TCRm. In some embodiments, the modification comprises introducing one or more polynucleotides encoding CAR, TCR or TCRm into the one or more cells. In some embodiments, the introduction of one or more polynucleotides comprises electroporation, transduction and / or transfection. In some embodiments, one or more polynucleotides comprise mRNA and / or DNA. In some embodiments, DNA comprises transposon DNA. In some embodiments, one or more polynucleotides comprise one or more vectors. In some embodiments, one or more vectors comprise one or more viral vectors. In some embodiments, one or more vectors comprise one or more lentiviral vectors or γ-retroviral vectors.

[0076] In some embodiments, 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 includes 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, blood includes peripheral blood and / or umbilical cord blood. In some embodiments, iNKT cells are isolated from one or more peripheral blood mononuclear cells.

[0077] In some embodiments of the methods described herein, iNKT cells are modified to express a chimeric antigen receptor (CAR) (e.g., CD7 CAR). In some embodiments, the cell comprises one or more polynucleotides encoding CAR. In some embodiments, CAR comprises at least an antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0078] In some embodiments, the antigen binding domain of 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.

[0079] 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.

[0080] 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 is capable of binding to CD70, and the cancer is T-cell leukemia and lymphoma, multiple myeloma, AML, diffuse large B-cell and follicular lymphoma, Hodgkin lymphoma, or a variety of 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 is capable of binding to PD-L1, and the cancer is T-cell leukemia and lymphoma, multiple myeloma, AML, or a variety of solid tumors (e.g., glioma, lung cancer, breast cancer).

[0081] 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 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.

[0082] In some embodiments, the antigen binding domain of CAR includes an antibody or its antigen binding fragment, 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 (referred to as a nanobody). In some embodiments, the antigen binding fragment includes a single chain variable fragment (scFv).

[0083] In some embodiments, the intracellular signaling domain of CAR includes a functional signaling domain of at least one stimulatory molecule. In some embodiments, at least one stimulatory molecule comprises a ζ chain associated with a T cell receptor complex. In some embodiments, at least one stimulatory molecule comprises a CD3ζ chain. In some embodiments, the intracellular signaling domain also includes a functional signaling domain of at least one costimulatory molecule. In some embodiments, at least one costimulatory molecule includes 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10 or DAP12.

[0084] In some embodiments of the methods described herein, iNKT cells modified with CD7 CAR are further modified to express a T cell receptor (TCR). In some embodiments, the cell comprises one or more polynucleotides encoding TCR. In some embodiments, the TCR comprises at least an 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 Wilm's tumor 1 (WT1), an antigen preferentially expressed in melanoma (PRAME), a minor histocompatibility antigen (MiHA, such as HA-1), or a mutated nucleolar phosphoprotein 1 (ΔNPM1).

[0085] In some embodiments, the α chain and / or β chain of the TCR can bind to WT1, PRAME, HA-1 or ΔNPM1. In some embodiments, the α chain, β chain and / or TCR can bind to WT1. In some embodiments, the α chain, β chain and / or TCR can bind to PRAME. In some embodiments, the α chain, β chain and / or TCR can bind to HA-1. In some embodiments, the α chain, β 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.

[0086] In some embodiments of the methods described herein, iNKT cells modified by CD7 CAR are further modified to express T cell receptor mimic antibodies (TCRm). In some embodiments, the cells include one or more polynucleotides encoding TCRm. In some embodiments, TCRm at least includes an antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.

[0087] In some embodiments, the antigen binding domain of the TCRm is capable of binding to a composite antigen. In some embodiments, the composite antigen comprises a peptide and a human leukocyte antigen (HLA) molecule.

[0088] In some embodiments, the HLA molecule is an HLA class I molecule. In some embodiments, the HLA molecule is an HLA class II molecule.

[0089] In some embodiments, the peptide comprises a WT1 peptide. In some embodiments, the WT1 peptide comprises the 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.

[0090] In some embodiments, the peptide comprises an antigen preferentially expressed in a melanoma (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*0201 molecule. 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.

[0091] 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.

[0092] 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.

[0093] In some embodiments, the antigen binding domain of TCRm includes an antibody or its antigen binding fragment 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 includes a single chain variable fragment (scFv).

[0094] In some embodiments, the intracellular signaling domain of TCRm includes a functional signaling domain of at least one stimulatory molecule. In some embodiments, at least one stimulatory molecule comprises a ζ chain associated with a T cell receptor complex. In some embodiments, at least one stimulatory molecule comprises a CD3ζ chain. In some embodiments, the intracellular signaling domain also includes a functional signaling domain of at least one costimulatory molecule. In some embodiments, at least one costimulatory molecule includes 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10 or DAP12.

[0095] In some embodiments of the methods described herein, iNKT cells modified by CD7 CAR are further modified to include exogenous cytokines, growth factors, antibodies or antigen binding fragments, or any combination thereof. In some embodiments, the antibody or antigen binding fragment comprises a bispecific T cell engager (BiTE).

[0096] In some embodiments, the present invention also provides iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + In some embodiments, the present disclosure provides a pharmaceutical composition for treating or preventing CD7 + A pharmaceutical composition for cancer comprising iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) and at least one pharmaceutically acceptable carrier. In some embodiments, the iNKT cells modified by CD7 CAR in the pharmaceutical composition are CD3 + iTCR(Vα24-Jα18) + CD7CAR + iNKT cells (e.g., CD3 + CD4 + Cells, CD3 + CD4 - CD8 - 、CD3 + CD8 + Cells, CD3 + CD4 + CD8 +or a mixture thereof). In some embodiments, the iNKT cells modified with CD7 CAR in the pharmaceutical composition are CD3 + CD4 + In some embodiments, the iNKT cells modified by CD7CAR in the pharmaceutical composition are CD3 + In some embodiments, the iNKT cells modified with CD7 CAR in the pharmaceutical composition are CD3 + CD8 + In some embodiments, the iNKT cells modified with CD7 CAR in the pharmaceutical composition are CD3 + CD4 + CD8 + In some embodiments, the iNKT cells modified with CD7 CAR in the pharmaceutical composition are CD3 + CD4 + Cells, CD3 + CD4 - CD8 - Cells, CD3 + CD8 + and CD3 + CD4 + CD8 + cell.

[0097] In some embodiments, iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) or a pharmaceutical composition comprising the same for therapeutic use.

[0098] Certain Illustrative Embodiments

[0099] To facilitate understanding of the present disclosure, certain terms are defined throughout the detailed description. Unless otherwise defined herein, all scientific and technical terms used in connection with the present disclosure have the same meanings as commonly understood by one of ordinary skill in the art.

[0100] All references cited herein are also incorporated by reference in their entirety. To the extent a cited reference conflicts with the disclosure herein, the specification will control.

[0101] As used herein, the singular form of a word also includes the plural form, unless the context clearly dictates otherwise; as an example, the terms "a", "an", and "the" are understood to be singular or plural. For example, "element" means one or more elements. Unless the specific context indicates otherwise, the term "or" should mean "and / or". Unless the context indicates otherwise, all ranges include the endpoints and all points therebetween.

[0102] The term "about" or "approximately" as used herein in the context of numerical value and range refers to a value or range that is approximate or close to the value or range of the recorded value or range, so that the embodiment can be performed as expected, as apparent to the technician from the teachings contained herein. This is at least in part due to the different properties, age, race, sex, anatomical and physiological changes and the imprecision of biological systems of nucleic acid compositions. Therefore, the values ​​covered by these terms exceed those values ​​caused by systematic errors. In some embodiments, "about" or "approximately" means plus or minus (±) 10% of the numerical value, sometimes preferably ± 5%, and sometimes more preferably ± 2%.

[0103] NKT cells

[0104] In certain aspects, the present disclosure provides type I NKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells), and methods and compositions using the CD7CAR-modified iNKT cells described herein.

[0105] As used herein, the term "natural killer T cell" or "NKT cell" refers to a T cell or a population of T cells that exhibits the properties of both conventional T cells and natural killer (NK) cells. For example, in some embodiments, NKT cells are mature lymphocytes that carry both T cell receptors and NK cell receptors. In some embodiments, NKT cells are produced in the thymus by CD4 T cells that have undergone T cell receptor (TCR) gene rearrangement. + CD8 + Produced by cortical thymocytes.

[0106] There are two types of NKT cells in the literature. One classic classification of NKT cells refers to the NKT cells of a subgroup of unconventional T cells that recognize lipid antigens presented by MHC class I CD1d molecules, and NKT cells are divided 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). Another 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).

[0107] As used herein, the term "type I NKT cell" or "invariant NKT cell" or "iNKT cell" refers to a NKT cell or NKT cell population that expresses an invariant or semi-invariant TCR library and binds to a glycosphingolipid α-galactosylceramide (α-GalCer) associated with an MHC class I-like CD1d molecule. 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-Jα18TCR in mice, and Vα24-Jα18 in humans), which is paired with a limited library of Vβ chains (e.g., Vβ8.2, Vβ7, and Vβ2 in mice, and Vβ11 in humans). In some embodiments, type I NKT cells recognize sphingomyelin α-galactosylceramide (α-GalCer) or a synthetic analog thereof (e.g., 7DW8-5) when presented by an MHC class I-like CD1d molecule. In some embodiments, the terms "type I NKT cells" or "invariant NKT cells" or "iNKT cells" are used interchangeably.

[0108] As used herein, the term "iTCR" or "invariant TCR" refers to an invariant TCR expressed on iNKT cells and includes, but is not limited to, an invariant TCR α chain and a limited number of non-invariant TCR β chains that are paired with a limited library of V β chains (e.g., V β11 in humans) (e.g., V α24-J α18, V α24-J αQ, V α24).

[0109] 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 in the genome. Unlike lentiviral transduction expression of CARs that randomly insert genetic material into the host genome, genome editing locates the insertion or deletion targeting site to a specific location. 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.

[0110] In some embodiments, NKT cells (e.g., iNKT cells) are unicellular. In some embodiments, NKT cells (e.g., iNKT cells) are homogeneous cell populations. In some embodiments, NKT cells (e.g., iNKT cells) are heterogeneous cell populations. In some embodiments, NKT cells (e.g., iNKT cells) cause, stimulate and / or contribute to the production of at least one cytokine (e.g., IL-4 and / or IFN-γ). In some embodiments, NKT cells (e.g., iNKT cells) are cytotoxic. In some embodiments, NKT cells (e.g., iNKT cells) can show cytotoxicity to various cells (including cancer cells or cell lines (e.g., T-ALL cells or cell lines)), as described herein and exemplified.

[0111] 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, 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 + etc.) in combination.

[0112] In some embodiments, the iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + iNKT cells) can be obtained or isolated from a biological sample. In some embodiments, the iNKT cells of the present disclosure can be obtained or isolated from one or more peripheral blood mononuclear cells.

[0113] In some embodiments, the biological sample is from a human (e.g., a fetus, a newborn, a child, or an adult). In some embodiments, the biological sample is from a non-human animal. Non-human animals include all vertebrates (e.g., mammals and 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, such as 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 includes blood (e.g., peripheral blood and / or 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, biological samples and / or blood include peripheral blood and / or cord blood. In some embodiments, biological samples and / or blood are collected by separation transfusion and / or leukocyte removal (e.g., from a subject or a donor).

[0114] In some embodiments, the iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + iNKT cells) can be isolated, for example, from a human biological sample. In some embodiments, the iNKT cells are isolated iNKT cells.

[0115] As used herein, the term "isolated" refers to material that is 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 a living organism is not isolated, but the same polynucleotide, polypeptide, or cell, separated from some or all of the coexisting materials in the living organism, is isolated.

[0116] As used herein, "separated cells" refer to cells or cell groups (e.g., type I NKT cells or cell groups) that have been identified and separated from one or more (e.g., most) components of their source environment (e.g., components from cell culture or biological samples). In some embodiments, separation is performed so that components that otherwise may interfere with the suitability of the cell for a desired application (e.g., for therapeutic uses of type I NKT cells or cell groups) are fully removed. In some embodiments, separation is performed so that cells expressing a specific marker or marker set (e.g., iTCR (Vα24-Jα18)) are fully separated from cells expressing an alternative marker or marker set. Methods for separating cells are known in the art, and include, but are not limited to, separation by positive and / or negative selection techniques or by cell sorting, such as using antibody-conjugated microbeads, using a mixture of monoclonal antibodies for cell surface markers for flow cytometry, etc. Exemplary separation and separation techniques are described and illustrated herein.

[0117] In some embodiments, the iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + iNKT cells) can be separated or detached by affinity-based detachment methods. In some embodiments, exemplary techniques for affinity detachment may include magnetic detachment (e.g., using antibody-coated magnetic beads), affinity chromatography, cytotoxic agents (e.g., complement and cytotoxins) conjugated to or used in conjunction with monoclonal antibodies, and "panning" of antibodies attached to a solid matrix (e.g., a plate), or any other suitable technique. In some embodiments, detachment techniques may also include the use of fluorescence-activated cell sorting agents, which may have varying degrees of complexity, such as multicolor channels, low-angle and obtuse-angle scattering detection channels, impedance channels, and the like. It should be understood that any method capable of detaching iNKT cells (e.g., iTCR (Vα24-Jα18)) may be used. + iNKT cells) isolation or separation technology.

[0118] In some embodiments, the affinity reagents used for various separation or detachment methods can be specific receptors or ligands for cell surface markers on iNKT cells. In some embodiments, the antibody can be conjugated to a label, which in some embodiments can be used for separation or detachment. In some embodiments, the label can include magnetic beads (e.g., which can allow direct detachment), biotin (e.g., which can be removed together with avidin or streptavidin bound to, for example, a support; for example, biotin-conjugated anti-TCRVα24-Jα18 (6B11 clone) or anti-Vα24 antibody combined with streptavidin microbeads), fluorescent dyes (e.g., which can be used with fluorescence-activated cell sorting agents such as phycoerythrin, fluorescein, Texas red, or a combination thereof), etc.

[0119] In some embodiments, the cell detachment using an antibody may include adding the antibody to a cell suspension, for example, continuing a time period sufficient to bind available cell surface markers. Incubation may continue for different time periods. For example, in some embodiments, incubation may continue for about 2 minutes, about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes or longer. It may be considered that any time length resulting in antibody specific labeling and having minimum non-specific binding is used for this aspect of the present disclosure. In some embodiments, the staining intensity of iNKT cells may be monitored by flow cytometry, for example, wherein the quantitative level of a laser-detected fluorescent dye (which may be proportional to the amount of the cell surface antigen bound by the antibody) is proportional. In some embodiments, flow cytometry or FACS may also be used to separate cell populations based on the intensity of antibody staining and other parameters (such as cell size and light scattering).

[0120] In some embodiments, iNKT cells are separated based on the expression of at least one cell surface marker. The separated cells can be collected in any suitable culture medium that maintains cell viability. In some embodiments, the culture containing cells may contain serum, cytokines or growth factors to which the cells respond. In some embodiments, cytokines or growth factors can promote cell survival, growth, function or a combination thereof. In some embodiments, cytokines and growth factors can include polypeptides and non-polypeptide factors.

[0121] As used herein, the term "activated" refers to a process in which the separated iNKT cells or conventional T cells are active (e.g., proliferative, cytokine-releasing or cytolytic). In some embodiments, the separated iNKT cells or conventional T cells are activated by co-culturing with a T cell receptor (TCR) engagement reagent (e.g., microbeads coated with antibodies to CD2, CD3 and CD28 or soluble anti-CD3 antibodies). In some embodiments, the separated iNKT cells are activated by co-culturing with an irradiated autologous PBMC negative fraction, αGalCer or 7DW8-5, IL-2, IL-15 and / or IL-7 in human T cell culture medium.

[0122] In some embodiments, iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + iNKT cells) are modified to express a CD7CAR construct capable of binding to a CD7 target antigen. In some embodiments, the iNKT cells modified with CD7 CAR of the present disclosure are further modified to express a chimeric antigen receptor (CAR), a T cell receptor (TCR), a T cell receptor mimetic antibody (TCRm), or any combination thereof, to target CD7 + Additional antigens on cancer.

[0123] In some embodiments, iNKT cells are modified to express chimeric antigen receptors (CAR). In some embodiments, CAR can be engineered using antigen binding domains so that when CAR is expressed on cells (e.g., iNKT cells), CAR and / or cells bind to target antigens (e.g., CD7 or another exemplary antigen described herein). In some embodiments, the CAR sequence is cloned into cells or cell groups (e.g., iNKT cells or iNKT cell groups), and amplified using our own irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5 and mixed cytokines (IL-2, IL-15 or IL-2, IL-7 and IL-15) and / or currently available schemes. In some embodiments, cells or cell groups include one or more polynucleotides encoding CAR. In some embodiments, cells or cell groups are from donors or patients (e.g., patients with or suspected of having cancer, such as T-ALL or another exemplary cancer described herein). In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a patient, the CAR-modified cell or cell group can be administered to the same patient and / or another patient in need of such treatment. In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a donor, the CAR-modified cell or cell group can be administered to any patient in need of such treatment.

[0124] 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 a sequence of a CAR peptide and can transcribe, translate, and express the CAR peptide on the cell surface. In some embodiments, the cell or cell population expressing CAR includes an iNKT cell or cell population. In some embodiments, the cell or cell population expressing CAR includes an iTCR (Vα24-Jα18) + iNKT cells or cell populations. In some embodiments, the cells or cell populations expressing CAR include CD3 + iTCR(Vα24-Jα18) + iNKT cells or cell populations. In some embodiments, the cells or cell populations expressing CAR include CD3 + iTCR(Vα24-Jα18) + CD4 + Cell or cell population. In some embodiments, the cell or cell population expressing CAR includes CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 -Cell or cell population. In some embodiments, the cell or cell population expressing CAR includes CD3 + iTCR(Vα24-Jα18) + CD8 + Cell or cell population. In some embodiments, the cell or cell population expressing CAR includes CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + Cell or cell population. In some embodiments, the cell or cell population expressing CAR includes a mixture of cells, such as 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 +Mixture of cells. In some embodiments, when used as a therapeutic agent, the CAR-expressing cells or cell groups administered to the subject may include CAR-modified NKT cells or CAR-modified NKT cell groups from the subject. In some embodiments, when used as a therapeutic agent, the CAR-expressing cells or cell groups administered to the subject may include CAR-modified NKT cells or CAR-modified NKT cell groups from a donor.

[0125] In some embodiments, CAR-modified cells or cell groups can engage with cells (e.g., malignant cancer cells) expressing target antigens (e.g., CD7) and kill the cells. Provided herein are methods and compositions for carrying out and administering disclosed CAR-based immunotherapies. Exemplary methods for carrying out CAR-based immunotherapies are also disclosed in, for example, U.S. Publication Nos. 2014 / 0271635 and 2016 / 0310532, both of which are incorporated herein by reference for such methods.

[0126] As used herein, the terms "chimeric antigen receptor" and "CAR" refer to a polypeptide or a group of polypeptides that, when expressed by a cell, provide the cell with specificity and intracellular signal generation for target antigen-expressing cells (e.g., malignant cancer cells). In some embodiments, CAR comprises at least an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, CAR comprises at least an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain includes a functional signal transduction domain derived from a stimulatory molecule and / or a costimulatory molecule. These domains may be present in a single polypeptide or a group of polypeptides. In some embodiments, the stimulatory molecule is a ζ chain associated with a T cell receptor complex. In some embodiments, the costimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, and / or DAP12.

[0127] In some embodiments, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain, the 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 co-stimulatory molecule; or (iii) a functional signaling domain derived from a stimulatory molecule and at least two functional signaling domains derived from one or more co-stimulatory molecules. In some embodiments, CAR includes an optional leader sequence at the N-terminus of the CAR fusion protein. In some embodiments, CAR also includes a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein during cell processing and CAR localization to the cell membrane, the optional leader sequence is cut from the antigen binding domain.

[0128] In some embodiments, the antigen binding domain of CAR includes an antibody or an antigen binding fragment thereof. In some embodiments, the antigen binding domain and / or antibody includes a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody or a single domain antibody (also referred to as a nanobody). In some embodiments, the antigen binding domain and / or antigen binding fragment includes a single chain variable fragment (scFv) or a Fab fragment. In some embodiments, the antigen binding domain and / or antigen binding fragment includes a scFv.

[0129] As used herein, the term "antibody" refers to any functional immunoglobulin molecule that recognizes and binds (e.g., specifically binds) a target (such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. The term "antibody" encompasses antibodies with sequences from any source species, such as mice, rabbits, goats, llamas, alpacas, non-human primates, and humans. The term also encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecules containing antigen recognition sites, as long as they exhibit the desired binding and / or biological activity. In some embodiments, the antibody has the ability to bind (e.g., specifically bind) to a target antigen expressed on a cancer cell (e.g., CD7). Antibodies can be produced using any suitable technology, such as recombinant expression, hybridoma technology, ribosome display, phage display, gene shuffling libraries, semisynthetic or fully synthetic libraries, or any combination thereof. The term "antibody" includes full-length antibodies and antigen-binding domains and antigen-binding fragments thereof. In some embodiments, the antibodies used in CAR and / or other constructs described herein are full-length or complete antibodies. In some embodiments, the antibodies used in 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 CAR and / or other constructs described herein are antigen binding domains or antigen binding fragments of antibodies.

[0130] "Full-length" or "complete" antibodies typically comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The recognized classes of immunoglobulin genes encoding antibody chains include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. In some embodiments, the antibody comprises a κ light chain. In some embodiments, the antibody comprises a λ light chain. The κ or λ light chain can be selected from any κ or λ light chain sequence from any species. The heavy chain is classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin class: 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.

[0131] Antibodies that can be used in CAR and / or other constructs described herein also include antigen binding fragments. As used herein, the term "antigen binding fragment" or "antigen binding portion" of an antibody refers to the ability of a full-length antibody to retain binding to a target antigen (e.g., CD7) (e.g., specific binding) and / or provide the function of a full-length antibody (e.g., ability to specifically bind to CD7) One or more fragments. The antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Fragments can also be present in larger macromolecules, such as bispecific antibodies. Examples of such antibody fragments include Fab fragments (monovalent fragments comprising at least the VL, CL, VH and CH1 domains); F(ab)2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region); Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; single domain antibody (SdAb) fragments, which consist of either a VH domain or a VL domain; isolated complementarity determining regions (CDRs); and hemibodies, which contain only one heavy chain and one light chain rather than the typical pairing of two heavy chains and two light chains on separate arms. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be made into a single protein chain using recombinant methods, such as by an artificial peptide linker, which enables them to be made into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (called a single-chain variable fragment (scFv)) (see, for example, 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), which are composed 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 scFv. In some embodiments, the antigen-binding fragment is a scFv.

[0132] In some embodiments, the antigen binding domain of CAR includes a cell binding agent. In some embodiments, the antigen binding domain and / or cell binding agent of CAR include DARPin, duobody, bicyclic peptide, nano antibody, centyrin, MSH (melanocyte stimulating hormone), receptor-Fc fusion molecule, T cell receptor structure, natural ligand (e.g., receptors expressed in mature non-malignant and / or malignant B cells, including plasma cells, such as exemplary ligands of B cell maturation antigen (BCMA), including but not limited to B cell activation factor (BAFF) and proliferation inducing ligand (APRIL)), steroid hormones (e.g., androgen or estrogen), growth factors, colony stimulating factors (e.g., EGF) or other non-antibody scaffolds. In some embodiments, non-antibody scaffolds can be broadly divided into two structural categories, namely, compounds of domain size (about 6-20kDa) and constrained peptides (about 2-4kDa). Exemplary domain-sized scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (e.g., adnectin and centyrin), Fynomers, Kunitz domains, probentins, O-bodies, and receptor-Fc fusion proteins, and exemplary restricted peptides include high-affinity polymers (avimers), bicyclic peptides, and cysteine-disulfide knots (Cys-knots). In some embodiments, the antigen binding domains and / or cell binding agents of CAR include affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds such as adnectin and centyrin, Fynomers, Kunitz domains, probentins, O-bodies, and receptor-Fc fusion proteins, high-affinity polymers (avimers), bicyclic peptides, and / or cysteine-sulfur knots (Cys-knots). Non-antibody scaffolds are reviewed, for example, in Vazquez-Lombardi et al., Drug Dis Today 2015; 20(10): 1271-83.

[0133] In some embodiments, the antigen binding domain of 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.

[0134] 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 (e.g., CD7).The term encompasses 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: P01850; 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), CD10 (e.g., UniProt reference sequence: P10692; SEQ ID NO: 10), CD11 (e.g., UniProt reference sequence: P11814; SEQ ID NO: 11), CD12 (e.g., UniProt reference sequence: P10693; SEQ ID NO: 12), CD13 (e.g., UniProt reference sequence: P11829; SEQ ID NO: 13), CD14 (e.g., UniProt reference sequence: P11830; SEQ ID NO: 14), CD15 (e.g., UniProt reference sequence: P11831; SEQ ID NO: 15 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: 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: O43699; SEQ ID NO: 17), CD70 (e.g., UniProt Reference Sequence: P32970; SEQ ID NO: 18), 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 the antigen (e.g., CD7) that can be produced by cell processing. The term also encompasses 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 encompassed unless the context indicates that the term is used to refer only to the wild-type protein).The antigen (eg, CD7) can be isolated from a human, or can be produced recombinantly or by synthetic methods.

[0135] In some embodiments, iNKT cells modified by CD7 CAR are modified to express T cell receptors (TCR). In some embodiments, antigen binding α chains and / or β chains can be used to engineer TCR so that when TCR is expressed on cells (e.g., iNKT cells modified by CD7 CAR), TCR and / or cells bind to target antigens (e.g., WT1 or another exemplary antigen described herein). In some embodiments, TCR sequences are cloned into cells or cell populations (e.g., iNKT cells or iNKT cell populations modified by CD7 CAR), and amplified using our own irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5, and mixed cytokines (IL-2, IL-15 or IL-2, IL-7, and IL-15) and / or currently available protocols. In some embodiments, cells or cell populations contain one or more polynucleotides encoding TCR. In some embodiments, the cell or cell group is from a donor or patient (e.g., a patient suffering from or suspected of having cancer, such as T-ALL or another exemplary cancer described herein). In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a patient, the TCR-modified cell or cell group can be administered to the same patient and / or another patient in need of such treatment. In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a donor, the TCR-modified cell or cell group can be administered to any patient in need of such treatment.

[0136] 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 a sequence of a TCR peptide and can transcribe, translate, and express the TCR peptide on the cell surface. In some embodiments, the cell or cell population expressing the TCR includes an iNKT cell or cell population modified with a CD7 CAR. In some embodiments, the cell or cell population expressing the TCR includes a CD3 + iTCR(Vα24-Jα18) + CD4 + In some embodiments, the cell or cell population expressing the TCR comprises CD3 + iTCR(Vα24-Jα18) + CD4 - CD8 - In some embodiments, the cell or cell population expressing the TCR comprises CD3 +iTCR(Vα24-Jα18) + CD8 + In some embodiments, the cell or cell population expressing the TCR comprises CD3 + iTCR(Vα24-Jα18) + CD4 + CD8 + In some embodiments, the cell or cell population expressing a TCR comprises a mixture of cells, such as 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 group administered to the subject may include a TCR-modified NKT cell or a TCR-modified NKT cell group from the subject. In some embodiments, when used as a therapeutic agent, the TCR-expressing cell or cell group administered to the subject may include a TCR-modified NKT cell or a TCR-modified NKT cell group from a donor.

[0137] In some embodiments, cells or cell groups modified by TCR can engage with cells (e.g., malignant cancer cells) expressing target antigens (e.g., WT1) and kill the cells. Provided herein are methods and compositions for conducting and administering disclosed TCR-based immunotherapies. Exemplary methods for conducting TCR-based immunotherapies are also disclosed in, for example, U.S. Patent No. 9,115,372, which is incorporated herein by reference for such methods.

[0138] As used herein, the terms "T cell receptor" and "TCR" refer to a polypeptide or a group of polypeptides that, when expressed by a cell, provide the cell with specificity and intracellular signal generation for a target antigen expressing cell (e.g., a malignant cancer cell). In some embodiments, the TCR comprises at least an alpha chain and a beta chain. These chains may be present in a single polypeptide or a group of polypeptides. In some embodiments, the alpha chain and / or the beta chain are capable of binding to an antigen.

[0139] In some embodiments, TCR comprises α chain and β chain. In some embodiments, both α chain and β chain comprise constant region (c) and variable region (v). In some embodiments, variable region determines antigen specificity. In some embodiments, variable region recognizes target antigen, for example, comprises antigen ligand of short continuous amino acid sequence of protein presented on target cell by major histocompatibility complex (MHC) molecule (also referred to as human leukocyte antigen (HLA) molecule). In some embodiments, it is also related to auxiliary adhesion molecules expressed by T cells, such as CD4 for MHC class II and CD8 for MHC class I. In some embodiments, signal transduction of TCR is by the constant CD3 complex of association. In some embodiments, CD3 complex comprises different CD3 proteins forming two heterodimers (CD3δε and CD3γε) and one homodimer (CD3ζζ).

[0140] In some embodiments, the α chain and / or β chain of the TCR is capable of binding to an antigen. In some embodiments, the α chain and / or β chain is capable of binding to WT1, PRAME, HA-1 or ΔNPM1.

[0141] As used herein, the terms "WT1, PRAME, HA-1 and ΔNPM1" refer to any natural form of human WT1, PRAME, HA-1 and ΔNPM1. The terms encompass 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 form of human WT1, PRAME, HA-1 and ΔNPM1 that can be produced by cell processing. The term also encompasses 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 encompassed unless the context indicates 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 can be produced recombinantly or by synthetic methods.

[0142] In some embodiments, iNKT cells expressing CD7 CAR are further modified to express T cell receptor mimic antibodies (TCRm). In some embodiments, antigen binding domains can be used to engineer TCRm so that when TCRm is expressed on cells (e.g., iNKT cells), TCRm and / or cells bind to target antigens (e.g., composite antigens, e.g., composite antigens comprising peptides and human leukocyte antigen (HLA) molecules, as described herein). In some embodiments, TCRm sequences are cloned into cells or cell groups (e.g., iNKT cells or iNKT cell groups), and amplified using our own irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5 and mixed cytokines (IL-2, IL-15 and / or IL-7) and / or currently available schemes. In some embodiments, cells or cell groups include one or more polynucleotides encoding TCRm. In some embodiments, the cell or cell group is from a donor or patient (e.g., a patient with or suspected of having cancer, such as T-ALL or another exemplary cancer described herein). In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a patient, the cell or cell group modified by TCRm can be administered to the same patient and / or another patient in need of such treatment. In some embodiments, when used as a therapeutic agent, and when the cell or cell group is from a donor, the cell or cell group modified by TCRm can be administered to any patient in need of such treatment.

[0143] 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 a sequence of a TCRm peptide and can transcribe, translate, and express the TCRm peptide on the cell surface. In some embodiments, the cell or cell population expressing TCRm includes an iNKT cell or cell population modified with a CD7 CAR. In some embodiments, the cell or cell population expressing TCRm includes a CD3 + iTCR(Vα24-Jα18) + CD4 + In some embodiments, the cell or cell population expressing TCR m comprises CD3 + iTCR(Vα24-Jα18) + CD4-CD8 - In some embodiments, the cell or cell population expressing TCR m comprises CD3 + iTCR(Vα24-Jα18) + CD8 + In some embodiments, the cell or cell population expressing TCRm comprises a mixture of cells, such as 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 cells or cell groups expressing TCRm administered to the subject may include NKT cells modified by TCRm from the subject or NKT cell groups modified by TCRm. In some embodiments, when used as a therapeutic agent, the cells or cell groups expressing TCRm administered to the subject may include NKT cells modified by TCRm from the donor or NKT cell groups modified by TCRm.

[0144] In some embodiments, cells or cell groups modified by TCRm can engage with and kill cells (e.g., malignant cancer cells) expressing target antigens (e.g., composite antigens, e.g., composite antigens comprising peptides and human leukocyte antigen (HLA) molecules, as described herein). Provided herein are methods and compositions for conducting and administering disclosed TCRm-based immunotherapies. Exemplary methods for conducting TCRm-based immunotherapies are also disclosed, for example, in U.S. Publication No. 2019 / 092876, which is incorporated herein by reference for such methods.

[0145] As used herein, the terms "T cell receptor mimicking antibody" and "TCRm" refer to a polypeptide or a group of polypeptides that provide cells with specificity and intracellular signal generation for target antigen expressing cells (e.g., malignant cancer cells) when expressed by cells. In some embodiments, TCRm at least comprises an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain. In some embodiments, TCRm at least comprises an extracellular antigen binding domain, a hinge domain, a transmembrane domain, and an intracellular signal transduction domain, and the intracellular signal transduction domain includes a functional signal transduction domain derived from a stimulatory molecule and / or a costimulatory molecule. These domains may be present in a single polypeptide or a group of polypeptides. In some embodiments, the stimulatory molecule is a ζ chain associated with a T cell receptor complex. In some embodiments, the costimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10, and / or DAP12.

[0146] In some embodiments, the antigen binding domain of TCR m includes antibody or its antigen binding fragment. In some embodiments, antigen binding domain and / or antibody include monoclonal antibody, polyclonal antibody, synthetic antibody, human antibody, humanized antibody or single domain antibody. In some embodiments, antigen binding domain and / or antigen binding fragment include single chain variable fragment (scFv) or Fab fragment. In some embodiments, antigen binding domain and / or antigen binding fragment include scFv.

[0147] In some embodiments, the antigen binding domain of TCRm comprises a cell binding agent. In some embodiments, the antigen binding domain of TCRm and / or the cell binding agent include DARPin, duobody, bicyclic peptide, nano antibody, centyrin, MSH (melanocyte stimulating hormone), receptor-Fc fusion molecule, T cell receptor structure, natural ligand (e.g., receptor expressed in mature non-malignant and / or malignant B cells, including plasma cells, such as exemplary ligands of B cell maturation antigen (BCMA), including but not limited to B cell activation factor (BAFF) and proliferation inducing ligand (APRIL)), steroid hormones (e.g., androgen or estrogen), growth factors, colony stimulating factors (e.g., EGF) or other non-antibody scaffolds. In some embodiments, the antigen binding domain and / or cell binding agent of TCRm includes an affibody, an afrin, an anticalin, an atretibody, a DARPin, a FN3 scaffold such as an adnectin and a centyrin, a phenobody, a Kunitz domain, a pronectin, an O-body and a receptor-Fc fusion protein, a high affinity multimer, a bicyclic peptide and / or a cysteine-sulfur knot.

[0148] In some embodiments, the antigen binding domain of a TCRm is capable of binding to a complex antigen comprising a peptide and a human leukocyte antigen (HLA) molecule.

[0149] In some embodiments, the HLA molecule is a class I HLA molecule. In some embodiments, the HLA molecule is a class I HLA binding peptide. In some embodiments, the class I HLA binding peptide is about 9 or 10 amino acids in length. Exemplary class I HLA binding peptides include, but are not limited to: HLA-A*0201 binding peptide RMFPNAPYL derived from WT1 (SEQ ID NO: 26); HLA-A*2402 binding peptide CMTWNQMNL derived from WT1 (SEQ ID NO: 27); HLA-A*0201 binding peptide VLDGLDVLL derived from PRAME (SEQ ID NO: 28); HLA-A*0201 binding peptide ALYVDSLEFL derived from PRAME (SEQ ID NO: 29); HLA-A*0201 binding peptide SLYSFPEPEA derived from PRAME (SEQ ID NO: 30); HLA-A*0201 binding peptide SLLQHLIGL derived from PRAME (SEQ ID NO: 31); HLA-A*2402 binding peptide LYVDSLFFLC derived from PRAME (SEQ ID NO: 32); HLA-A*0201 binding peptide VLHDDLLEA derived from HA-1 (SEQ ID NO: 33); NO: 33); HLA-A*0201 binding peptide CLAVEEVSL (SEQ ID NO: 34) derived from ΔNPM1.

[0150] In some embodiments, the antigen binding domain of TCRm is capable of binding to a complex antigen comprising an HLA molecule having the following amino acid sequence: RMFPNAPYL (SEQ ID NO: 26); CMTWNQMNL (SEQ ID NO: 27); VLDGLDVLL (SEQ ID NO: 28); ALYVDSLEFL (SEQ ID NO: 29); SLYSFPEPEA (SEQ ID NO: 30); SLLQHLIGL (SEQ ID NO: 31); LYVDSLFFLc (SEQ ID NO: 32); VLHDDLLEA (SEQ ID NO: 33); and / or CLAVEEVSL (SEQ ID NO: 34).

[0151] In some embodiments, the HLA molecule is a class II HLA molecule. In some embodiments, the HLA molecule is a class II HLA binding peptide. In some embodiments, the class II HLA binding peptide is about 13 to about 25 amino acids in length. Exemplary class II HLA binding peptides include, but are not limited to: HLA-DRB1*0405 derived from WT1, HLA-DRB1*1501 derived from WT1, HLA-DRB1*1502 derived from WT1, HLA-DPB1*0501 derived from WT1, and HLA-DPB1*0901 binding peptide KRYFKLSHLQMHSRKH (SEQ ID NO: 35) derived from WT1.

[0152] In some embodiments, the antigen binding domain of the TCRm is capable of binding to a composite antigen comprising an HLA molecule having the amino acid sequence of KRYFKLSHLQMHSRKH (SEQ ID NO: 35).

[0153] In some embodiments, the iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) are further modified to contain exogenous cytokines, growth factors, antibodies or antigen binding fragments, or any combination thereof. In some embodiments, the antibody or antigen binding fragment comprises a bispecific T cell engager (BiTE).

[0154] Table 1. Exemplary target antigen amino acid and ScFv sequences

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Methods of treatment, uses and compositions

[0165] iNKT cells modified with CD7 CAR described herein (e.g., CD3 + iTCR(Vα24-Jα18) + , or CD3 + Vα24 + iNKT cells, also known as type I NKT cells, can be used for various therapeutic and preventive applications. For example, in some embodiments, the iNKT cells modified with CD7 CAR described herein can be used to treat or prevent CD7 + Cancer. The CD7 CAR-modified iNKT cells described herein may be administered by themselves or in any suitable pharmaceutical composition.

[0166] Thus, in certain aspects, the present disclosure provides methods for treating a subject with a therapeutically effective amount of iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) to treat or prevent CD7 + In certain aspects, the present disclosure also provides a method for treating or preventing CD7 in a subject in need thereof by: + Method for treating cancer by: (a) isolating one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells); (b) using, but not limited to, irradiated autologous PBMC negative fractions and / or whole PBMCs, α-galactosylceramide (αGalCer) or 7DW8-5, IL-2, IL-15 and / or IL-7 in growth medium, one or more iTCR cells are enabled or activated to proliferate; (c) using lentiviral vectors to engineer activated or proliferated iNKT cells to express CAR (e.g., CD7); and (d) using our own irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5 and mixed cytokines (IL-2, IL-15 and / or IL-7) in growth medium, one or more CAR (e.g., CD7) engineered iNKT cells are expanded. In certain aspects, the present disclosure also provides a method of administering a therapeutically effective amount of an expanded cell population (e.g., iNKT cells modified with CD7 CAR (e.g., CD3) as described herein and / or prepared by the methods described herein) to a subject.+ iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) to treat or prevent CD7 + Also provided are methods for treating or preventing CD7 cancer by using the disclosed iNKT cells modified with CD7 CAR, for example + Also disclosed is a method of treating a patient with a CD7 CAR-modified iNKT cell (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The invention relates to a pharmaceutical composition for the treatment of NKT cells, and the pharmaceutical composition can be used in the methods of treatment and uses provided herein.

[0167] One exemplary embodiment is to treat or prevent CD7 in a subject in need thereof. + A method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells), or a therapeutically effective amount of iNKT cells modified with CD7CAR (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + A pharmaceutical composition comprising iNKT cells) and at least one pharmaceutically acceptable carrier.

[0168] Another exemplary embodiment is a method for treating or preventing CD7 in a subject in need thereof. + A method for treating cancer, the method comprising: (a) isolating one or more iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + (b) using, but not limited to, irradiated autologous PBMC negative fractions and / or whole PBMCs, α-galactosylceramide (αGalCer) or 7DW8-5, IL-2, IL-15 and / or IL-7 in the growth medium to one or more iTCRs +iNKT cells are enabled or activated to proliferate; (c) the activated or proliferating iNKT cells are engineered to express a CAR (e.g., CD7) using a lentiviral vector; and (d) one or more iNKT cells engineered with a CAR (e.g., CD7) are expanded using our own regimen of irradiated allogeneic PBMCs and EBV-B cells, αGalCer or 7DW8-5, and a cocktail of cytokines (IL-2, IL-15, and / or IL-7) in growth medium.

[0169] Another exemplary embodiment is to treat or prevent CD7 in a subject in need thereof. + A method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of an expanded cell population (e.g., comprising iNKT cells modified with a CD7 CAR (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells)), or a therapeutically effective amount of an expanded cell population (e.g., comprising iNKT cells modified with a CD7 CAR (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + A pharmaceutical composition comprising an expanded cell population of iNKT cells) and at least one pharmaceutically acceptable carrier.

[0170] Another exemplary embodiment is an isolated iNKT cell (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) and modified to express CD7 CAR for treating or preventing CD7 in a subject in need thereof + Cancer. In some embodiments, the use comprises administering to a subject a therapeutically effective amount of cells, or a pharmaceutical composition comprising a therapeutically effective amount of cells and at least one pharmaceutically acceptable carrier.

[0171] Another exemplary embodiment is an isolated iNKT cell (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) to treat or prevent CD7+ Use in cancer. In some embodiments, the use comprises administering to a subject a therapeutically effective amount of cells, or a pharmaceutical composition comprising a therapeutically effective amount of cells and at least one pharmaceutically acceptable carrier.

[0172] Another exemplary embodiment is an isolated iNKT cell (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells) in the preparation for treating or preventing CD7 + Use in a cancer medicine. In some embodiments, the medicine comprises a therapeutically effective amount of cells, or a pharmaceutical composition comprising a therapeutically effective amount of cells and at least one pharmaceutically acceptable carrier.

[0173] As used herein, the term "treatment" and its homonyms refer to the improvement of a disease, disorder or condition (e.g., cancer) or at least one perceptible symptom thereof. The term "treatment" includes, but is not limited to, complete treatment or complete improvement of one or more symptoms of cancer. In some embodiments, "treatment" refers to at least a partial improvement of at least one measurable physical parameter, which may not necessarily be perceptible to the subject. In some embodiments, "treatment" refers to physically (e.g., stabilizing perceptible symptoms), physiologically (e.g., stabilization of physical parameters), or both to inhibiting the progression of a disease, disorder or condition. In some embodiments, "treatment" refers to slowing the progression of a disease, disorder or condition or reversing the progression of a disease, disorder or condition. As used herein, "treatment" and its homonyms also encompass delaying the onset or reducing the risk of suffering from a given disease, disorder or condition. In some embodiments, "treatment" refers to administering the iNKT cells, cell populations, or compositions modified by CD7 CAR disclosed herein to a subject suspected of having a disease, disorder, or condition (e.g., cancer or a precancerous condition). In some embodiments, a subject suspected of having CD7 + Subjects with cancer and / or precancerous conditions and / or from individuals suspected of having CD7 + A sample from a subject with cancer and / or a precancerous condition may contain one or more abnormal, malignant and / or precancerous cells.

[0174] The terms "subject" and "patient" are used interchangeably herein and 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.

[0175] As used herein, the term "donor" refers to any human or non-human animal that donates a biological sample (e.g., a blood sample) for a subject in need of treatment and / or for preparing a therapeutic agent (e.g., a CD7 CAR-modified iNKT cell therapeutic disclosed herein) for a subject in need of treatment. In some embodiments, the donor is a human.

[0176] As used herein, the term "cancer" refers to the presence of cells with typical properties of cells that cause cancer (such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and / or certain morphological characteristics). Cancer cells can be in the form of tumors or masses, but such cells can exist alone in a subject, or can circulate in the bloodstream as independent cells (such as leukemia or lymphoma cells). The term "cancer" includes all types of cancer and cancer metastasis, including hematological malignancies, solid tumors, sarcomas, carcinomas and other solid and non-solid tumor cancers.

[0177] As used herein, the term "CD7 + The term "cancer" refers to cancer cells that express the CD7 antigen on the cell surface, and includes, but is not limited to, T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL, precursor T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and a subset of 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 malignancies or CD7 + CD1d + In some embodiments, the CD7+ cancer is T-ALL or AML. In some embodiments, the CD7+ cancer is + 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.

[0178] As used herein, the term "target antigen" refers to any antigen targeted by a construct expressed by iNKT cells modified by CD7 CAR and / or by iNKT cells (e.g., CAR, TCR or TCR). As used herein, the term "antigen" is synonymous with "antigenic determinant" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a continuous stretch of amino acids or a conformational configuration consisting of different regions of non-continuous amino acids), and an antigen binding portion (e.g., an antigen binding portion of CAR, TCR or TCR) binds thereto to form an antigen binding portion-antigen complex. For example, useful antigenic determinants can be found 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).

[0179] 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. The target antigen may include a full-length antigen (e.g., any exemplary antigen disclosed herein), as well as any form of antigen that can be produced by cell processing. The target antigen also encompasses functional variants or fragments of an antigen (e.g., any exemplary antigen 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 encompassed unless the context indicates that the term is only used to refer to a wild-type antigen). In some embodiments, the target antigen is a functional fragment of a full-length antigen.

[0180] In some embodiments of the methods and uses described herein, iNKT cells modified with CD7 CAR are formulated and / or used as pharmaceutical compositions. Thus, in certain aspects, the present disclosure provides pharmaceutical compositions comprising iNKT cells modified with CD7 CAR. An exemplary embodiment is, for example, for treating or preventing CD7 in a subject in need thereof. + A pharmaceutical composition for cancer comprising iNKT cells (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 +iNKT cells). In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise one or more additional therapeutic agents suitable for treating or preventing, for example, cancer (e.g., anticancer agents, standard care agents for the specific cancer being treated, etc.). The pharmaceutical composition may also comprise one or more inactive carriers, excipients, and / or stabilizer components, etc. Methods for preparing 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.

[0181] As used herein, a "pharmaceutical composition" refers to a NKT cell or cell population (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + The pharmaceutical compositions provided herein are in a form that allows administration and subsequently provides the expected biological activity of the active ingredient and / or achieves a therapeutic effect. The pharmaceutical compositions provided herein do not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0182] As used herein, the phrases "pharmaceutically acceptable carrier" and "physiologically acceptable carrier", which can be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the administered iNKT cells or cell populations or any other therapeutic agent in the composition. A pharmaceutically acceptable carrier can enhance or stabilize the composition and / or can be used to facilitate the preparation of the composition. A pharmaceutically acceptable carrier can include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. The carrier can be selected to minimize adverse side effects to the subject and / or minimize degradation of the active ingredient. An adjuvant may also be included in any of these formulations.

[0183] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of an active ingredient. Formulations for parenteral administration may, for example, contain an excipient such as sterile water or saline, a polyalkylene glycol (such as polyethylene glycol), a vegetable oil, or hydrogenated naphthalene. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and various 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 to be pharmaceutically acceptable carriers or excipients.

[0184] The pharmaceutical compositions of the present disclosure can be administered by various methods known in the art. The route and / or mode of administration may vary depending on the desired result. In some embodiments, the 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., by injection or infusion).

[0185] iNKT cells or cell populations modified with CD7 CAR (e.g., CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells or cell groups) can be administered alone or in combination with at least one additional therapeutic agent (e.g., an anticancer agent, a standard of care agent for a specific cancer being treated, etc.), and can be administered in any acceptable formulation, dosage, or dosing regimen. When administered in combination with an additional therapeutic agent, the additional therapeutic agent can be administered according to its standard dose and / or dosing regimen. Alternatively, the additional therapeutic agent can be administered in a higher or lower amount and / or frequency compared to the standard dose and / or dosing regimen of the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered in a lower amount and / or frequency.

[0186] As used herein, the term "preparation" refers to a compound, a mixture of compounds, a biomacromolecule, or an extract made of a biological material. The term "therapeutic agent" refers to a preparation that can regulate a biological process and / or has biological activity. The iNKT cells and cell populations modified by CD7 CAR described herein are exemplary therapeutic agents. Additional therapeutic agents (e.g., those therapeutic agents that can be administered in combination with iNKT cells or cell populations modified by CD7 CAR described herein) may include any active ingredient suitable for the specific indication (e.g., cancer) to be treated, for example, those therapeutic agents that have complementary activities and do not adversely affect each other.

[0187] Typically, a therapeutically effective dose of iNKT cells or cell populations modified by CD7 CAR is used in the pharmaceutical composition of the present disclosure. iNKT cells or cell populations modified by CD7 CAR can be formulated into pharmaceutically acceptable dosage forms by conventional methods known in the art.

[0188] The dosage regimen of iNKT cells or cell groups modified by CD7 CAR alone or in combination with at least one additional therapeutic agent can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus of one or both preparations can be administered at one time, multiple separate doses can be administered within a predetermined time period, or the dosage of one or both preparations can be proportionally reduced or increased, as indicated by the emergency state of the treatment situation. For any particular subject, the specific dosage regimen can be adjusted over time according to the needs of the individual and the professional judgment of the treating clinician. For ease of administration and dosage uniformity, the parenteral composition can be formulated into a dosage unit form. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound, which is calculated to produce the desired therapeutic effect in combination with the desired pharmaceutically acceptable carrier.

[0189] The dosage value of the composition comprising iNKT cells or cell groups modified by CD7 CAR and / or any additional therapeutic agent can be selected based on the unique characteristics of the active agent and the specific therapeutic effect to be achieved. The starting dose of iNKT cells or cell groups modified by CD7 CAR that a physician or veterinarian can use in a pharmaceutical composition is lower than the level required to achieve the desired therapeutic effect, and the dose is gradually increased until the desired effect is achieved. Generally, the effective dose of the composition of the present disclosure for treating cancer may vary depending on many different factors, including the mode of administration, the target site, the physiological state of the subject, whether the subject is a human or an animal, other drugs administered, and whether the treatment is preventive or therapeutic. The selected dosage level may also depend on various pharmacokinetic factors, including the activity of the specific composition of the present disclosure used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health and previous medical history of the subject treated. The therapeutic dose can be titrated to optimize safety and efficacy.

[0190] 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, so as to normalize physical functions in a disease or condition that causes a specific physical function to be impaired, or to provide an improvement in one or more clinically measured parameters of a disease or to slow the progression of one or more clinically measured parameters of a disease. The therapeutically effective amount may be, for example, sufficient to treat cancer, prevent cancer, reduce cancer severity, delay cancer onset, and / or reduce the risk of one or more symptoms of cancer. The therapeutically effective amount and 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, iNKT cells or cell populations modified by CD7 CAR are administered in a therapeutically effective amount when administered as a single preparation. In some other embodiments, iNKT cells or cell populations modified by CD7 CAR and at least one additional therapeutic agent are each administered in a therapeutically effective amount when the preparation is used in combination. In some embodiments, the therapeutically effective amount of iNKT cells or cell populations modified by CD7 CAR is the amount required to kill a cancer cell population or a portion thereof in a subject. In some embodiments, the therapeutically effective amount of iNKT cells or cell populations modified by CD7 CAR is the amount required to reduce or slow the expansion of cancer cell populations in a subject. In some embodiments, the therapeutically effective amount of iNKT cells or cell populations modified by CD7 CAR is the amount required to reduce or slow tumor growth in a subject.

[0191] In some embodiments, the therapeutically effective amount of the CD7 CAR-modified iNKT cell or cell population is about 1×10 7 About 5×10 9 In some embodiments, the therapeutically effective amount of the iNKT cell or cell population modified with CD7 CAR is about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 or about 9×10 7 In some embodiments, the therapeutically effective amount of the iNKT cell or cell population modified with CD7 CAR is about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 , about 6×10 8 , about 7×108 , about 8×10 8 or about 9×10 8 In some embodiments, the therapeutically effective amount of the iNKT cell or cell population modified with CD7 CAR is about 1×10 9 , about 2×10 9 , about 3×10 9 , about 4×10 9 or about 5×10 9 In some embodiments, the therapeutically effective amount of the CD7 CAR-modified iNKT cells or cell population is less than about 1×10 7 In some embodiments, the therapeutically effective amount of the CD7 CAR-modified iNKT cells or cell population is greater than about 5×10 9 In some embodiments, cells, cell groups, or pharmaceutical compositions are administered to a subject at a single time. In some embodiments, cells, cell groups, or pharmaceutical compositions are administered to a subject at multiple times (e.g., hourly, daily, weekly, biweekly, monthly, or annually).

[0192] The therapeutically effective dose of iNKT cells or cell groups modified by CD7 CAR described herein generally provides therapeutic benefits without causing significant toxicity. The toxicity and therapeutic efficacy of iNKT cells or cell groups modified by CD7 CAR can be determined by standard drug procedures, such as in cell culture or animal models. Cell culture assays and animal studies can be used to determine LD50 (a dose lethal to 50% of the population) and ED50 (a dose effective for the treatment of 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as a ratio LD50 / ED50. In some embodiments, iNKT cells or cell groups modified by CD7 CAR exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range suitable for humans. In some embodiments, the dose is within a circulating concentration range including ED50, with minimal toxicity or no toxicity. The dose can vary within this range, depending on various factors, such as the dosage form used, the route of administration used, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be selected by the attending physician in view of the subject's condition.

[0193] In some embodiments, iNKT cells, cell groups or pharmaceutical compositions modified by CD7 CAR are administered at a single opportunity. In some embodiments, iNKT cells, cell groups or pharmaceutical compositions modified by CD7 CAR are administered at multiple opportunities. The interval between single doses may be, for example, hourly, daily, weekly, biweekly, monthly or annually. Based on the measured level of the preparation (e.g., iNKT cells or cell groups modified by CD7 CAR) administered in the subject, the interval may also be irregular, so as to maintain the relatively consistent concentration of the preparation. The dosage and frequency of iNKT cells or cell groups modified by CD7 CAR may also vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses may 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 may sometimes be administered at relatively short intervals until disease progression is reduced or terminated, and / or until the subject shows partial or complete improvement of one or more symptoms of the disease. Thereafter, a lower dosage regimen, such as a preventive dosage regimen, may be administered to the subject.

[0194] In some embodiments, kits and products for the treatment and prevention applications described herein are also provided. In some embodiments, the present disclosure provides a kit or product comprising iNKT cells or cell groups modified by CD7 CAR. In some embodiments, the kit or product also includes one or more additional components, including but not limited to: instructions for use; other reagents, such as therapeutic agents (such as anticancer agents); devices, containers or other materials for preparing iNKT cells or cell groups modified by CD7 CAR for administration; pharmaceutically acceptable carriers; and devices, containers or other materials for administering iNKT cells or cell groups modified by CD7 CAR to subjects. Instructions for use may include guidance for therapeutic applications, including, for example, recommended dosages and / or modes of administration in subjects suffering from or suspected of having cancer. In some embodiments, the kit includes iNKT cells or cell groups modified by CD7 CAR, and instructions for treating and / or preventing cancer by iNKT cells or cell groups modified by CD7 CAR.

[0195] Example

[0196] The following examples provide illustrative embodiments of the present disclosure. The examples provided are not intended to limit the present disclosure in any way.

[0197] Materials and methods

[0198] Cell culture: K562 (chronic myeloid leukemia, CML), Daudi (B-cell Burkitt's lymphoma), Raji (B-cell Burkitt's lymphoma), Nalm-6 (B-cell precursor leukemia), EBV-B (B-cell line transformed by Epstein-Barr virus), HSB2 (T-cell acute lymphoblastic leukemia, T-ALL), Jurkat (T-ALL), MOLT13 (T-ALL), KG1 (acute myeloid leukemia) Disease, AML), Kasumi-1 (AML), Kasumi-6 (AML), Molm13 (AML), MV4:11 (AML), U937 (AML), SaOS2 (osteosarcoma), Rh30 (alveolar rhabdomyosarcoma), and TC71 (Ewing sarcoma) cell lines and their luciferase-expressing counterparts were maintained in RPMI 1640 medium (Corning, catalog number 10-040-CM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Corning, catalog number 35-015-CV), 2 mM L-glutamine (Corning, catalog number 25-005-CI), 50 U / mL penicillin, and 50 μg / mL streptomycin (Corning, catalog number 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 and 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 (American Type Culture Collection (ATCC), catalog number 30-2003) and F12 medium (American Type Culture Collection, catalog number 30-2006) supplemented with 10% FBS (Corning Incorporated, catalog number 35-015-CV), and Dulbecco's Modified Eagle's Medium (DMEM) (Corning Incorporated, catalog number 10-013-CM) supplemented with 10% FBS, 0.1 mM non-essential amino acids (Corning Incorporated, catalog number 45000-700).

[0199] K562, Daudi, Raji, Nalm-6, BE(2)C and SKNFI were purchased from the American Type Culture Collection. EBV-transformed B (EBV-B) cell lines were provided by the Ludwig Cancer Research Branch in 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. Identification of luciferase and NGFR (hfflucN) expressing HSB2, Jurkat, MOLT13 and KG1 was performed by the Oregon Health Science University Integrated Genomics Laboratory for 10 short tandem repeat (STR) loci plus sex determination loci and sex identification loci.

[0200] Peripheral blood mononuclear cell isolation, CAR-T production and culture: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood units purchased from the New York Blood Center using Ficoll Paque Plus (Cytiva, catalog number GE17-1440-03) and cryopreserved. PBMCs were thawed and cultured at 1×10 6Cells / well were cultured in 24-well plates in human T cell culture medium consisting of RPMI 1640 (Corning, catalog number 10-040-CM), 10% FBS (Corning, catalog number 35-015-CV), 2 mM L-glutamine (Corning, catalog number 25-005-CI), 50 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich, catalog number 60-24-2), 100 IU / mL penicillin and 100 μg / mL streptomycin (Corning, catalog number 30-002-CI) with Dynabeads Human T-Activator CD3 / CD28 (Invitrogen, catalog number 11131D). On the third day, the culture was transduced with CD19, ROR1, CD7 CAR lentivirus (infection multiplicity (MOI) = 9-20) on a 24-well plate coated with recombinant human fibronectin (retronectin) (Takara Bio, catalog number T100B) by centrifugation at 1,902g (3000rpm) for 2 hours at 32°C and incubated at 37°C for 48 hours. After removing the CD3 / CD28 beads, the transduced T cells or untransduced mock T cells were cultured in human T cell culture medium supplemented with human IL-2 (50 IU / mL, aldesleukin, Novartis Pharmaceuticals), IL-7 (10 ng / mL, Peprotech, catalog number 200-07) and IL-15 (10 ng / mL, Peprotech, catalog number 200-15). Cell numbers were determined on days 11 and 16 by trypan blue staining on a hemacytometer.

[0201] iNKT cell isolation, CAR transduction and expansion: iNKT cells (1-2×10 per well) were isolated from fresh PBMCs using anti-iNKT microbeads (Miltenyi Biotec, catalog number 130-094-842) that positively select for human cells expressing TCRVα24-Jα18. 5 After iNKT isolation, the negative fraction of autologous PBMCs (3-×10 per well) was cultured in 1 mL of human T cell culture medium. 6) were cultured in 48-well plates, and the human T cell culture medium was supplemented with α-GalCer (200ng / mL, DiagnoCine, catalog number KRN7000; FlycoFineChem, catalog number FC-070) or 7DW8-5 glycolipid analogs (200ng / mL, provided by Dr. Moriya Tsuji of Columbia University; DiagnoCine, catalog number 7DW8-5), 250IU / mL IL-2 (aldesleukin, Novartis Pharmaceuticals) and 10ng / mL IL-15 (Peptech, catalog number 200-15). On day 3 or 4, iNKT cell cultures were transduced with CAR lentivirus (MOI = 20-200) on 24-well plates coated with recombinant human fibronectin (Takara Bio, catalog number T100B) by centrifugation at 1,902g (3000rpm) for 2 hours at 32°C. After 48 hours, the transduced iNKT cells were transferred to 48-well plates, fed with human T cell medium supplemented with IL-2 and IL-15, and split into new wells when they became confluent. At 2-3 weeks, iNKT cells were initially expanded in 24-well plates and then transferred to T75 flasks. Briefly, iNKT cells (5 × 10 cells per well) were plated and cultured in 24-well plates. 5 ) with irradiated autologous PBMC negative fraction and / or whole PBMC (regularly autologous) or pooled PBMC from 3-5 allogeneic donors (3.5 × 10 per well) 6 40 Gy) and irradiated EBV-B cells (5 × 10 5 iNKT cells (5 × 10 cells per flask) were co-cultured in 24-well plates in 2 mL of human T cell culture medium supplemented with α-GalCer or 7DW8-5 (100-200 ng / mL), IL-2 (250 IU / mL), and IL-15 (10 ng / mL). 5 ), irradiated pooled PBMCs from 3-5 allogeneic donors (3×10 per flask) 7 40 Gy) and irradiated EBV-B cells (3 × 10 per flask) 6iNKT cells were expanded in T25 flasks by co-culturing with 8×10 cells / mL of 7DW8-5 (100-200 ng / mL), IL-2 (250 IU / mL) and IL-15 (10 ng / mL) or anti-human CD3 monoclonal antibody (OKT3, 30 ng / mL, Miltenyi Biotech, catalog number 130-093-387), IL-2 (50 IU / mL), IL-7 (10 ng / mL) and IL-15 (10 ng / mL). The expanded iNKT cells were cultured at 8×10 7 Cells / vial were cryopreserved in 1 mL of 90% FBS and 10% dimethyl sulfoxide (DMSO, MPBio, SKU NO.021960559). αGalCer or 7DW8-5 was dissolved in DMSO at 1 mg / mL or 4 mg / mL, heated in a 60°C-80°C water beaker for 10 minutes, vortexed for 30 seconds, and aliquoted in 10 μl or 2 μl per tube and stored at -20°C. When used, γGalCer (10 μl per tube) or 7DW8-5 (2 μl per tube) was heated in a 60-80°C water beaker for 10 minutes, vortexed for 30 seconds, and diluted with T cell culture medium at 100 μg / mL.

[0202] Lentivirus production and tumor cell transduction: HIV-1-based bidirectional vectors expressing humanized firefly luciferase and NGFR (hffluc N) were constructed as described previously (Huang et al., Mol Ther. 2008; 16(3):580-9; Huang et al., PLoS ONE. 2015; 10(7):e0133152). A lentiviral vector expressing CD7 CAR, CD19 CAR and ROR1 was constructed based on a previously described lentiviral vector, which contained 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, CD8α hinge and transmembrane regions, and the intracellular domain of 4-1BB and CD3ζ chain (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 CAR was produced by commercial gene synthesis using the anti-CD7 single-chain variable fragment (scFv) sequence found in patent WO2003051926A2 (Cooper et al., Leuk. 2018; 32 (9): 1970-1983). Lentivirus supernatant was harvested 48 hours and 96 hours after transfection in Lenti-X 293T cell line (Takara Bio, catalog number 632180) with 4 plasmids (pLVCARsin, pMDLg / pRRE, PRSV-REV, pMD2.G) and Lipofectamine 2000 (Thermo Fisher, catalog number 11668019) and concentrated using Lenti-X concentrator (TakaraBio, catalog number 631231). Virus titer was determined in Lenti-X 293T cell line, ranging from 2.3×10 7 -2×10 8Transduction units / mL. Leukemia cell lines were transduced with hfflucN lentivirus at 1170 g for 1 hour at 32°C in the presence of polybrene (8 μg / mL) and then sorted by FACS or enriched for NGFR expression by biotin anti-human CD271 (NGFR, BD Biosciences, catalog number 557195) and anti-biotin microbeads (Miltenyi Biotech, catalog number 130-090-485) or CD271 microbeads (Miltenyi Biotech, catalog number 130-099-023). All transduced cell lines were verified by flow cytometric analysis of NGFR expression and hffluc bioluminescent activity using a Synergy2 microplate reader (Bio Tek).

[0203] Luciferase-based killing assay: Target cells (2 × 10 5 αGalCer or DMSO-pulsed target cells were prepared by incubating αGalCer (200 ng / mL) or DMSO (1:500 of 10% stock solution) overnight and used for cytotoxicity assays after spinning to remove the medium containing αGalCer or DMSO. Luciferase-expressing target cells (1 × 10 in 50 μL per well) were plated at different effector T cell:target cell (E / T) ratios in 96-well flat-bottom white polystyrene microplates (Corning, catalog number 3912). 4 ) were incubated with 50 μL of iNKT cells per well in quadruplicate. Spontaneous or maximum killing was established by adding 50L culture medium or 1% Triton X-100 to each well instead of iNKT cells. After incubation standard for 4 hours or 16 hours (as indicated) at 37°C, 10 μL of D-luciferin (1:10 of 30 mg / mL stock solution) or 50 μL (1:50 of 30 mg / mL) (Caliper LifeSciences, catalog number XR-1001) was added to each well. Luciferase activity was measured using Synergy2 microplate reader (Bio Tek). The specific lysis percentage was calculated as follows:

[0204] % specific lysis = (spontaneous death RLU - sample RLU) / (spontaneous death RLU - maximum death RLU) x 100. RLU = relative luminescence unit.

[0205] Antibodies and flow cytometry: FITC anti-human TCR Vα24-Jα18 (iNKT cells, clone 6B11 that recognizes the invariant CDR3 region of TCRVα24-Jα18 and TCRVα24-JQ, catalog number 342906); 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 (catalog number 405204); PE mouse IgG1, Isotype control (clone MOPC-21, catalog number 981804); APC mouse IgG1, Isotype control (clone MOPC-21, catalog number 981806); PE mouse IgG2a, Isotype control (clone MOPC-173, catalog number 400246); and PE mouse IgG2b, Isotype control (clone MPC-11, catalog number 400314) was 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 UCHT2, catalog number 555353); =HIT8a (clone RPA-T8, catalog number 555635); APC anti-human CD8 (clone RPA-T8, catalog number 555369); V450 anti-human CD7 (clone M-T701, catalog number 642916); PE anti-human CD7 (clone M-T701, catalog number 555361); PE anti-human CD271 (NGFR) (clone C40-1457, catalog number 557196); PE mouse IgG1, Isotype control (clone MOPC-21, catalog number 555749); APC mouse IgG1, Isotype control (clone MOPC-21, catalog number 555751) and V450 mouse IgG1, Isotype control (clone MOPC-21, catalog number 560373) was purchased from BD Biosciences. Biotinylated human CD19 protein (catalog number 11880-H08H-B); biotinylated human ROR1 protein (catalog number 13968-HCCH1-B) and human CD7 protein (His tag, catalog number 11028-H08H) were purchased from SinoBiological. Surface CAR expression was detected with biotinylated human CD19 protein or biotinylated human ROR1 and PE streptavidin or human CD7 protein His tag and PE anti-His tag or goat anti-mouse IgG F(ab')2 AlexaFluor647 (AF-647)-conjugated F(ab')2 fragment (anti-CAR, Jackson ImmunoResearch, catalog number 115-606-006) and ChromPure goat IgG isotype control Alexa Fluor647-conjugated F(ab')2 fragment (catalog number 015-600-006). Flow cytometry analysis was performed on a BD FACSCelesta or BD Accuri C6 blood cell counter. Data were analyzed with Flowjo software 7.2.2 or V10.

[0206] Cytokine release assay: Place 1-2 × 10 5 T cells and 2×10 4 Target cells were co-cultured in duplicate in 96-well flat-bottom plates for cytokine release assays. After 24 hours, supernatants were assayed using the LEGENDMAX Human IFN-γ ELISA Kit (BioLegend, catalog number 430107).

[0207] In vivo antitumor assay: NOD.Cg-Prkdc scid IL tm1WjI / SzJ (NSG, accession number 005557) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in the specific pathogen-free facility of New York Medical College. To establish T-ALL and AML xenograft models, 5 × 10 56-7 week old NSG mice were intravenously (iv) inoculated with 100 HSB2-hfflucN or KG1-hfflucN cells. On day 0, the human leukemia grafts of the mice were examined by bioluminescence imaging (BLI) using the Perkin Xenogen IVIS Spectrum Imaging System. Mice with T-ALL or AML were randomly divided into three or two groups (n=5 per group) based on imaging intensity. On days 0, 3, and 6, PBS, thawed mimic iNKT55 cells, and thawed CD7 CAR-iNKT55 cells (5×10 6 / 0.2mL / mouse) was intravenously infused into mice carrying HSB2-hfflucNT-ALL. Mice carrying KG1-hfflucN AML were intravenously infused with thawed mimics iNKT55 and CD7CAR-iNKT55 on days 0 and 3. Mimics iNKT55 and CD7 CAR-iNKT55 cells were thawed from liquid nitrogen Dewar flasks, washed twice with human T cell culture medium and PBS, and resuspended in PBS. Mice were also injected intraperitoneally (ip) every three days with a cytokine mixture (IL-22,000IU / mouse, IL-152μg / mouse) or PBS (Tian et al., J Clin Invest.2016; 126(6):2341-55; Xu et al., Clin Cancer Res.2019; 25(23):7126-38) for two weeks. After the first T cell infusion, tumor BLI was performed at the 3rd, 6th, 10th, 17th and 24th days in mice with T-ALL, or at the 0th, 3rd, 6th, 10th, 17th, 24th, 31st, 38th, 45th days in mice with AML. After intraperitoneal injection of D-fluorescein (Caliper Life Sciences, catalog number XR-1001), BLI was performed under isoflurane anesthesia. Images were collected and analyzed using IVIS imaging system and Living Image 4.7.3 software (Perkin Elmer). Constant regions of interest (ROI) were drawn on the whole mouse body, and the intensity of the signal was measured as the total photon flux normalized for exposure time and surface area, and in units of photons / second / cm 2 / Steradian(p / sec / cm 2 The mice were then monitored for survival without imaging manipulation.

[0208] Statistical analysis: Data were calculated as mean ± standard deviation (SD) and analyzed using one-way ANOVA analysis, followed by Tukey's Honestly Significant Difference (Tukey's HSD) post hoc test for pairwise comparisons. The statistical significance of Kaplan-Meier survival curves was assessed using the Mantel-Cox log-rank test. All p values ​​were calculated using Prism7 software (Graph Pad).

[0209] result

[0210] Figure 1 It is shown that CD1d is expressed in most AML cell lines (including HL60, KG1, Molm13, MV4:11, THP-1 and U937), but not in the Mo7eAML cell line. CD1d expression in KG1 is weakly positive. CD1d expression in B cell tumor cell lines (Daudi, Raji, Nalm6) and CML cell lines (K562, K562CD19) is negative. All tumor cell lines transduced with luciferase express high levels (>90%) of NGFR, indicating that they can all express luciferase.

[0211] Figure 2A The results show that compared with iNKT1(auto) cells expanded with irradiated autologous PBMC negative fraction and / or whole PBMC and αGalCer (which is a common approach in iNKT cell expansion), the CD1d sensitized cells were expanded with irradiated allogeneic PBMC and EBV-B cells and αGalCer antigen-specific lysis. + (Molm13, THP-1, U937) but not CD1d - iNKT1(allo) cells were amplified by (Daudi, Nalm6) target cells. + and CD1d- target cells were not lysed by iNKT1(auto) and iNKT1(allo).

[0212] Figure 2B The results show that iNKT2(auto) cells expanded with irradiated allogeneic PBMCs and EBV-B cells and αGalCer-sensitized CD1d cells lysed with αGalCer antigen-specific lysis compared with iNKT2(auto) cells expanded with irradiated autologous PBMC negative fractions and / or whole PBMCs and αGalCer. +iNKT2(allo) cells that expand on CD1d-(KG1, Molm13, MV4:11, THP-1) but not CD1d-(Daudi, Nalm6) target cells. Figure 2A and Figure 2B These results indicate that iNKT cells expanded with irradiated allogeneic PBMCs and EBV-B cells and αGalCer antigen retained αGalCer-CD1d specificity compared with iNKT cells expanded with irradiated autologous PBMC negative fractions and / or whole PBMCs and αGalCer.

[0213] Figure 3A It was shown that iNKT1 (OKT3) cells expanded with irradiated allogeneic PBMCs and EBV-B cells and a soluble anti-CD3 monoclonal antibody (OKT3) surprisingly recognized CD1d + As a control, iNKT1(αGalCer) cells expanded with irradiated allogeneic PBMCs and EBV-B and αGalCer antigens recognized αGalCer but not DMSO-treated CD1d + Target cells. iNKT1(OKT3) and iNKT1(αGalCer) did not recognize CD1d-K562 cells, regardless of whether they were treated with GalCer or DMSO.

[0214] Figure 3B iNKT2 (OKT3) cells expanded with irradiated allogeneic PBMC and EBV-B cells and soluble anti-CD3 monoclonal antibody (OKT3) recognize CD1d + As a control, iNKT2(αGalCer) cells expanded with irradiated allogeneic PBMC and EBV-B cells and αGalCer antigen recognized αGalCer but not DMSO-treated CD1d + iNKT2(OKT3) and iNKT2(αGalCer) did not recognize CD1d regardless of whether they were treated with GalCer or DMSO. - (K562, Daudi) cells. The weak recognition of iNKT2 (OKT3) on DMSO-treated KG1 cells may be due to the low expression of CD1d in KG1 cells ( Figure 1 ).

[0215] Figure 3CFigure 2 shows that iNKT12 (OKT3) cells expanded with irradiated allogeneic PBMC and EBV-B cells and soluble anti-CD3 monoclonal antibody (OKT3) recognize CD1d + As a control, iNKT12(αGalCer), iNKT2(αGalCer), and iNKT1(αGalCer) cells expanded with irradiated allogeneic PBMCs and EBV-B cells and αGalCer antigen recognized αGalCer but not DMSO-treated CD1d + iNKT12(OKT3) but not iNKT12(αGalCer) and iNKT2(αGalCer) also showed CD1d - (Daudi, Raji) Low background recognition of cells.

[0216] Figure 4A Figure 3 shows that iNKT cells from four donors (iNKT1, iNKT2, iNKT11, iNKT12) expanded with irradiated allogeneic PBMCs and EBV-B cells, αGalCer, and a cocktail of cytokines (IL-2, IL-7, and IL-15) recognized αGalCer but not DMSO-treated CD1d + (THP1, U937) target cells.

[0217] Figure 4B Figure 2 shows CD3 expression of iNKT1 and iNKT2 cells expanded with OKT3 or αGalCer. + iTCR + More than 92% and in CD3 + iTCR + Phenotypically similar in expression. All these results based on iNKT cells (iNKT1, iNKT2, iNKT12) derived from three donors expanded with OKT3 and iNKT cells (iNKT1, iNKT 2, iNKT 11, iNKT 12) derived from four donors expanded with GalCer indicate that OKT3 expansion can change the antigen specificity of iNKT cells. GalCer stimulation of iNKT cells is essential for maintaining their antigen specificity.

[0218] Next, we tested the function, phenotype, and expansion rate of iNKT cells expanded with irradiated allogeneic PBMCs and EBV-B cells and αGalCer antigen (regularly, iNKT(allo)) in comparison with iNKT cells expanded with irradiated autologous PBMC negative fractions and / or whole PBMCs and αGalCer (regularly, iNKT(auto)), all supplemented with IL-2 and IL-15. Figure 5A It is shown that iNKT45(auto), iNKT45(allo), iNKT46(auto), and iNKT46(allo), isolated from healthy blood donors 45 and 46 and generated using either the auto or allo expansion protocol, are reactive only to αGalCer but not to DMSO-treated CD1d + (MV4:11, U937) target cells were not reactive. In addition, those iNKT45 and iNKT46 indiscriminately recognized CD1d-(Nalm6) target cells, regardless of DMSO or αGalCer.

[0219] Figure 5B It was shown that iNKT45 and iNKT46 cells showed more than 96% CD3 + iTCR + Homogeneous groups. Figure 5C As shown in Figure 2, iNKT45 and iNKT46 cells expanded under auto or allo conditions displayed more than 94% CD3+ expression after secondary stimulation or expansion with irradiated autologous PBMC negative fractions and / or whole PBMC and αGalCer or irradiated allogeneic PBMC and EBV-B cells and αGalCer. + iTCR + Homogeneous population of cells.

[0220] To demonstrate that iNKT cells expanded with irradiated allogeneic PBMCs and EBV-B cells and αGalCer can be used to prepare CAR-modified iNKT cells, iNKT47 and iNKT48 cells isolated from donors 47 and 48 were lentivirally transduced to express CD19 CAR or CD19 CAR / GFP and expanded under auto or allo conditions. Figure 6 Figure 3 shows that iNKT47 and iNKT48 cells modified with CD19CAR or CD19 CAR / GFP expanded under auto or allo conditions specifically killed CD19 in an E / T ratio-dependent manner. +B cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji) but not CD19 - CML (K562) cells. As expected, the mimic iNKT47 and iNKT48 cells were unable to kill CD19 + In addition, CD19 CAR-modified or CD19 CAR / GFP-modified iNKT47 and iNKT48 cells expanded under auto or allo conditions were responsive to αGalCer but not to DMSO-treated CD1d + (Molm13, U937) target cells were not reactive. CD1d treated with DMSO or αGalCer - Mo7e was not recognized by all iNKT47 and iNKT48.

[0221] Figure 7 Figure 2 shows that iNKT47 and iNKT48 cells modified with CD19 CAR or modified with CD19CAR / GFP expanded under auto or allo conditions responded to CD19 + B-cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji) but not CD19 - CML (K562) and AML (Mo7e) cells specifically produced higher amounts of IFN-γ compared to their mock counterparts. + THP1 cells significantly enhanced IFN-γ production by all iNKT47 and iNKT48 cells compared with DMSO-treated THP1 cells, indicating that CD19 CAR-modified iNKT cells expanded under allo conditions are cytotoxic and responsive to CD19 + Tumor cells produce IFN-γ while they remain specific for αGalCer.

[0222] Figure 8 Figure 3 shows that more than 93% of CD19 CAR-modified iNKT47 and iNKT48 cells expanded under auto or allo conditions expressed CD3 compared to their mock counterparts. + iTCR + population, indicating that iNKT cells expanded under allo conditions possess a subset of their phenotypes.

[0223] Fig. 9 It is shown that CD19CAR or CD19 CAR / GFP expression was confirmed in iNKT47 and iNKT48 cells expanded under auto or allo conditions, indicating that auto or allo culture conditions do not affect CAR expression levels.

[0224] Fig.10 Figure 3 shows that iNKT50 cells modified with CD19 CAR or modified with CD19 CAR / GFP isolated from donor 50 and expanded under auto or allo conditions specifically killed CD19 in an E / T ratio-dependent manner. + B-cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji) and CD19-transfected K562 (K562CD19), but not CD19-CML (K562) and AML (HL60, KG1, Molm13, THP1) cells. As expected, mock cells were unable to kill CD19 + B cell leukemia and lymphoma cell lines and K562CD19. In addition, CD19 CAR-modified or CD19 CAR / GFP-modified iNKT50 cells expanded under auto or allo conditions were reactive to αGalCer but not to DMSO-treated CD1d + (MV4:11, U937) target cells are not reactive. CD1d treated with DMSO or αGalCer - Mo7e cells were not recognized by all iNKT50 cells.

[0225] Fig.11A Figure 3 shows that more than 87% of CD19CAR-modified iNKT50 cells expanded under auto or allo conditions were CD3 + iTCR + , indicating that iNKT cells expanded under allo conditions maintained phenotypically intact.

[0226] Fig. 11B It is shown that CD19 CAR or CD19 CAR / GFP expression was confirmed in transduced iNKT50 but not expanded mock cells under auto or allo culture conditions, indicating that auto or allo culture conditions do not affect CAR expression levels.

[0227] Fig.12The expansion rates of non-transduced iNKT45 and iNKT46 and iNKT47, iNKT48, and iNKT50 modified with CD19 CARs expanded under auto or allo culture conditions are shown. In iNKT45 and iNKT46, approximately 245-fold and 76-fold expansions were achieved on day 13, respectively, after primary stimulation with irradiated autologous PBMC negative fractions and αGalCer. On day 26, 10,800-fold and 2,800-fold expansions were obtained in iNKT45 and iNKT46 expanded under allo conditions, compared to 6,080-fold and 2,106-fold expansions in iNKT45 and iNKT46 cultured under auto conditions. It appears that allo culture conditions are as effective as auto culture conditions in iNKT cell expansion.

[0228] Next, we tested the expansion of CD19CAR-modified iNKT cells in iNKT47, iNKT48, and iNKT50 using auto and allo expansion conditions. Fig.12 As shown, at the end of culture day 35, 4,160-fold and 4,065-fold expansions were demonstrated in iNKT47CD19 CAR(allo) and iNKT47CD19 CAR / GFP(allo) cells expanded under allo conditions, compared to 2,250-fold and 2,680-fold expansions in iNKT47CD19 CAR(auto) and iNKT47CD19CAR / GFP(auto), respectively. For iNKT48 expansion, at the end of culture day 35, 4,417-fold and 3,650-fold expansions were recorded in iNKT48CD19 CAR(allo) and iNKT48CD19 CAR / GFP(allo) cells expanded under allo conditions, compared to 441-fold and 985-fold expansions in iNKT48CD19 CAR(auto) and iNKT48CD19 / GFP(auto), respectively.

[0229] We also expanded iNKT cells modified with CD19 CAR in iNKT50 cells. Fig.12As shown, on day 36, 18,500-fold, 8,400-fold, and 10,680-fold expansions were recorded for iNKT50 mimic (allo), iNKT50 CD19 CAR (allo), and iNKT50 CD19 CAR / GFP (allo) cells expanded under allo conditions, compared to 14,666-fold, 5,200-fold, and 15,353-fold expansions for iNKT50 mimic (auto), iNKT50 CD19 CAR (auto), and iNKT50 CD19 CAR / GFP (auto), respectively. At the end of the culture, 14,666-fold, 5,200-fold, and 15,353-fold expansions for iNKT50 mimic (auto), iNKT50 CD19 CAR (allo), and iNKT50 CD19 CAR / GFP (allo) cells expanded under auto conditions, respectively. 5 (simulant) and 0.75×10 5 iNKT50 cells isolated by CAR generated 4.4×10 8 iNKT50 mimics (auto), 5.5×10 8 iNKT50 mimics (allo), 3.9×10 8 iNKT50 CD19 CAR (auto), 6.3×10 8 iNKT50CD19CAR (allo), 1.1×10 9 iNKT50 CD19 CAR / GFP (auto) and 8.0×10 8 iNKT50CD19 CAR / GFP (allo). It appears that 5,863 (5,863±2,858, n=6)-fold expansion rates and 4,484 (4,484±5,576, n=6)-fold expansion rates of CD19 CAR-modified and CD19 CAR / GFP-modified iNKT cells were demonstrated under allo or auto conditions, respectively. Allo conditions may be more advantageous than auto conditions in iNKT expansion. For example, allo feeder cells can be pre-made, grouped, and used for all iNKT expansions, while auto feeder cells are prepared individually, thus potentially simplifying the regulatory process and reducing manufacturing costs and product variations.

[0230] Fig.13A It was shown that thawed and cultured iNKT47 CD19 CAR (allo) and iNKT48 CD19 CAR (allo) were able to kill CD19 + B-cell leukemia and lymphoma cells (Daudi, Raji, Nalm6) and K562 transfected with CD19 (K562CD19) but without killing of CD19 -CML(K562) (even at a low E / T ratio of 2.2:1 in the 16-hour assay), indicating that freezing and thawing conditions minimally affect the cytotoxicity of CAR-modified iNKT cells. Fig. 13B The results show that thawed and cultured iNKT47 CD19CAR cells (allo) killed CD1d cells treated with 7DW8-5 or αGalCer in a dose-dependent manner. + Molm13 and THP1 target cells. It appears that 7DW8-5 plays a role in sensitizing CD1d + The potency of the target cells was at least 16-fold and 64-fold higher than that of αGalCer when used in Molm13 and THP1. Fig. 13C It is shown that 7DW8-5 is more effective than αGalCer in stimulating iNKT47 CD19 CAR (allo) and iNKT48 CD19 CAR / GFP (allo) cultured for 3 months to produce IFN-γ, thereby 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, iNKT47 cells modified with CD19 CAR (allo) and iNKT48 cells modified with CD19 CAR / GFP (allo) instead of the mimetic iNKT47 (auto) responded to CD19 + Target cells (K562CD19, Nalm6) produce IFN-γ.

[0231] Fig.14A shows that when compared with more than 95% of single CD2 + or CD5 + When comparing populations, we unexpectedly found that iNKT cells from one representative blood donor showed CD7 + (about 32%) and CD7 - 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).

[0232] Fig. 14BThe results showed that approximately 56±17.96% and 42.96±17.21% of iNKT cells from nine healthy blood donors (n=9) were CD7+, CD8+, CD9+, CD10+, CD21+, CD22+, CD31+, CD4+, CD51+, CD61+, CD7+, CD81+, CD91+, CD10+, CD11+, CD12+, CD13+, CD14+, CD15+, CD16+, CD17+, CD18+, CD19+, CD21+, CD22+, CD19+, CD23+, CD18+, CD19+, CD24+, CD19+, CD25+, CD19+, CD26+, CD19+, CD27+, CD19+, CD28+, CD19+, CD29+, CD29+, CD19+, CD29+, CD21+, CD19+, CD29+, CD21+, CD19+, CD2 + and CD7 - Similarly, the majority of iNKT cells (>96%) were CD2 + (n=7) and CD5 + (n=9). Therefore, we hypothesized that CD7 in iNKT cells expressing CD7 CAR - Subsets can partially avoid cannibalism in the absence of nuclease (e.g., CRISPR)-mediated genome editing of CD7 knockout and can be used in our study for CD7 + Adoptive cell therapy of cancer is carried out under allo culture conditions.

[0233] Fig.15 Figure 2 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, showed CD7, CD1d, and NGFR, respectively. - and CD7 + , and CD1d - .

[0234] Fig.16 It is shown that peripheral blood T cells transduced by CD7 CAR induce substantial fratricide and cannot be amplified. In order to confirm that T cells transduced by CD7 CAR induce fratricide and prevent CAR-T cell amplification, activated human T cells from blood donors (PBL36) are transduced with lentiviral CD7CAR constructs. CAR-T targeting CD19 or ROR1 or untransduced analogs T cells are used as controls. After T cell transduction, on the 11th and 16th days, CD7 CAR-T is less than CD19CAR-T or ROR1CAR-T or analogs T cells by about 19 times-30 times and 41 times-59 times, thereby confirming that CD7 antigen-driven fratricide prevents CD7 CAR-T amplification (Gomes-Silva et al., Blood 2017; 130 (3): 285-296; Cooper et al., Leuk. 2018; 32 (9): 1970-1983).

[0235] Fig.17 It is shown that iNKT cells modified with CD7 CAR can be expanded in culture. On days 22 and 41, expansion rates of 46-fold and 6,407-fold were obtained in iNKT55 cells modified with CD7 CAR (iNKT55 CD7 CAR), while expansion rates of 334-732-fold and 25,661-50,492-fold were recorded in iNKT55 cells modified with CD19 CAR (iNKT55 CD19CAR) and iNKT55 mimic cells. At the end of the culture, 1.3 × 10 cells were grown from their starting number using our allo expansion protocol. 5 iNKT cells generated 6.5×10 9 iNKT55 mimics, 3.30×10 9 iNKT55 CD19 CAR and 8.3×10 8 In addition, CD7 CAR-modified iNKT68 cells from donor 68 could also be expanded in culture. On days 24 and 39, 176-fold and 7,470-fold expansion rates were obtained in iNKT68 cells modified with CD7 CAR (iNKT68 CD7 CAR), while 600-fold and 27,120-fold expansion rates were recorded in iNKT68 mimic cells. At the end of the culture, 2 × 10 5 The starting number of iNKT cells generated was 5.4 × 10 9 iNKT68 mimics and 1.5×10 9 It appears that approximately 6,000-fold expansion of iNKT cells modified with either CD7 CAR or CD19 CAR can be achieved after two rounds of stimulation. On average, 10 8 2.526±1.495×10 5 iNKT cells (n = 27 donors). 8 PBMC or a leukopak (containing about 5-8×10 9 PBMCs) produced 70%-90% CAR + About 1.5×10 9 or 50×10 9 Therefore, based on the current Food and Drug Administration (FDA)-approved Yescarta (axicabtagene ciloleucel, CD19 CAR-T) of 2×10 per kg body weight, 6 CAR+ T cells, for patients with large B-cell lymphoma, a maximum of 2 × 10 8 CAR + -T cell dose, which can achieve approximately 175 infusions of CD7 CAR-modified iNKT cells from one leukopak.

[0236] Fig.18 A representative cytotoxicity graph is shown, where iNKT55 modified with CD7 CAR is able to kill luciferase-expressing CD7 cells in an E / T ratio-dependent manner. + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + AML (KG1) cell line, but no killing of CD7 - Tumor cell lines (including AML (Molm13, MV4:11, U937)), soft tissue sarcoma (Rh30, TC71, SaOS2), neuroblastoma (BE(2)C, SKNFI) and CML (K562). iNKT55 modified with CD7 CAR showed low background cytotoxicity against B cell leukemia and lymphoma cell lines (Nalm6, Raji) compared with mock cells. As a control, iNKT55 modified with CD19 CAR specifically cleaved CD19 + Malignant B cells (Nalm6, Raji), but do not specifically cleave CD19 - T-ALL, AML, sarcoma, and neuroblastoma. Untransduced mock iNKT cells did not kill all target cells tested, including CD1d + AML (KG1, Molm13, MV4:11, U937), but kills CD1d + Jurkat cells, confirming previous studies demonstrating that many human leukemic T cells express CD1d and can be induced by Va24 + NKT cells directly kill) (Takahashi et al., Br J Haematol. 2003; 122(2):231-239).

[0237] Fig.19 Figure 2 shows a combined cytotoxicity plot with statistical analysis, where iNKT cells modified with CD7 CAR (n=5 donors) specifically killed luciferase-expressing CD7 cells at all E / T ratios (20:1, 6.7:1, and 2.2:1) compared to mock iNKT cells and iNKT cells modified with CD19 CAR (p<0.0001). + T-ALL (HSB2, Jurkat, MOLT13) and CD7 +AML (KG1). CD7 CAR iNKT cells appeared to show a positive effect on CD7 when compared to mock iNKT cells (n=5 donors) (p<0.05). - Low background cytotoxicity of B-cell leukemia and lymphoma cells (Nalm6, Raji) and U937 (E / T ratio of 6.7:1 and 2.2:1 but not 20:1). As a control, iNKT cells modified with CD19 CAR (n=3 donors) specifically killed CD19 + B cell tumor cells, not CD19 - T-ALL and AML cells (p<0.0001). + All target cells except Jurkat T-ALL cells showed minimal cytotoxicity.

[0238] To demonstrate that CD7 CAR-modified iNKT cells also respond to CD7 + Tumor cells were stimulated to secrete IFN-γ, and the IFN-γ levels of the co-culture supernatants were examined by ELISA. Fig. 20 A shows that CD7 CAR-modified iNKT55 cells from a representative donor were more potent than CD19 CAR-modified iNKT55 and mock iNKT55 cells in comparison with CD7 + T-ALL (HSB2, MOLT13) and CD7 + AML (KG1, Kasumi-6) produced at least 4,000-fold higher amounts of IFN-γ when co-cultured. iNKT55 cells modified with CD7 CAR secreted 1,000-fold more IFN-γ for Jurkat T-ALL than CD19 CAR iNKT and mock iNKT cells. As expected, iNKT cells modified with CD19-CAR produced at least 2,500-fold more IFN-γ when co-cultured with CD19+ leukemia and lymphoma cells (Daudi, Nalm6, Raji) and EBV-transformed B cells (EBV-B). CD7 CARiNKT and CD19 CAR iNKT cells secreted 1,000-fold more IFN-γ for Jurkat T-ALL than CD19 CAR iNKT and mock iNKT cells. -AML (Kasumi-1, U937), sarcomas (Rh30, TC71), neuroblastomas (BE(2)C, SKNFI), and CML (K562) produced minimal amounts of IFN-γ. Untransduced mock iNKT cells produced negligible amounts of IFN-γ for all tumor cells tested. Of note, CD7 CAR iNKT or mock cells expanded with irradiated allogeneic PBMCs and EBV-B, αGalCer or 7DW8-5, IL2, and IL-15 did not recognize the EBV-B cells used for iNKT expansion, indicating that CD7 CAR iNKT cells expanded under allo conditions are CD7 specific and may not be alloreactive.

[0239] To demonstrate that iNKT cells modified with CD7 CAR can reproducibly produce IFN-γ in an antigen-specific manner, we examined IFN-γ production in CD7 CAR iNKT cells from multiple donors (n=5). Fig. 20 (B) CD7 CAR-modified iNKT cells from all five donors (n=5) responded to CD7 compared to mock iNKT (n=5, p<0.001) and CD19 CAR iNKT cells (p<0.05 (except Jurkat), p<0.01). + T-ALL (HSB2, Jurkat, MOLT13) and CD7 + AML (KG1, Kasumi-6) produced significant amounts of IFN-γ. Control iNKT cells modified with CD19 CAR (n=3) showed a significant increase in CD19 expression compared to mock iNKT and CD7 CAR iNKT cells (p<0.0001). + B cell leukemia and lymphoma cell lines (Daudi, Nalm6, Raji) produce significant amounts of IFN-γ. CD7 CARiNKT and CD19 CAR iNKT cells recognize CD7 - or CD19 - The mimetic iNKT cells showed minimal IFN-γ production for all target cells tested, even for the cytotoxic sensitive CD1d + The same is true for Jurkat (T-ALL) cells ( Fig.18 and Fig.19 , Takahashi et al., Br J Haematol. 2003; 122(2):231-239).

[0240] Fig.21It is shown that the expression of CD7 CAR in transduced iNKT cells (n=5) ranged from 64.6% to 98.8%, while the expression of CD19 CAR or ROR1 CAR varied from 5.37% to 43.9%. It seems that the transduction efficiency of CD7 CAR in iNKT cells is higher than that of CD19 CAR or ROR1 CAR, because CD7 antigen-mediated cannibalism can enrich CD7 CAR + iNKT cell population.

[0241] To confirm that cannibalism leads to CD7 + iNKT cells were completely depleted by iNKT cells modified with CD7 CAR, and flow cytometric dot plot analysis of the expression of CD2 relative to CD7 and CD5 relative to CD7 was performed. Fig.22A Data from one representative donor 55 are shown, where 45%-50% of CD7 + The cells were depleted to 0.4% in CD7 CAR iNKT cells. Fig. 22B The results showed that CD7 CAR iNKT cells significantly eliminated almost all CD7 CAR iNKT cells compared with mock iNKT (61.6% ± 24.4, n = 4) and CD19 or ROR1 CAR iNKT cells (69.9% ± 24.4, n = 3) (p < 0.0001). + cells (mean ± SD 0.4% ± 0.1, n = 4).

[0242] Phenotypically, CD3 in CD7 CAR iNKT cells was significantly higher than that in mock iNKT and CD19 CAR iNKT cells. + iTCR + The expression remains unchanged, such as Fig.23A Similar to mimic iNKT and CD19CAR iNKT cells, CD7 CARiNKT cells consist of CD4 + 、CD4-CD8-、CD8 + and CD4 + CD8 + Group composition ( Fig. 23B ). It seems that CD7 CAR iNKT cells are more potent than CD8 + Cells differentiate into more CD4 + , and expressed less cell surface PD-1 inhibitory molecules ( Fig. 23B , Fig.23C ).

[0243] The anti-leukemic effect of iNKT cells modified by CD7 CAR was evaluated using temporal dynamic bioluminescence imaging (BLI) technology in living mice. For the xenograft T-ALL model, NSG mice were intravenously injected 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 intravenously infused with thawed CD7 CAR iNKT cells, mock iNKT cells, or PBS ( Fig.24A Administration of three infusions of thawed CD7 CAR iNKT cells to leukemia-bearing mice significantly inhibited 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 with PBS and mock controls. Fig. 24B , Fig.24C , Fig.24D A significant survival advantage was observed after CD7-CAR-iNKT cell treatment compared with PBS and mock control groups (p=0.0009, Fig.24E ).

[0244] We also evaluated the effect of CD7 CAR-modified iNKT cells on CD7 + Anti-leukemic effects in AML models. Fig.25A The experimental scheme of tumor cell injection, CD7 CAR iNKT cell infusion and BLI monitoring is shown. KG1-hfflucNAML cells were injected intravenously into NSG mice, followed by thawed CD7 CAR iNKT cells and mock iNKT cells (n=5 mice per group) ( Fig.25A ). No significant difference in tumor mass was found between the two groups before treatment (p=0.3787). Compared with mock controls, two infusions of thawed CD7 CAR iNKT cells into pre-established AML mice significantly inhibited AML growth at days 3 (p=0.0096), 6 (p<0.0001), 10 (p=0.0026), 17 (p<0.0001), 24 (p<0.0001), 31 (p<0.0001), 38 (p<0.0001) and 45 (p=0.0003). Fig.25B , Fig.25C , Fig.25D CD7 CAR iNKT cell therapy also significantly extended animal survival to more than one year in 50% of treated mice, while mock control mice died around day 74 (p=0.0069, Fig.25ETogether, these results demonstrate that CD7 CAR-modified iNKT cells expanded in the absence of genome editing and under our allo culture conditions are able to enhance anti-CD7 + Tumor response.

[0245] The foregoing embodiments and examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Based on the present disclosure, many variations of the above-mentioned embodiments and examples are possible and obvious to those skilled in the art. Such modifications and variations are within the spirit and scope of the present invention. All patents or non-patent documents cited are incorporated herein by reference in their entirety without admitting that they are prior art.

Claims

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

2. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to claim 1, wherein the cells are isolated from a biological sample and the CD3+ activated cells are activated in the presence of α-galactosylceramide (αGalCer) or 7DW8-5 or other glycolipid analogs. + iTCR(Vα24-Jα18) + or CD3 + Vα24 + ; and wherein the cells are modified to express a CD7 chimeric antigen receptor (CAR) via a lentiviral vector and the cells are 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. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to claim 1, wherein the CD3 + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells have CD3 + iTCR + CD4 + Cells, CD3 + iTCR + CD8 + Cells, CD3 + iTCR + CD4 - CD8 - cells or CD3 + iTCR + CD4 + CD8 + The phenotype of a cell or mixture thereof.

4. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to claim 2 or 3, 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 wherein optionally the blood is peripheral blood and / or umbilical cord blood.

5. The isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to any one of claims 1 to 4, wherein the cells are isolated from one or more peripheral blood mononuclear cells.

6. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell according to any one of claims 1-5, wherein the cell comprises one or more polynucleotides encoding a CD7-specific CAR.

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

8. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell according to claim 7, wherein the antigen binding domain is capable of binding CD7.

9. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell according to claim 7 or 8, 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 a 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.

10. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell of claim 9, wherein the at least one stimulatory molecule comprises a zeta chain or a CD3 zeta chain associated with a T cell receptor complex; wherein the intracellular signaling domain further comprises a functional signaling domain of at least one co-stimulatory molecule; and wherein optionally, the at least one co-stimulatory molecule is 4-1BB, CD28, CD27, CD134 (OX40), ICOS, DAP10 or DAP12.

11. The isolated and activated invariant natural killer T (iNKT) cell or type I natural killer T (NKT) cell of any one of claims 1-10, wherein the cell is further modified to comprise an exogenous cytokine, a growth factor, an antibody or an antigen binding fragment, or any combination thereof, wherein the antibody or antigen binding fragment optionally comprises a bispecific T cell engager (BiTE).

12. A pharmaceutical composition comprising the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A method for treating or preventing CD7 in a subject in need thereof + A method for treating cancer, comprising administering to the subject a therapeutically effective amount of the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12.

14. The method of claim 13, wherein the cancer is selected from the group consisting of CD7 + Hematological malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL (Pro-T-ALL), precursor T-ALL (Pre-T-ALL), cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and subsets of 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 malignancies or CD7 + CD1d + A malignant tumor, and wherein optionally, the cancer is resistant or refractory to treatment in the absence of the cells.

15. The method according to claim 13 or 14, wherein the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells are isolated from a biological sample of the subject or donor and are activated by using a cell culture medium having CD3 + iTCR + CD4 + Cells, CD3 + iTCR + CD8 + Cells, CD3 + iTCR + CD4 - CD8 - cells, or CD3 + iTCR + CD4 + CD8 + Synthetic receptor activation and engineered CD3 of cell or mixture phenotypes + iTCR(Vα24-Jα18) + or CD3 + Vα24 + iNKT cells.

16. Use of the isolated and activated invariant natural killer T (iNKT) cells or type I natural killer T (NKT) cells according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing cancer in a subject in need thereof.

17. The use according to claim 15 or 16, wherein the cancer is CD7 + Hematologic malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL, precursor T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and subsets of 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 malignancies or CD7 + CD1d + A malignant tumor, and wherein optionally, the cancer is resistant or refractory to treatment in the absence of the cells.

18. A method for treating or preventing CD7 in a subject in need thereof + A method of treating cancer comprising: a. isolating one or more invariant natural killer T cells from a biological sample based on the expression of the TCR α-chain Vα24-Jα18 (iTCR); b. 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 in the growth medium to one or more iTCR + Unchanged natural killer T cells are primed or activated to proliferate; c. Lentivirally engineering activated invariant natural killer T cells to express CAR (e.g., CD7); d. Expanding one or more CAR (e.g., CD7) engineered invariant natural killer T cells 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 in growth medium.

19. The method of claim 18, further comprising modifying the one or more invariant natural killer T cells to express CAR or TCR or TCRm, wherein the modification comprises introducing one or more polynucleotides encoding the 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 wherein optionally, the DNA comprises transposon DNA.

20. The method of claim 19, wherein the one or more polynucleotides comprise one or more vectors, wherein the one or more vectors comprise one or more viral vectors or lentiviral vectors or gamma-retroviral vectors.

21. The method of any one of claims 18-20, wherein the cancer is CD7 + Hematologic malignancies; T-cell lymphoblastic leukemia (T-ALL) and T-ALL subtypes, including early thymic precursor (ETP)-ALL (ETP-ALL), early precursor T-ALL, precursor T-ALL, cortical T-ALL, and mature T-ALL; peripheral T-cell lymphoma (PTCL) and subsets of 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 malignancies or CD7 + CD1d + A malignant tumor, and wherein optionally, the cancer is resistant or refractory to treatment in the absence of the cells.

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