Nucleic acid, engineered immune cells containing the same, and preparation method and use thereof

Abstract

The present invention relates to a nucleic acid, an engineered immune cell comprising the nucleic acid, and a preparation method and use thereof. The nucleic acid comprises a nucleotide sequence encoding an anti-CD7 antibody, the nucleotide sequence encoding the anti-CD7 antibody is connected to a nucleotide sequence encoding a localization domain, and the localization domain comprises an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, or a proteasome localization sequence. The engineered immune cell comprising the nucleic acid of the present invention has enhanced therapeutic efficacy.

Description

[0001] This application is a divisional application of a Chinese patent application filed on February 5, 2016, with application number 201680018962.7 and invention name “Method for enhancing the efficacy of therapeutic immune cells”. Background Art

[0002] Immune cells can be powerful and specific "living drugs." Immune cells have the potential to target tumor cells while sparing normal tissue; several clinical observations indicate that they may have major anti-cancer activity. Thus, in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT), T cell-mediated graft-versus-host disease (GvHD) (Weiden, PL et al., N. Engl. J. Med. 1979; 300(19): 1068-1073; Appelbaum, FR Nature, 2001; 411(6835): 385-389; Porter, DL et al., N. Engl. J. Med. 1994; 330(2): 100-106; Kolb, HJ et al., Blood. 1995; 86(5): 2041-2050; Slavin, S. et al., Blood. 1996; 87(6): 2195-2204), as well as donor natural killer (NK) cell alloreactivity (Ruggeri et al., 2001; 330(2): 100-106) are common. L et al. Science. 2002; 295(5562): 2097-2100; Giebel S et al. Blood. 2003; 102(3): 814-819; Cooley S et al. Blood. 2010; 116(14): 2411-2419) is negatively correlated with leukemia relapse. In addition to the HSCT setting, administration of antibodies that release T cells from inhibitory signals (Sharma P et al., Nat Rev Cancer. 2011; 11(11): 805-812.; Pardoll DM., Nat Rev Cancer. 2012; 12(4): 252-264) or bridge them to tumor cells (Topp MS et al. J. Clin. Oncol. 2011; 29(18): 2493-2498) produces major clinical responses in patients with solid tumors or leukemias. Finally, infusion of genetically modified autologous T lymphocytes induced complete and durable remissions in patients with refractory leukemias and lymphomas (Maude SL et al. N Engl J Med. 2014; 371(16): 1507-1517).

[0003] However, there is a significant need for improving immune cell therapies by broadening their applicability and enhancing their efficacy. Summary of the invention

[0004] The present invention relates to engineered immune cells with enhanced therapeutic efficacy for, for example, cancer treatment. In certain embodiments, the present invention provides engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain.

[0005] In other embodiments, the present invention provides the use of engineered immune cells for treating cancer, wherein the engineered immune cells comprise a gene encoding an immune activation receptor and a gene encoding a target binding molecule linked to a localization domain, and the use comprises administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0006] In various embodiments, the present invention also provides a method for producing an engineered immune cell, the method comprising introducing into an immune cell a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, thereby producing an engineered immune cell.

[0007] In some embodiments, the engineered immune cells have enhanced therapeutic efficacy due to one or more of reduced graft-versus-host disease (GvHD) in the host, reduced or eliminated rejection by the host, prolonged survival in the host, reduced inhibition by tumors in the host, reduced autokilling in the host, reduced inflammatory cascades in the host, or sustained natural / artificial receptor-mediated (e.g., CAR-mediated) signal transduction in the host. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing will be apparent from the following more particular description of exemplary embodiments of the present invention, as shown in the accompanying drawings, in which like reference numerals refer to like parts throughout the different views. The accompanying drawings are not necessarily drawn to scale, emphasis instead being placed on illustrating embodiments of the present invention.

[0009] Figure 1 is a graphical representation of the strategy employed in the present invention. Figure 1 A in FIG is the overall mechanism of CAR-mediated cancer cell killing. Figure 1 B shows the combined expression of CAR with different forms of compartment-directed scFv (an example of a target binding molecule linked to a localization domain) and examples of possible targets. CAR can be replaced with other receptors that can enhance the capabilities of immune cells.

[0010] Figure 2It is a schematic diagram of the construct containing scFv together with the domain that localizes them to a specific cell compartment. Abbreviations: β2M, β-2 microglobulin; SP, signal peptide; VL, variable light chain; VH, variable heavy chain; TM, transmembrane domain; HA, human influenza hemagglutinin. Other constructs not listed in the figure include membrane-bound (mb) myc EEKKMP, mb myc KKTN, mb mycYQRL, mb TGN38 cytoplasmic domain, mb myc RNIKCD, linker (20 amino acids) mb EEKKMP, and construct variants without signal peptide and with different numbers of amino acids in the CD8 transmembrane domain. The nucleotide sequence of the linker of 10 amino acids is GGTGGTGGCGGCAGTGGTGGCGGTGGCTCA (SEQ ID NO: 61); the amino acid sequence is GGGGSGGGGS (SEQ ID NO: 62). The nucleotide sequence of the 20 amino acid linker is GGTGGTGGCGGCAGTGGTGGCGGTGGCTCAGGCGGTGGTGGCTCCGGTGGCGGTGGCTCT (SEQ ID NO:63); the amino acid sequence is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:41). Various localization domains are indicated under the heading "Localization Domains", and linkers are described in some examples, as shown. The constructs "myc KDEL" and "PEST KDEL" show the use of more than one localization domain in a single construct.

[0011] Figures 3A-3C Downregulation of CD3 / TCR in T cells by scFv targeting of CD3ε is shown. Figure 3A Shown is the surface CD3ε expression in Jurkat cells transduced with a retroviral vector containing green fluorescent protein (GFP) alone ("mock"), or with a vector containing GFP plus different constructs, as indicated. CD3ε expression on the cell membrane was compared to that of mock-transduced cells 1 week after transduction using an anti-CD3 antibody conjugated to allophycocyanin (BD Biosciences). All comparisons were performed after gating on GFP-positive cells. Figure 3B Similar experiments performed with peripheral blood T lymphocytes expanded with anti-CD3 / CD28 beads (Lifesciences) are described. Staining was performed 1 week after transfection. Figure 3C Shown are flow cytometry graphs showing downregulation of membrane CD3ε in Jurkat cells following transduction with the indicated constructs. The dashed rectangle on the upper right quadrant of each graph encloses GFP+CD3+ cells.

[0012] Figure 4Shown is down-regulation of CD3ε and TCRαβ on the cell membrane in Jurkat T cells after transduction with anti-CD3εscFv-KDEL or –PEST or –mb EEKKMP. Membrane marker expression was measured 1 week after transduction using anti-CD3 antibody conjugated to allophycocyanin (BD Biosciences) or anti-TCRαβ conjugated to phycoerythrin (Biolegend). The line marked “control” indicates the labeling of mock transduced cells. The dashed vertical line indicates the upper limit of staining obtained with isotype-matched non-reactive antibodies.

[0013] Figure 5 It is shown that anti-scFv and CAR can be expressed simultaneously. Flow cytometry dot plots represent staining of Jurkat cells (top row) or peripheral blood lymphocytes (bottom row) with anti-CD3 allophycocyanin antibody and goat anti-mouse Fab2 biotin plus streptavidin conjugated to phycoerythrin (to detect CAR). Cells were transduced with anti-CD3 scFv-myc KDEL constructs, anti-CD19-4-1BB-CD3 ζ constructs or both. After gating GFP-positive cells, those transduced with anti-CD3 scFv-mycKDEL down-regulated CD3 (left column, lower left quadrant), and those transduced with anti-CD19-4-1BB-CD3 ζ constructs expressed CAR (middle column, upper right quadrant). A large proportion of cells transduced with two constructs were CD3 negative and CAR positive (right column, upper left quadrant).

[0014] Figure 6 It is shown that anti-CD19 CAR triggers T cell activation and degranulation, which is independent of CD3 / TCR downregulation. Jurkat cells are transduced with anti-CD3 scFv-myc KDEL constructs, anti-CD19-4-1BB-CD3ζ constructs, or both. T cell activation and degranulation are compared with those of simulated transduced cells. Cells are co-cultured alone or with CD19+ leukemia cell line OP-1 at a ratio of 1: 1. After 18 hours, the expression of CD69 and CD25 is tested by flow cytometry using specific antibodies (from BD Biosciences); the expression of CD107a is tested after 6 hours (antibodies from BD Biosciences). In the presence of OP-1 cells, whether or not the cells are also transduced with anti-CD3 scFv-KDEL, CD69 and CD25 expression in CAR-expressing cells occurs; activation does not occur in cells transduced with simulation or anti-CD3 scFv-myc KDEL or in the absence of OP-1 cells. CAR stimulation enhances CD107 expression independent of CD3 / TCR downregulation.

[0015] Figure 7It is shown that anti-CD19 CAR expressed in T cells causes T cell proliferation, regardless of CD3 / TCR downregulation. Peripheral blood T lymphocytes were transduced with both anti-CD3 scFv-myc KDEL constructs and anti-CD19-4-1BB-CD3ζ constructs. Transduced T lymphocytes were co-cultured with OP-1 cells treated with Streck (Omaha, NE) to inhibit their proliferation within the indicated time. The amplification of CD3-positive and CD3-negative T lymphocytes expressing anti-CD19 CAR was compared with the amplification of T cells transduced by simulation. Each symbol shows the average cell count of two parallel cultures. Regardless of CD3 / TCR expression, CAR T cells are equally well amplified.

[0016] Figure 8 CD7 expression on the membrane of peripheral blood T lymphocytes transduced with a retroviral vector containing GFP alone ("mock"), or a vector containing GFP plus and anti-CD7 scFv-myc KDEL constructs is shown. CD7 expression on the cell membrane was compared to that of mock-transduced cells 1 week after transduction using an anti-CD7 antibody conjugated to phycoerythrin (BD Biosciences). The dashed rectangle on the upper right quadrant of each figure encloses GFP+CD7+ cells.

[0017] Fig. 9 Depicts downregulation of HLA class I in T cells by scFv targeting of β2-microparticle protein. Jurkat T cells were transduced with anti-β2M scFv-myc KDEL. HLA-ABC expression on the cell membrane was compared with that of mock-transduced cells 1 week after transduction using anti-HLA-ABC antibody conjugated to phycoerythrin (BD Biosciences). Staining with an isotype-matched control antibody is also shown. Analysis was performed after gating on GFP-positive cells.

[0018] Fig.10Depicted is downregulation of killer cell immunoglobulin-like receptor (KIR) 2DL1 and KIR2DL2 / DL3 in human NK cells by scFv targeting of KIR2DL1 and KIR2DL2 / DL3. NK cells were transduced with anti-KIR2DL1-KIR2DL2 / DL3 scFv-linkers (20) AEKDEL or -EEKKMP, which were expanded ex vivo and selected for KIR2DL1 expression. 8 days after transduction using anti-KIR2DL1 antibodies conjugated to allophycocyanin (R&D Systems) or anti-KIR2DL2 / DL3 antibodies conjugated to phycoerythrin (BD Biosciences), the corresponding KIR expression on the cell membrane was compared to that of mock-transduced cells. Staining with isotype-matched control antibodies is also shown. Analysis was performed after gating on GFP-positive cells.

[0019] Fig.11 Described is the downregulation of NKG2A in human NK cells by scFv targeting. NK cells expanded in vitro were transduced with anti-NKG2A scFv-EEKKMP. 8 days after transduction using NKG2A antibody (Beckman Coulter) conjugated to phycoerythrin, NKG2A expression on the cell membrane was compared with that of mock transduced cells. Also shown was staining with an isotype-matched control antibody. Analysis was performed after gating on GFP-positive cells. DETAILED DESCRIPTION

[0020] The following is a description of example embodiments of the invention.

[0021] In recent years, the growth in knowledge about the molecular pathways regulating immune cells has been paralleled by significant changes in the ability to manipulate them (including their expansion and genetic engineering) in vitro. It is now possible to reliably prepare highly complex clinical-grade immune cell products in a timely manner. The main example of how the anti-cancer activity of immune cells can be guided and amplified by ex vivo cell engineering is the development of chimeric antigen receptor (CAR) T cells (Eshhar, Z. et al., PNAS. 1993; 90 (2): 720-724).

[0022] CAR is an artificial multimolecular protein, which has been described previously (Geiger TL et al., J Immunol. 1999; 162 (10): 5931-5939; Brentjens RJ et al., Nat Med. 2003; 9 (3): 279-286; Cooper LJ et al., Blood. 2003; 101 (4): 1637-1644). CAR comprises an extracellular domain, a transmembrane domain and a cytoplasmic domain that bind to a specific target. The extracellular domain and the transmembrane domain can be derived from any desired source for such a domain, as described in, for example, U.S. Patent No. 8,399,645, which is incorporated herein by reference in its entirety. In short, CAR can be designed as a single-chain variable region (scFv) containing an antibody that specifically binds to a target. ScFv can be connected to a signaling molecule such as CD3ζ associated with a T cell receptor (TCR) via a transmembrane domain and a hinge domain. The connection of scFv to the cognate antigen triggers signal transduction. Therefore, CAR can transiently redirect cytotoxic T lymphocytes toward cancer cells and trigger tumor cell lysis (Eshhar, Z. et al., PNAS.1993; 90(2):720-724; Geiger TL et al., J Immunol.1999; 162(10):5931-5939; Brentjens RJ et al., Nat Med.2003; 9(3):279-286; Cooper LJ et al., Blood.2003; 101(4):1637-1644; Imai C et al., Leukemia.2004; 18:676-684). Because CD3ζ signaling alone is not enough to continuously activate T cells (Schwartz RH.Annu Rev Immunol.2003; 21: 305-334; Zang X and Allison JP.Clin Cancer Res.2007; 13 (18Pt 1): 5271-5279), costimulatory molecules such as CD28 and 4-1BB (or CD137) have been incorporated into CAR constructs to promote signal transduction. This dual signaling design ("second generation CAR") can be used to elicit effective anti-tumor activity from T cells (Imai C et al., Leukemia.2004; 18: 676-684; Campana D et al., Cancer J.2014; 20 (2): 134-140).

[0023] A specific CAR containing both 4-1BB and CD3ζ, anti-CD19 CAR, has been described in U.S. Pat. No. 8,399,645. Infusion of autologous T cells expressing anti-CD19-4-1BB-CD3ζ CAR resulted in a dramatic clinical response in patients with chronic lymphocytic leukemia (CLL) (Porter DL et al., Chimeric antigen receptor-modified T cells in chronic lymphoidleukemia; 2011: N Engl J Med. 2011; 365(8): 725-733; Kalos M et al., Sci Transl Med. 2011; 3(95): 95ra73) and acute lymphoblastic leukemia (ALL) (Grupp SA et al., N Engl J Med. 2013; 368(16): 1509-1518; Maude SL et al., N Engl J Med. 2014; 371(16): 1507-1517). These studies and studies using CARs with different signaling modules (Till BG et al., Blood. 2012; 119(17):3940-3950; Kochenderfer JN et al., Blood. 2012; 119(12):2709-2720; Brentjens RJ et al., Blood. 2011; 118(18):4817-4828; Brentjens RJ et al., Sci Transl Med. 2013; 5(177):177ra138) provide strong confirmation of the clinical potential of this technology and immunotherapy in general.

[0024] Methods described herein allow for the rapid removal or inactivation of specific proteins in immune cells redirected by natural or artificial receptors (such as CAR), thus widening the application potential and significantly improving the function of engineering cells. The method is partially dependent on a single construct or multiple constructs, which contain immune activation receptors such as CAR (which include an extracellular domain (such as scFv), a transmembrane domain and a cytoplasmic domain for binding specific targets) together with a target binding molecule, and the target binding molecule binds to a target (such as protein) to be removed or neutralized; depending on the application, the target binding molecule is connected to a domain (i.e., a localization domain) to which it is directed to a specific cell compartment (such as a Golgi apparatus or endoplasmic reticulum, a proteasome or a cell membrane). For simplicity, the target binding molecule connected to a localization domain (LD) is sometimes referred to herein as "the target binding molecule connected by LD".

[0025] As is apparent from the teachings herein, various immune activation receptors may be suitable for the methods of the present invention. That is, any receptor comprising a molecule can be used according to the present method, and after binding (connecting) a ligand (eg, a peptide or antigen) expressed on a cancer cell, the molecule is capable of activating an immune response. For example, as described above, an immune activation receptor may be a chimeric antigen receptor (CAR); methods for designing and manipulating CAR are known in the art (see, Geiger TL et al., J Immunol. 1999; 162 (10): 5931-5939; Brentjens RJ et al., Nat Med. 2003; 9 (3): 279-286; Cooper LJ et al., Blood. 2003; 101 (4): 1637-1644). In addition, a receptor with antibody binding ability (e.g., CD16-4-1BB-CD3ζ receptor-Kudo K et al. Cancer Res. 2014; 74 (1): 93-103) can be used, which is similar to CAR, but replaces scFv with an antibody binding molecule (e.g., CD16, CD64, CD32). In addition, according to the present method, a T cell receptor comprising T cell receptor α and β chains can also be used, and the T cell receptor α and β chains bind to peptides expressed on tumor cells in the context of tumor cell HLA. In addition, other receptors with molecules that activate immune responses by binding to ligands expressed on cancer cells-e.g., NKG2D-DAP10-CD3ζ receptors, which bind to NKG2D ligands expressed on tumor cells (see, e.g., Chang YH et al., Cancer Res. 2013; 73 (6): 1777-1786). As used herein, all of these suitable receptors are collectively referred to as "immune activation receptors" or "receptors that activate immune responses after binding to cancer cell ligands". Thus, immune activating receptors with molecules activated by cancer cell ligands can be expressed along with LD-linked target binding molecules according to the present methods.

[0026] Based on the infusion of immune cells redirected by artificial receptors, this method significantly expands the potential application of immunotherapy. The method is practical and can be easily incorporated into clinical-grade cell processing. For example, by inserting an internal ribosome entry site (IRES) or a 2A peptide coding region site between 2 cDNAs encoding the target binding molecules connected by CAR and LD, a single bicistronic construct containing target binding molecules such as scFv-myc KDEL (or PEST or transmembrane) connected by CAR and LD can be prepared. It should also be feasible to design a tricistronic delivery system to lack more than one target. Alternatively, the separate transcription of 2 genes can be performed (simultaneously or sequentially). In the context of cancer cell therapy, CAR can be replaced by antibody binding signaling receptors (Kudo K et al., Cancer Res.2014; 74 (1): 93-103), T cell receptors for specific HLA-peptide combinations, or any receptors activated by contact with cancer cells (Chang YH et al., Cancer Res.2013; 73 (6): 1777-1786). The results of the studies described herein using simultaneous anti-CD19-4-1BB-CD3ζCAR and anti-CD3εscFv-KDEL demonstrated that the signaling capacity of the CAR was not impaired.

[0027] Both anti-CD3εscFv-KDEL (and – PEST) tested herein stably downregulate CD3 and TCR expression. Residual CD3+T cells can be removed using CD3 beads, which is also a method available in clinical-grade forms. The ability to generate CD3 / TCR-negative cells that respond to CAR signaling represents a major advance. Clinical studies using CAR T cells have generally been performed using autologous T cells. Therefore, the quality of cell products varies from patient to patient, and the response is heterogeneous. Due to the stimulation of endogenous TCR by the recipient's tissue antigens, the infusion of allogeneic T cells is currently impossible because it has an unacceptably high risk of potentially fatal GvHD. Downregulation of CD3 / TCR opens up the possibility of infusing allogeneic T cells because the lack of endogenous TCR eliminates the ability of GvHD. Allogeneic products can be prepared and selected with optimal cell composition (e.g., T cells rich in highly cytotoxicity, exhausted regulatory T cells, etc.) so that the infused cells have high CAR expression and functional efficacy. In addition, fully standardized products can be cryopreserved and can be used, regardless of the patient's immune cell status and his / her adaptability to undergoing apheresis or large-volume blood draws. Gene editing tools such as nucleases have been used to solve the removal of TCR expression (Torikai H et al. Blood, 2012; 119 (24): 5697-5705). Although this is an effective method, it is difficult to implement in a clinical setting because it requires several rounds of cell selection and amplification, accompanied by extended culture. The method described herein has considerable practical advantages.

[0028] In addition, the target binding molecules (e.g., scFv-myc KDEL, scFv-EEKKMP or scFv-PEST, wherein scFv targets specific proteins / molecules) connected by LD can be used to delete HLA class I molecules according to the present invention to reduce the possibility of allogeneic cell rejection. Although the infusion of allogeneic T cells is the future goal of CAR T cell therapy, the infusion of allogeneic natural killer (NK) cells has been used to treat patients with cancer. The key factor that determines the success of NK cell-based treatment is that NK cells must continue to have a sufficient number to achieve an effector: target ratio that may produce a reduction in tumor cells (Miller JS. Hematology Am Soc Hematol Educ Program. 2013; 2013: 247-253). However, when allogeneic cells are infused, their persistence is limited. Immunosuppressive chemotherapy given to patients allows transient engraftment of the infused NK cells, but these are rejected within 2-4 weeks of infusion (Miller JS et al. Blood. 2005; 105: 3051-3057; Rubnitz JE et al., J Clin Oncol. 2010; 28 (6): 955-959). In contrast to organ transplantation, continued immunosuppression is not an option because immunosuppressive drugs also inhibit NK cell function. Because rejection is primarily mediated by the recognition of HLA class I molecules by the recipient's CD8+ T lymphocytes, removing HLA class I molecules from the infused NK cells (or T cells) will reduce or eliminate the rejection rate, prolong the survival of allogeneic cells, and therefore prolong their anti-tumor ability.

[0029] In addition, the target binding molecules connected by LD can be used to target inhibitory receptors according to the present invention. Specifically, the administration of antibodies such as anti-PD1 or anti-CTLA-4 that release T cells from inhibitory signals has produced significant clinical responses (Sharma P et al., Nat Rev Cancer. 2011; 11 (11): 805-812; Pardoll DM. Nat Rev Cancer. 2012; 12 (4): 252-264). CAR-T cells, especially those against solid tumors, can be inhibited by similar mechanisms. Thus, the expression of target binding molecules (e.g., scFv or ligands) for PD1, CTLA-4, Tim3, or other inhibitory receptors will prevent the expression of these molecules (if connected to, for example, KDEL (SEQ ID NO: 4), EEKKMP (SEQ ID NO: 64), or PEST motif SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7)), or prevent the binding of the receptor to its ligand (if connected to the transmembrane domain), and continue CAR-mediated signal transduction. In NK cells, examples of inhibitory receptors include killer cell immunoglobulin-like receptors (KIR) and NKG2A (Vivier E et al., Science, 2011; 331(6013): 44-49).

[0030] The method of the present invention also enables targeting of more targets that are compliant with CAR-guided T cell therapy. One of the main limitations of CAR-guided therapy is the lack of specific antigens expressed by tumor cells. In the case of hematological malignancies such as leukemias and lymphomas, molecules that are not expressed in non-hematopoietic cells may be potential targets, but cannot be used as CAR targets because they are also expressed on T cells and / or NK cells. Expressing this CAR on immune cells may cause the death of the immune cells themselves due to a "cannibalism" mechanism, rendering their anti-cancer ability ineffective. If the target molecule can be removed from the immune cell without adverse functional effects, a CAR with corresponding specificity can be expressed. This opens up many new opportunities for targeting hematological malignancies. Examples of possible targets include CD38 expressed in multiple myeloma, CD7 expressed in T cell leukemia and lymphoma, Tim-3 expressed in acute leukemia, CD30 expressed in Hodgkin's disease, CD45 and CD52 expressed in all hematological malignancies. These molecules are also expressed in a large proportion of T cells and NK cells.

[0031] In addition, it has been shown that cytokines secreted by activated immune cells trigger cytokine release syndrome and macrophage activation syndrome, presenting serious adverse reactions to immune cell therapy (Lee DW et al., Blood. 2014; 124 (2): 188-195). Therefore, LD-linked target binding molecules can be used according to the present invention to block cytokines such as IL-6, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, IL-35, interferon (IFN) -γ, IFN-β, IFN-α, tumor necrosis factor (TNF) -α and transforming growth factor (TGF) -β, which can contribute to this inflammatory cascade.

[0032] Accordingly, in one embodiment, the present invention relates to an engineered immune cell comprising a nucleic acid comprising a nucleotide sequence encoding an immune activating receptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain.

[0033] As used herein, "engineered" immune cells include immune cells that have been genetically modified compared to naturally occurring immune cells. For example, the engineered T cells produced according to the inventive method carry nucleic acids comprising nucleotide sequences that are not naturally present in the T cells from which it is derived. In some embodiments, the engineered immune cells of the present invention include a chimeric antigen receptor (CAR) and a target binding molecule (LD-connected target binding molecule) connected to a localization domain. In a specific embodiment, the engineered immune cells of the present invention include anti-CD19-4-1BB-CD3ζCAR and an anti-CD3scFv connected to a localization domain.

[0034] In certain embodiments, the engineered immune cell is an engineered T cell, an engineered natural killer (NK) cell, an engineered NK / T cell, an engineered monocyte, an engineered macrophage, or an engineered dendritic cell.

[0035] In certain embodiments, as used herein, "immune activation receptor" refers to a receptor that activates an immune response after binding to a cancer cell ligand. In some embodiments, the immune activation receptor comprises a molecule that can activate an immune response after binding to a ligand (e.g., a peptide or antigen) expressed on a cancer cell. In one embodiment, the immune activation receptor is a chimeric antigen receptor (CAR); methods for designing and manipulating CAR are known in the art. In other embodiments, the immune activation receptor is an antibody binding receptor, which is similar to CAR, but replaces scFv with an antibody binding molecule (e.g., CD16, CD64, CD32) (see, e.g., CD16-4-1BB-CD3ζ receptor-Kudo K et al. Cancer Res. 2014; 74 (1): 93-103). In various embodiments, according to the present method, a T cell receptor comprising a T cell receptor α and a β chain can also be used, and in the context of tumor cell HLA, the T cell receptor α and β chains bind to peptides expressed on tumor cells. In certain embodiments, other receptors with molecules that activate immune responses by binding to ligands expressed on cancer cells may also be used—for example, the NKG2D-DAP10-CD3ζ receptor, which binds to NKG2D ligands expressed on tumor cells (see, e.g., Chang YH et al., Cancer Res. 2013; 73(6): 1777-1786). All of these suitable receptors that are capable of activating immune responses after binding (linking) to ligands (e.g., peptides or antigens) expressed on cancer cells are collectively referred to as "immune activating receptors." As will be appreciated by those skilled in the art, immune activating receptors need not contain antibodies or antigen binding fragments (e.g., scFvs); rather, a portion of an immune activating receptor that binds to a target molecule may be derived from, for example, a receptor in a receptor-ligand pair, or a ligand in a receptor-ligand pair.

[0036] In some aspects, immune activation receptors bind to molecules expressed on the surface of tumor cells, including but not limited to CD20, CD22, CD33, CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD52, CD38, CS-1, TIM3, CD123, mesothelin, folate receptor, HER2-neu, epidermal-growth factor receptor and epidermal growth factor receptor. In some embodiments, immune activation receptor is CAR (eg, anti-CD19-4-1BB-CD3ζCAR). In certain embodiments, immune activation receptors include antibodies or their antigen binding fragments (eg, scFv), which bind to molecules expressed on the surface of tumor cells, including but not limited to CD20, CD22, CD33, CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD52, CD38, CS-1, TIM3, CD123, mesothelin, folate receptor, HER2-neu, epidermal-growth factor receptor and epidermal growth factor receptor. Antibodies to these molecules expressed on the surface of tumor cells are known and available in the art. For example, antibodies to CD3 and CD7 are commercially available and known in the art. Such antibodies and antibody fragments derived therefrom (e.g., scFv) can be used in the present invention, as shown herein. In addition, methods for producing antibodies and antibody fragments to target proteins are well known and conventional in the art.

[0037] According to the transmembrane domain of the immune activation receptor (eg, CAR) of the present invention, it can be derived from a single transmembrane protein, including but not limited to CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (eg, CD16A or CD16B), OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32 (eg, CD32A or CD32B), CD64 (eg, CD64A, CD64B or CD64C), VEGFR2, FAS and FGFR2B. In some examples, the membrane protein is not CD8α. The transmembrane domain can also be a non-naturally occurring hydrophobic protein segment.

[0038] The hinge domain of the immune activation receptor (eg, CAR) may be derived from a protein such as CD8α or IgG. The hinge domain may be a fragment of the transmembrane domain or hinge domain of CD8α, or a non-naturally occurring peptide, such as a polypeptide consisting of hydrophilic residues of varying lengths, or (GGGGS) n (SEQ ID NO: 8) polypeptide, wherein n is an integer, e.g., 3-12 (inclusive).

[0039] The signaling domain of an immune activation receptor (e.g., CAR) may be derived from CD3ζ, FcεRIγ, DAP10, DAP12, or other molecules known to deliver activation signals in immune cells. At least one co-stimulatory signaling domain of a receptor may be a co-stimulatory molecule, such as 4-1BB (also known as CD137), CD28, CD28 LL→GG Variants, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 or CD2. Such molecules are readily available and known in the art.

[0040] As will be appreciated by those skilled in the art, the components of immune activation receptors can be modified to include many functional combinations as described herein to produce the desired results. Using specific CAR anti-CD19-4-1BB-CD3 ζ as an example, the antibody of the binding molecule (e.g., or its antigen binding fragment, such as scFv) can replace the antibody binding different molecules, as described herein (e.g., anti-CD20, anti-CD33, anti-CD123, etc., instead of anti-CD19). In other embodiments, costimulatory molecules (4-1BB in this specific example) can also be changed with different costimulatory molecules such as CD28. In some embodiments, the stimulatory molecule (CD3 ζ in this specific example) can be replaced by another known stimulatory molecule. In various embodiments, the transmembrane domain of the receptor can also be changed as needed. The functional design, production and testing of this immune activation receptor can be easily determined by those skilled in the art. Similarly, the design, delivery and expression of nucleic acids encoding this immune activation receptor are easily known and available in the art.

[0041] As used herein, the term "nucleic acid" refers to a polymer comprising a plurality of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules may be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules may be single-stranded, double-stranded, or triple-stranded. In some embodiments, nucleic acid molecules may be modified. In the case of a double-stranded polymer, "nucleic acid" may refer to either or both strands of the molecule.

[0042] The term "nucleotide sequence" referring to a nucleic acid refers to a continuous series of nucleotides linked by covalent bonds, such as phosphate bonds (e.g., phosphodiester, alkyl and aryl-phosphonates, phosphorothioate, phosphotriester bonds), and / or non-phosphate bonds (e.g., peptide and / or sulfamate bonds). In certain embodiments, the nucleotide sequence encoding a target binding molecule, such as one linked to a localization domain, is a heterologous sequence (e.g., a gene having an origin in a different species or cell type).

[0043] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotides or deoxyribonucleotide monomers, and non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides may include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine or deoxycytidine), as well as nucleotides comprising modified bases known in the art.

[0044] As will be appreciated by those skilled in the art, in some aspects, the nucleic acid further comprises a plasmid sequence. The plasmid sequence may include, for example, one or more sequences selected from a promoter sequence, a selection marker sequence, and a locus targeting sequence.

[0045] As used herein, a gene encoding a target binding molecule linked to a localization domain is sometimes referred to as a "LD-linked target binding molecule."

[0046] In certain embodiments, the target binding molecule is an antibody or an antigen binding fragment thereof. As used herein, "antibody" refers to an intact antibody or an antigen binding fragment of an antibody, including intact antibodies or antigen binding fragments that have been modified or transformed, or that are human antibodies. Examples of antibodies that have been modified or transformed are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen binding fragments include Fab, Fab', F(ab')2, Fv, single-chain antibodies (e.g., scFv), mini antibodies, and diabodies.

[0047] The "Fab fragment" contains a light chain and a heavy chain C H 1 and variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0048] The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0049] "A Fab' fragment contains one light chain and a portion of one heavy chain, which portion contains the VH domain and the CH1 domain, as well as a region between the CH1 and CH2 domains that allows the formation of an interchain disulfide bond between the two heavy chains of the two Fab' fragments to form a F(ab')2 molecule.

[0050] The "F(ab')2 fragment" contains two light chains and two heavy chains. H 1 and C H 2A portion of the constant region between the domains allows the formation of an interchain disulfide bond between the two heavy chains. The F(ab')2 fragment is therefore composed of two Fab' fragments held together by the disulfide bonds between the two heavy chains.

[0051] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0052] In a specific embodiment, the target binding molecule is a single-chain Fv antibody ("scFv antibody"). ScFv refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide also comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun (1994) The Pharmacology Of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315. See also PCT Publication No. WO88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. For example, the linker between the VH and VL domains of the scFv disclosed herein comprises, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGSGGGGS (SEQ ID NO: 43). As will be appreciated by those skilled in the art, various suitable linkers can be designed and tested for optimal function, as provided in the art and as disclosed herein.

[0053] The scFv that is part of the target binding molecule that is LD-linked is not necessarily the same as the scFv that exists in the context of, for example, a chimeric antigen receptor (CAR) or similar antibody binding signaling receptor. In some embodiments, the scFv that is part of the target binding molecule that is LD-linked is the same as the scFv that exists in the context of, for example, a chimeric antigen receptor (CAR) or similar antibody binding signaling receptor.

[0054] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., a scFv in the context of an LD-linked target binding molecule) comprises one or more sequences having at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one or more of SEQ ID NO: 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, or 39.

[0055] The term "sequence identity" means that when, for example, the default gap weight is used to carry out the best comparison by the program GAP or BESTFIT, two nucleotides or amino acid sequences share for example at least 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity or more. For sequence comparison, usually a sequence serves as the reference sequence (for example parental sequence) that a test sequence is compared with it. When using a sequence comparison algorithm, test sequence and reference sequence are input in the computer, and when necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Based on the program parameters specified, the sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence.

[0056] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., Current Protocols in Molecular Biology). An example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (National Center for Biotechnology Information) (available for public access through the NCBI Internet server of the National Institutes of Health). Typically, default program parameters can be used to perform sequence comparisons, although custom parameters can also be used. For amino acid sequences, the BLASTP program uses word length (W) 3, expected value (E) 10, and BLOSUM62 scoring matrix as default values ​​(see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0057] In certain embodiments, the antibody (e.g., scFv) comprises a VH and a VL having the amino acid sequences shown in SEQ ID NOs: 12 and 13; SEQ ID NOs: 16 and 17; SEQ ID NOs: 20 and 21; SEQ ID NOs: 24 and 25; SEQ ID NOs: 28 and 29; SEQ ID NOs: 32 and 33; or SEQ ID NOs: 36 and 37, respectively. In some embodiments, the antibody (e.g., scFv) comprises a VH and a VL having sequences that each have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH and VL sequences shown in SEQ ID NOs: 12 and 13; SEQ ID NOs: 16 and 17; SEQ ID NOs: 20 and 21; SEQ ID NOs: 24 and 25; SEQ ID NOs: 28 and 29; SEQ ID NOs: 32 and 33; or SEQ ID NOs: 36 and 37, respectively.

[0058] "Diabodies" are small antibody fragments with two antigen binding sites. The fragment contains a heavy chain variable region (VH) connected to a light chain variable region (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and produce two antigen binding sites. Diabodies are described in, for example, patent documents EP 404,097; WO 93 / 11161; and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

[0059] In certain embodiments, the antibody is a triabody or a tetrabody. Methods for designing and producing triabodies and tetrabodies are known in the art. See, for example, Todorovska et al., J. Immunol. Methods 248(1-2):47-66, 2001.

[0060] "Domain antibody fragments" are immunoglobulin fragments that contain only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to produce a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

[0061] In some embodiments, the antibody is modified or engineered. Examples of modified or engineered antibodies include chimeric antibodies, multiparatopic antibodies (eg, biparatopic antibodies), and multispecific antibodies (eg, bispecific antibodies).

[0062] As used herein, a "multi-paratopic antibody" means an antibody comprising at least two single domain antibodies, wherein at least one single domain antibody is directed against a first antigenic determinant on an antigen, and at least one other single domain antibody is directed against a second antigenic determinant on the same antigen. Thus, for example, a "biparatopic" antibody comprises at least one single domain antibody directed against a first antigenic determinant on an antigen, and at least one further single domain antibody directed against a second antigenic determinant on the same antigen.

[0063] As used herein, a "multispecific antibody" means an antibody comprising at least two single domain antibodies, wherein at least one single domain antibody is directed against a first antigen and at least one other single domain antibody is directed against a second antigen (different from the first antigen). Thus, for example, a "bispecific" antibody is a "bispecific" antibody comprising at least one single domain antibody directed against a first antigen and at least one further single domain antibody directed against a second antigen (e.g., different from the first antigen).

[0064] In some embodiments, the antibodies disclosed herein are monoclonal antibodies, such as mouse monoclonal antibodies. Methods for producing monoclonal antibodies are known in the art. See, for example, Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315.

[0065] In various embodiments, the target binding molecule in the context of the LD-linked target binding molecule is a receptor or ligand that binds to the target molecule. For example, the target binding molecule may be a ligand that binds to PD-1 (e.g., PD-L1 or PD-L2). Therefore, as will be appreciated by those skilled in the art, the target binding molecule may be an antibody or ligand / receptor that binds to the target molecule.

[0066] As used herein, "connected" in the context of LD-linked target binding molecules refers to a gene encoding a target binding molecule that is directly adjacent in frame (e.g., without a linker) to one or more genes encoding one or more localization domains. Alternatively, as described herein, a gene encoding a target binding molecule can be connected to one or more genes encoding one or more localization domains via a linker sequence. Various suitable linkers known in the art can be used to tether a target binding molecule to a localization domain. For example, non-naturally occurring peptides, such as polypeptides composed of hydrophilic residues of varying lengths, or (GGGGS) can be used according to the present invention. n (SEQ ID NO:8) polypeptide, wherein n is an integer such as 3-12 inclusive. In a specific embodiment, the joint comprises, for example, GGGGSGGGGS (SEQ ID NO:62). In some embodiments, the joint comprises, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:41). In various embodiments, peptide joints with a length of about 5 to about 100 amino acids (including the end values) can be used in the present invention. In certain embodiments, peptide joints with a length of about 20 to about 40 amino acids (including the end values) can be used in the present invention. In some embodiments, peptide joints with a length of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, or at least 40 amino acids can be used in the present invention. As will be appreciated by those skilled in the art, such joint sequences and variants of such joint sequences are known in the art. Methods for designing constructs incorporating joint sequences and methods for evaluating functionality are readily available to those skilled in the art.

[0067] In certain embodiments, the target binding molecules connected by LD bind to a target expressed on the surface of an immune cell. In some embodiments, the target binding molecules connected by LD inhibit the activity or function of the target molecule. For example, as disclosed herein, the target binding molecules connected by LD can be designed to bind to, for example, CD3, CD7, CD45, hB2MG, KIR2DL1, KIR2DL2 / DL3 or NKG2A, thereby down-regulating the cell surface expression of these molecules. The down-regulation of these molecules can be achieved by, for example, positioning / targeting the molecules for degradation and / or internalization. In other embodiments, the target binding molecules connected by LD render the target inactive (e.g., the target can no longer interact and / or bind to its cognate ligand or receptor).

[0068] In certain embodiments, the engineering immune cells of the present invention have enhanced therapeutic efficacy. As used herein, "enhanced therapeutic efficacy" refers to reduced graft-versus-host disease (GvHD) in the host, reduced or eliminated by host rejection, extended survival in the host, reduced suppression of tumors in the host, reduced autokilling in the host, reduced inflammatory cascades in the host, or continued CAR-mediated signal transduction in the host One or more.

[0069] In certain embodiments of the invention, the target binding molecule in the context of a LD-linked target binding molecule binds to a molecule in the CD3 / T cell receptor (TCR) complex, a cytokine, a human leukocyte antigen (HLA) class I molecule, or a receptor that downregulates an immune response.

[0070] In certain embodiments, the molecule in the CD3 / TCR complex may be CD3ε, TCRα, TCRβ, TCRγ, TCRδ, CD3δ, CD3γ, or CD3ζ. In a specific embodiment, the molecule is CD3ε.

[0071] In another embodiment, the HLA class I molecule is beta-2 microglobulin, alpha 1-microglobulin, alpha 2-microglobulin, or alpha 3-microglobulin.

[0072] In other embodiments, the receptor that downregulates the immune response is selected from, for example, PD-1, CTLA-4, Tim3, killer cell immunoglobulin-like receptors (KIRs—e.g., KIR2DL1 (also known as CD158a), KIR2DL2 / DL3 (also known as CD158b)), CD94 or NKG2A (also known as CD159a), protein tyrosine phosphatases such as Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2. Thus, such receptors can be targeted by partially LD-linked target binding molecules as described herein.

[0073] In various embodiments, examples of cytokines that can be targeted using a target binding molecule that can be linked to a partial LD ​​include, for example, interleukin (IL)-6, IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, IL-27, IL-35, interferon (IFN)-γ, IFN-β, IFN-α, tumor necrosis factor (TNF)-α, or growth factor (TGF)-β.

[0074] In a further aspect, the LD-linked target binding molecule binds a molecule selected from, e.g., CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127.

[0075] Methods for producing antibodies and antibody fragments thereof against any target protein are well known and conventional in the art. In addition, as exemplified herein, commercially available antibodies against various targets such as CD3 and CD7 can be used to generate target binding molecules connected to LDs as exemplified herein. Antibodies known in the art and antibody fragments derived therefrom (e.g., scFv) can be used in the present invention, as exemplified herein.

[0076] In other aspects, the localization domain of the LD-linked target binding molecule comprises the endoplasmic reticulum (ER) retention sequence KDEL (SEQ ID NO: 4), or other ER or Golgi retention sequences, such as KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10) (wherein X can be any amino acid - see Gao C et al., Trends in Plant Science 19: 508-515, 2014) and YQRL (SEQ ID NO: 11) (see Zhan J et al., Cancer Immunol Immunother 46: 55-60, 1998); comprising, for example, the "PEST" motif - SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO:7) proteasome targeting sequence; and / or a sequence that targets a target binding molecule to a cell membrane, such as a CD8α transmembrane domain or another single-pass transmembrane protein, as described herein (e.g., CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (e.g., CD16A or CD16B), OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32 (e.g., CD32A or CD32B), CD64 (e.g., CD64A, CD64B or CD64C), VEGFR2, FAS or FGFR2B). Examples of specific localization domains (sequences) exemplified herein are shown in Figure 2 Various other localization sequences are known and available in the art.

[0077] like Figure 2 As shown, the LD-linked target binding molecules of the present invention may comprise one or more localization domains. For example, the LD-linked target binding molecules may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten localization domains linked together. When more than one localization domain is used in a given LD-linked target binding molecule, each localization domain may be linked with or without any intervening linkers. For example, Figure 2As shown, the localization domains CD8 TM, PEST motif and EEKKMP can be used in a single LD-linked target binding molecule. Although this particular construct shows the localization domains without any intervening linkers, various intervening linkers can be incorporated between some or all of the localization domains. Other examples are shown in Figure 2 middle.

[0078] As will be appreciated by those skilled in the art, the target binding molecules connected to immune activation receptors and / or LDs can be designed to bind to targets disclosed herein, as well as variants of targets disclosed herein. For example, target binding molecules connected to immune activation receptors and / or LDs can be designed to bind to molecules in CD3 / TCR complexes, or naturally occurring variant molecules thereof. Such naturally occurring variants may have the same function as the wild-type form of the molecule. In other embodiments, variants may have a function that is changed relative to the wild-type form of the molecule (e.g., conferring a diseased state).

[0079] As will be appreciated by those skilled in the art, Figure 2 The various components of the LD-linked target binding molecule construct shown can be substituted in different combinations (e.g., containing different linkers, different positioning sequences, different scFvs, etc.), as long as the combination produces a functional LD-linked target binding molecule. Methods for evaluating the functionality of a particular construct are within the knowledge of those skilled in the art, as disclosed herein.

[0080] In a further aspect, the present invention relates to the use of engineered immune cells for treating cancer, wherein the engineered immune cells include a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor, and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., scFv) linked to a localization domain, the use comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0081] In another aspect, the present invention relates to the use of engineered immune cells for treating cancer, wherein the engineered immune cells include a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid comprising a nucleotide sequence encoding a single-chain variable fragment (scFv) connected to a localization domain, and the use includes administering a therapeutic amount of engineered immune cells to a subject in need thereof.

[0082] In other aspects, the present invention relates to the use of engineered immune cells for treating autoimmune disorders, wherein the engineered immune cells include a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., scFv) linked to a localization domain, the use comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0083] In other aspects, the present invention also relates to the use of engineered immune cells for treating infectious diseases, wherein the engineered immune cells include a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor, and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., scFv) linked to a localization domain, and the use includes administering a therapeutic amount of engineered immune cells to a subject in need thereof.

[0084] In various embodiments, the immune activating receptor is a CAR (eg, an anti-CD19-4-1BB-CD3ζ CAR).

[0085] In other embodiments, the single chain variable fragment (scFv) linked to the localization domain is selected from Figure 2 Any one or more of the constructs shown.

[0086] In some aspects, the engineered immune cells are administered by infusion into a subject. Methods for infusing immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient so as to ameliorate the symptoms of the disease. Typically, 10 7 Up to 10 10 A dose of 10 cells, for example 9 The infusion was a single 10 9 Cell doses can be divided into several 10 9 Cell dose. When needed or indicated, the frequency of infusion can be every 3 to 30 days or even longer intervals. The number of infusions is generally at least 1 infusion / subject, and preferably at least 3 infusions, as tolerated, or until the disease symptoms have improved. The cells can be infused intravenously at a rate of 50-250 ml / hour. Other suitable modes of administration include intra-arterial infusion, direct injection into the tumor and / or postoperative tumor bed perfusion, implantation in an artificial scaffold at the tumor site, intrathecal administration, and intraocular administration. Methods for adapting the present invention to this delivery mode are readily available to those skilled in the art.

[0087] In certain aspects, the cancer to be treated is a solid tumor or a hematological malignancy. Examples of hematological malignancies include acute myeloid leukemia, chronic myeloid leukemia, myelodysplasia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Examples of solid tumors include lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, neuroblastoma, rhabdomyosarcoma, brain tumors.

[0088] In another embodiment, the present invention relates to a method for producing an engineered immune cell of the present invention, the method comprising introducing into an immune cell a nucleic acid comprising a nucleotide sequence encoding an immune activation receptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain, thereby producing an engineered immune cell.

[0089] In certain embodiments, a nucleic acid comprising a nucleotide sequence is introduced into an immune cell ex vivo. In other embodiments, a nucleic acid comprising a nucleotide sequence is introduced into an immune cell in vivo.

[0090] In some embodiments, "immune cells" include, for example, T cells, natural killer (NK) cells, NK / T cells, monocytes, macrophages, or dendritic cells.

[0091] The nucleic acid comprising the nucleotide sequence to be introduced can be a single bicistronic construct, which contains an immune activation receptor as described herein and a target binding molecule (e.g., scFv) connected to a localization domain. As described herein, by inserting an internal ribosome entry site (IRES) or a 2A peptide coding region site between 2 cDNAs encoding immune activation receptors (e.g., CAR) and target binding molecules (e.g., scFv) as described herein, a single bicistronic construct can be prepared. It should also be feasible to design a tricistronic delivery system to lack more than one target. Alternatively, separate transcription (simultaneous or sequential) of each construct (e.g., target binding molecules connected by CAR and LD) can be performed. The method of introducing exogenous nucleic acids is exemplified herein, and is well known in the art.

[0092] As used herein, the indefinite articles "a" and "an" should be understood to mean "at least one", unless explicitly stated to the contrary.

[0093] Example

[0094] method

[0095] Cloning of scFv from mouse anti-human CD3 hybridoma

[0096] PLU4 hybridoma cells secreting anti-human CD3 monoclonal antibodies (IgG2a isotype; Creative Diagnostics, Shirley, NY) were cultured in IMDM plus GlutaMAX medium (Life Technologies, Carlsbad, CA) with 20% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and antibiotics. Total RNA was extracted using TRIzol reagent (Life Technologies) and expressed by M-MLV reverse transcriptase (Promega, Madison, WI) and oligo(dT) 15cDNA was synthesized using primers (Promega). IgG Library Primer Set Mouse BioGenomics (US Biological, Salem, MA) was used to amplify the variable regions of the heavy chain (VH) and light chain (VL); PCR products were cloned into a TOPO TA cloning kit for sequencing (Life Technologies). The VH and VL genes were assembled into scFv using splicing by overlap extension PCR, with a flexible linker sequence encoding (Gly4Ser)4. The signal peptide domain of CD8α was subcloned by PCR using cDNA derived from human activated T cells from healthy donors and attached to the 5' end of the VL fragment. A Myc tag (EQKLISEEDL; SEQ ID NO: 1) was added to the C-terminus of VH by PCR using a sense primer: 5'-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3' (SEQ ID NO: 2) and a reverse primer: 5'-CAGATCTTCTTCAGAAATAAGTTTTTGTTCGGCTGAGGAGACTGTGAGAG-3' (SEQ ID NO: 3). In addition, a KDEL (SEQ ID NO: 4) coding sequence was generated after the Myc tag by a sense primer: 5'-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3' (SEQ ID NO: 5) and a reverse primer: 5'-TATATACTCGAGTTACAACTCGTCCTTCAGATCTTCTTCAGAAATAAG-3' (SEQ ID NO: 6). A synthetic gene consisting of a CD8 signal peptide, a scFv against human CD3, a Myc tag, and a KDEL (SEQ ID NO: 4) sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which myc-KDEL was replaced with other sequences were also prepared, such as Figure 2 Listed in.

[0097] The sequence of the "PEST"-SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7) motif corresponding to amino acids 422-461 of mouse ornithine decarboxylase was obtained from GenBank (accession number NM_013614.2). Codon optimization and gene synthesis were performed by GenScript (Piscataway, NJ) and subcloned into the 3' end of VH by PCR. The construct was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

[0098] Cloning of scFv against human CD7

[0099] The sequence scFv derived from the mouse TH69 (anti-CD7) antibody was obtained from the literature (Peipp et al., Cancer Res 2002 (62): 2848-2855). After codon optimization, the synthetic gene consisting of the CD8 signal peptide, the scFv for human CD7, the Myc tag and the KDEL (SEQ ID NO: 4) sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which myc-KDEL was replaced with other sequences were also prepared, such as Figure 2 Listed in.

[0100] Cloning of scFv against human beta-2 microglobulin (hB2MG)

[0101] The sequence scFv derived from the mouse BBM.1 (anti-hB2MG) IgG2b antibody was obtained from the literature (Grovender, EA et al., Kidney Int. 2004; 65(1):310-322). After codon optimization, the synthetic gene consisting of the CD8 signal peptide, scFv for human B2MG, Myc tag and KDEL (SEQ ID NO: 4) sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

[0102] Cloning of scFvs against human KIR2DL1 and KIR2DL2 / DL3

[0103] The amino acid sequence of human monoclonal antibody I-7F9 (anti-KIR2DL1, KIR2DL2 and KIR2DL3) is derived from the published international patent application WO2006003179 A2 by Moretta et al. After codon optimization, the sequence of the scFv was designed by connecting the variable light (VL) region and the variable heavy (VH) region with a linker sequence. A synthetic gene consisting of a CD8 signal peptide, scFvs against human KIRs (KIR2DL1, KIR2DL2 and KIR2DL3), a CD8 hinge and transmembrane domain, and a KKMP sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP was replaced with other sequences, such as Figure 2 Listed in.

[0104] Cloning of scFv against human NKG2A

[0105] The sequence of the mouse antibody Z199 (anti-NKG2A) is derived from the patent disclosed by Spee et al. (EP2247619A1). After codon optimization, the sequence of the scFv was designed by connecting the variable light (VL) region and the variable heavy (VH) region with a linker sequence. The synthetic gene consisting of the CD8 signal peptide, the scFv for human NKG2A, the CD8 hinge and transmembrane, and the KKMP sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP was replaced with other sequences were also prepared, such as Figure 2 The sequence information for the scFv generated herein is shown in Table 1. Figure 2 The sequence information of the various components shown in is shown in Table 2.

[0106] Anti-CD19-4-1BB-CD3ζCAR

[0107] This CAR was generated as previously described (Imai, C. et al., Leukemia. 2004; 18:676-684; Imai, C. et al., Blood. 2005; 106:376-383).

[0108] Table 1. scFv sequence information

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Table 2. Figure 2 Sequence information of the components shown in

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Gene transduction, cell expansion, flow cytometry analysis, and functional studies

[0121] These were performed as previously described (Kudo, K et al., Cancer Res. 2014; 74(1):93-103).

[0122] result

[0123] Generation of scFv constructs

[0124] Figure 1 A schematic diagram of the technique is outlined in . A schematic diagram of the inhibitory constructs we generated is shown in Figure 2 In. The scFv portion can be derived by cloning cDNA encoding variable light (VL) and variable heavy (VH) immunoglobulin chain regions from a hybridoma cell line producing an antibody or a corresponding public sequence. According to standard techniques, VL and VH are connected to a short peptide sequence ("linker") to prepare a complete scFv. For expression, the scFv is connected to a signal peptide at the N-terminus; as confirmed in preliminary experiments, a signal peptide is required for the expression of scFv. Proteins containing scFv plus a signal peptide are generally released into the environment of the cell. For example, in preliminary experiments (not shown), anti-CD3εscFv plus a signal peptide expressed in Jurkat T cells was detected in the culture supernatant of the cells. By directing the scFv to a specific compartment and preventing its secretion, possible effects on other cells are prevented. To direct it to the endoplasmic reticulum (ER), the KDEL (SEQ ID NO: 4) motif was used (which retains the protein in the ER) (Strebe N. et al., J Immunol Methods. 2009; 341(1-2): 30-40). To promote degradation of the targeted protein, we linked it to the proteasome targeting PEST motif (Joshi, SN et al., MAbs. 2012; 4(6): 686-693). ScFv can also be directed to the cell membrane by linking it to the transmembrane domain and hinge of CD8α or another transmembrane protein.

[0125] Downregulation of T cell receptors in T lymphocytes expressing anti-CD19-BB-ζCAR

[0126] To determine whether the proposed strategy could be applied to generate immune cells expressing CAR and lacking one or more markers, T cell receptor (TCR) expression was downregulated in anti-CD19 CAR T cells.

[0127] In order to be expressed on the cell membrane, the CD3 / TCR complex requires the assembly of all its components (TCRα, TCRβ, CD3δ, CD3ε, CD3γ, CD3ζ). The lack of one component prevents CD3 / TCR expression and, therefore, antigen recognition. In preliminary studies, scFv from an anti-CD3ε hybridoma (purchased from Creative Diagnostics, Shirley, NY) was cloned and constructs containing KDEL (SEQ ID NO: 4), PEST, CD8α transmembrane domain or others were generated, such as Figure 2 shown.

[0128] The constructs disclosed herein were transduced in the CD3 / TCR+ Jurkat cell line using a murine stem cell virus (MSCV) retroviral vector containing green fluorescent protein (GFP). The percentage of GFP+ cells after transduction was >90% in all experiments. Figure 3A Results of staining with anti-CD3ε antibodies in GFP+ cells as measured by flow cytometry are shown. Antibody staining of CD3ε was reduced to varying degrees in cells transduced with the listed constructs. Similar downregulation of CD3ε was obtained with human peripheral blood T lymphocytes ( Figure 3B ). Figure 3C Shown are illustrative flow cytometry dot plots of CD3ε expression in GFP-positive Jurkat cells after transduction with different gene constructs compared to cells transduced with vectors containing GFP alone. Downregulation of CD3 does not affect the growth of Jurkat cells or the expression of all other cell markers tested (including CD2, CD4, CD8, CD45, CD25, CD69). The suppression of CD3 expression lasts for more than 3 months. Further enrichment of CD3 negative cells can be achieved by depletion of CD3+T cells with anti-CD3 magnetic beads (Dynal, Life Technologies, Carlsbad, CA).

[0129] Staining of Jurkat cells or human peripheral blood T lymphocytes with anti-TCRαβ antibody showed that the downregulation of CD3ε expression was associated with the downregulation of TCRαβ expression ( Figure 4 ).

[0130] Next, it was determined whether anti-CD3 scFv-myc KDEL could be expressed simultaneously with anti-CD19-4-1BB-CD3ζCAR. Figure 5 As shown, this resulted in T cells lacking CD3 expression while expressing anti-CD19 CAR. TCR was also absent on these cells (not shown).

[0131] To evaluate whether CAR could signal in Jurkat cells with downregulated CD3 / TCR, expression of activation markers CD69 and CD25 was tested, and exocytosis of lytic granules was measured by CD107a expression in Jurkat cells co-cultured with the CD19+ leukemia cell line OP-1. Figure 6 As shown, downregulation of CD3 / TCR with the anti-CD3 scFv-myc KDEL construct did not reduce the ability of the anti-CD19-4-1BB-CD3ζCAR to activate Jurkat cells. To further explore the effect of CD3 / TCR loss on CAR signaling, it was determined whether CD3-negative T lymphocytes expressing CAR could be stimulated by their ligation. Figure 7 As shown, co-culture of T lymphocytes expressing anti-CD19 CAR with CD19+ leukemia cells resulted in T cell proliferation regardless of whether CD3 was downregulated, indicating that CD3 / TCR downregulation did not reduce CAR proliferation stimulation.

[0132] Accordingly, the anti-CD3 scFv-myc KDEL construct can be used to effectively downregulate CD3 / TCR in CAR-T cells without affecting CAR-driven T cell activation, degranulation, and proliferation.

[0133] Downregulation of CD7

[0134] It was determined whether the strategy for successfully modulating CD3 / TCR expression could be applied to other surface molecules. To this end, CD7 expression was modulated. The scFv sequence was derived from that disclosed by Peipp et al. (Cancer Res 2002(62):2848-2855) and was linked to a CD8 signal peptide and a myc-KDEL sequence, such as Figure 2 Using the MSCV retroviral vector, the anti-CD7-myc KDEL construct was transduced in peripheral blood lymphocytes, which had high expression of CD7 as detected by anti-CD7 antibody conjugated to phycoerythrin (BD Bioscience). Figure 8 As shown, CD7 was virtually abolished in T lymphocytes transduced with the construct.

[0135] Downregulation of HLA class I

[0136] This strategy was then applied to downregulate another surface molecule, HLA class I.

[0137] HLA class I consists of a polymorphic α chain and a non-polymorphic chain called β2-microglobulin. Knockdown of the latter subunit leads to abolition of HLA (MHC in mice) class I expression (Koller, BH et al., Science. 1990; 248(4960): 1227-1230). ScFvs that react with β2-microglobulin are used to inhibit the expression of HLA class I in immune cells.

[0138] The scFv sequence was derived from that disclosed by Grovender et al. (Kidney Int. 2004; 65(1):310-322), which was linked to a CD8 signal peptide and a myc KDEL sequence, such as Figure 2 Using the MSCV retroviral vector, the anti-β2M-myc KDEL construct was transduced in Jurkat cells, which have a high expression of HLA class I, as detected by an anti-HLA-ABC antibody conjugated to phycoerythrin (BD Pharmingen). Fig. 9 As shown, Jurkat cells transduced with the construct had a significant downregulation of HLA-ABC expression. The cells maintained their morphology and growth capacity.

[0139] Downregulation of inhibitory receptors in NK cells

[0140] To determine whether the above strategy is also applicable to surface molecules expressed in other immune cells, functional downregulation of the inhibitory receptors KIR2DL1, KIR2DL2 / DL3, and NKG2A was tested in NK cells.

[0141] To downregulate KIR receptors, scFvs reactive with KIR2DL1 and KIR2DL2 / DL3 were used to inhibit their expression in NK cells. The scFv sequence was derived from that disclosed by Moretta et al. (patent WO2006003179A2), which was linked to a CD8 signal peptide and an ER retention sequence, such as Figure 2 As shown. Using the MSCV retroviral vector, the construct was transduced in NK cells expanded from human peripheral blood and selected for KIR2DL1 expression. These cells had high KIR2DL1 expression as detected by anti-KIR2DL1 antibody conjugated to allophycocyanin (R&D Systems), and these cells also had high KIR2DL2 / DL3 expression as detected by anti-KIR2DL2 / DL3 antibody conjugated to phycoerythrin (BD Bioscience). Fig.10 Shown are the results obtained using scFv-linker (20) AEKEDL and scFv-EEKKMP, with significant downregulation of the targeted KIR.

[0142] To downregulate NKG2A, a scFv reactive with NKG2A was used to inhibit its expression in NK cells. The scFv sequence derived from the published European patent application No. EP2247619 A1 by Scee et al. was linked to a CD8 signal peptide and an ER retention sequence, such as Figure 2 Using the MSCV retroviral vector, the construct was transduced in NK cells expanded from human peripheral blood, which have high NKG2A expression as detected by an anti-NKG2A antibody conjugated to phycoerythrin (Beckman Coulter). Fig.11 Significant downregulation of NKG2A obtained with scFv-EEKKMP is shown.

[0143] The teachings of all patents, published patent applications, and references cited herein are incorporated by reference in their entirety.

[0144] While the invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the following claims.

Claims

1. A nucleic acid comprising a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding an anti-CD7 antibody or an antigen-binding fragment thereof, and a nucleotide sequence encoding a localization domain, wherein the nucleotide sequence encoding the anti-CD7 antibody or an antigen-binding fragment thereof is linked to the nucleotide sequence encoding the localization domain, in, The anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region identical to the heavy chain complementary determining region of SEQ ID NO: 16, and a light chain complementary determining region identical to the light chain complementary determining region of SEQ ID NO: 17, The localization domain comprises an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, or a proteasome localization sequence, wherein the ER retention sequence or the Golgi retention sequence consists of an amino acid sequence of KDEL (SEQ ID NO: 4), KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10), EEKKMP (SEQ ID NO: 64), or YQRL (SEQ ID NO: 11), wherein X is any amino acid, and the proteasome localization sequence consists of a PEST motif; wherein the nucleic acid further comprises a nucleotide sequence encoding a linker between the nucleotide sequence encoding the anti-CD7 antibody or antigen-binding fragment thereof and the nucleotide sequence encoding the localization domain, and wherein the linker comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:50, and SEQ ID NO:

62.

2. The nucleic acid according to claim 1, wherein The nucleotide sequence encoding the anti-CD7 antibody or antigen-binding fragment thereof and the nucleotide sequence encoding the localization domain are codon-optimized.

3. The nucleic acid according to claim 1, wherein The anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) of the amino acid sequence of SEQ ID NO: 16 and a light chain variable region (VL) of the amino acid sequence of SEQ ID NO:

17.

4. The nucleic acid according to claim 1, wherein The ER retention sequence consists of the amino acid sequence KDEL (SEQ ID NO: 4).

5. The nucleic acid of claim 4, wherein the linker comprises SEQ ID NO: 1, SEQ ID NO: 62 or SEQ ID NO:

41.

6. The nucleic acid according to claim 1, wherein The linker comprises SEQ ID NO:1, SEQ ID NO:62 or SEQ ID NO:

41.

7. The nucleic acid according to claim 1, wherein The linker comprises SEQ ID NO:

1.

8. The nucleic acid according to claim 1, wherein The ER retention sequence or Golgi retention sequence consists of the amino acid sequence KKXX (SEQ ID NO: 9) or KXD / E (SEQ ID NO: 10), wherein X is any amino acid.

9. The nucleic acid according to claim 1, wherein The ER retention sequence consists of the amino acid sequence EEKKMP (SEQ ID NO: 64).

10. The nucleic acid according to claim 9, further comprising a nucleotide sequence encoding a CD8α transmembrane domain between the nucleotide sequence encoding an anti-CD7 antibody or an antigen-binding fragment thereof and the nucleotide sequence encoding the amino acid sequence EEKKMP (SEQ ID NO: 64).

11. The nucleic acid of claim 1, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:

50.

12. The nucleic acid of claim 1, wherein the linker comprises a human influenza hemagglutinin (HA) tag.

13. The nucleic acid according to claim 1 or 3, wherein the signal peptide is a CD8 signal peptide. An engineered immune cell comprising the nucleic acid of claim 1 .

15. The engineered immune cell according to claim 14, wherein The engineered immune cells are T cells.

16. The engineered immune cell according to claim 14, wherein The engineered immune cells are NK cells.

17. The engineered immune cell of claim 14, further comprising an immune activating receptor, wherein the immune activating receptor is a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular domain that binds to a specific target and a cytoplasmic domain that contains a CD3ζ signaling domain, wherein the specific target is CD19.

18. An in vitro method for preparing engineered immune cells, the method comprising: Introducing a nucleic acid into an immune cell, the nucleic acid comprising a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a localization domain, and a nucleotide sequence encoding an anti-CD7 antibody or an antigen-binding fragment thereof linked to the localization domain, wherein the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region identical to the heavy chain complementary determining region of SEQ ID NO: 16, and a light chain complementary determining region identical to the light chain complementary determining region of SEQ ID NO: 17, The localization domain comprises an endoplasmic reticulum (ER) retention sequence, a Golgi retention sequence, or a proteasome localization sequence, wherein the ER retention sequence or the Golgi retention sequence consists of an amino acid sequence of KDEL (SEQ ID NO: 4), KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10), EEKKMP (SEQ ID NO: 64), or YQRL (SEQ ID NO: 11), wherein X is any amino acid, and the proteasome localization sequence consists of a PEST motif; wherein the nucleic acid further comprises a nucleotide sequence encoding a linker between the nucleotide sequence encoding the anti-CD7 antibody or antigen-binding fragment thereof and the nucleotide sequence encoding the localization domain, and wherein the linker comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:50, and SEQ ID NO:

62.

19. The ex vivo method of claim 18, wherein the anti-CD7 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) of the amino acid sequence of SEQ ID NO: 16 and a light chain variable region (VL) of the amino acid sequence of SEQ ID NO:

17.

20. The ex vivo method according to claim 18 or 19, wherein the signal peptide is a CD8 signal peptide.

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