Methods for enhancing efficacy of therapeutic immune cells

Abstract

To provide methods for enhancing efficacy of therapeutic immune cells.SOLUTION: The present invention relates to a method of using a receptor (e.g., chimeric antigen receptor, CAR) that activates an immune response upon binding a cancer cell ligand in conjunction with a target-binding molecule that targets a protein or molecule for removal or neutralization to generate enhanced anti-cancer immune cells. The present invention also relates to engineered immune cells having enhanced therapeutic efficacy and uses thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [Background technology] Immune cells can be potent and specific "living drugs" that have the potential to target tumor cells while sparing normal tissue, and several clinical observations indicate that they may have major anti-cancer activity. Thus, in patients undergoing 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) and donor natural killer (NK) cell alloreactivity (Ruggeri L, et al. 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) are inversely associated with leukemia recurrence. In addition to the context of HSCT, 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 cross-link them to tumor cells (Topp MS, et al. J. Clin. Oncol. 2011;29(18):2493-2498) has produced major clinical responses in patients with either solid tumors or leukemia. Finally, infusion of genetically modified autologous T lymphocytes induced complete and durable remissions in patients with treatment-resistant leukemia and lymphoma (Maude SL, et al. N Engl J Med. 2014;371(16):1507-1517).

[0002] Nevertheless, there is a significant need to improve immune cell therapy by broadening its applicability and improving its efficacy. Summary of the Invention

[0003] The present invention relates to engineered immune cells with improved therapeutic efficacy, for example, for cancer treatment. In certain embodiments, the present invention provides engineered immune cells 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.

[0004] In other embodiments, the present invention provides a use of engineered immune cells comprising a gene encoding an immune activating receptor and a gene encoding a target binding molecule linked to a localization domain to treat cancer, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0005] In various embodiments, the present invention also provides methods for generating an engineered immune cell, comprising introducing into an immune cell 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, thereby generating the engineered immune cell.

[0006] In some embodiments, the engineered immune cells have improved therapeutic efficacy as a result of 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 tumor suppression in the host, reduced apoptosis in the host, reduced inflammatory cascades in the host, or sustained natural / artificial receptor-mediated (e.g., CAR-mediated) signal transduction in the host.

[0007] The foregoing will be apparent from the following more particular description of exemplary embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention. [Brief explanation of the drawings]

[0008] [Figure 1] Schematic diagram of the strategy used in the present invention. Figure 1A shows the overall mechanism of CAR-mediated killing of cancer cells. Figure 1B shows the combinatorial expression of CAR with different formats of compartment-oriented scFv (an example of a target-binding molecule linked to a localization domain) and examples of potential targets. CAR may be replaced by other receptors that can improve immune cell capacity. [Figure 2]Figure 1 shows a schematic diagram of constructs containing scFvs with domains that localize the scFv to specific cellular compartments. Abbreviations: β2M, β-2 microglobulin; SP, signal peptide; VL, variable light chain; VH, variable heavy chain; TM, transmembrane domain; HA, human influenza hemagglutinin. Additional constructs not listed in the figure include membrane-bound (mb) myc EEKKMP, mb myc KKTN, mb myc YQRL, mb TGN38 cytoplasmic domain, mb myc RNIKCD, linker (20-amino acid) mb EEKKMP, and variants of the construct without the signaling peptide and with various numbers of amino acids in the CD8 transmembrane domain. The nucleotide sequence of the 10-amino acid linker is GGTGGTGGCGGCAGTGGTGGCGGTGGCTCA (SEQ ID NO: 61), and 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), and the amino acid sequence is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41). Various localization domains are shown under the heading "Localization Domain," depicting the linker in some examples as shown. The constructs "myc KDEL" and "PEST KDEL" demonstrate the use of two or more localization domains in a single construct. [Figure 3A-3B]Figures 3A-3C show downregulation of CD3 / TCR in T cells by scFv targeting of CD3ε. Figure 3A shows surface CD3ε expression in Jurkat cells transduced with either a retroviral vector containing green fluorescent protein (GFP) alone ("mock") or a vector containing GFP plus the different constructs indicated. One week after transduction, CD3ε expression on the cell membrane was compared to that of mock-transduced cells using an anti-CD3 antibody conjugated to allophycocyanin (BD Biosciences). All comparisons were performed after gating on GFP-positive cells. Figure 3B depicts a similar experiment performed on peripheral blood T lymphocytes expanded with anti-CD3 / CD28 beads (Lifesciences). Staining was performed one week after transduction. [Figure 3C] Figure 3C shows flow cytometry plots illustrating downregulation of membrane CD3ε in Jurkat cells after transduction with the indicated constructs. The dashed box in the upper right quadrant of each plot encloses GFP+CD3+ cells. [Figure 4] Figure 1 shows downregulation of CD3ε and TCRαβ on the plasma membrane in Jurkat T cells upon transduction with anti-CD3ε scFv-KDEL or -PEST, or -mb EEKKMP. Membrane marker expression was measured 1 week after transduction using anti-CD3 antibodies conjugated to allophycocyanin (BD Biosciences) or anti-TCRαβ conjugated to phycoerythrin (Biolegend). The line labeled "Control" represents labeling of mock-transduced cells. The vertical dashed line represents the upper limit of staining obtained with an isotype-matched nonreactive antibody. [Figure 5]Figure 1 shows 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 the anti-CD3scFv-myc KDEL construct, the anti-CD19-4-1BB-CD3ζ construct, or both. After gating on GFP-positive cells, those transduced with the anti-CD3scFv-myc KDEL construct downregulated CD3 (left row, lower left quadrant), while those transduced with the anti-CD19-4-1BB-CD3ζ construct expressed CAR (middle row, upper right quadrant). A significant proportion of cells transduced with both constructs were CD3 negative and CAR positive (right row, upper left quadrant). [Figure 6] We demonstrate that anti-CD19 CAR induces T cell activation and degranulation regardless of CD3 / TCR downregulation. Jurkat cells were transduced with the anti-CD3scFv-myc KDEL construct, the anti-CD19-4-1BB-CD3ζ construct, or both. T cell activation and degranulation were compared to those of mock-transduced cells. Cells were cultured alone or cocultured with the CD19+ leukemia cell line OP-1 at a 1:1 ratio. After 18 hours, CD69 and CD25 expression was examined by flow cytometry using specific antibodies (from BD Biosciences), and after 6 hours, CD107a expression was examined (antibody from BD Biosciences). In the presence of OP-1 cells, CD69 and CD25 expression in CAR-expressing cells occurred regardless of whether the cells were also transduced with anti-CD3scFv-KDEL, and activation did not occur in mock- or anti-CD3scFv-mycKDEL-transduced cells or in the absence of OP-1 cells. CAR stimulation improved CD107 expression, which was not affected by CD3 / TCR downregulation. [Figure 7]This figure shows that anti-CD19 CAR expressed in T cells induces T cell proliferation regardless of CD3 / TCR downregulation. Peripheral blood T lymphocytes were transduced with both the anti-CD3scFv-myc KDEL construct and the anti-CD19-4-1BB-CD3ζ construct. The transduced T lymphocytes were co-cultured with Streck (Omaha, NE)-treated OP-1 cells to suppress their proliferation for the indicated times. The proliferation of CD3-positive and CD3-negative T lymphocytes expressing the anti-CD19 CAR was compared to that of mock-transduced T cells. Each symbol represents the average cell number of two parallel cultures. CAR T cells proliferated equally well regardless of CD3 / TCR expression. [Figure 8] Figure 1 shows CD7 expression on the membrane of peripheral blood T lymphocytes transduced with either a retroviral vector containing GFP alone ("mock") or a vector containing GFP plus the anti-CD7scFv-myc KDEL construct. One week after transduction, CD7 expression on the cell membrane was compared to that of mock-transduced cells using an anti-CD7 antibody conjugated to phycoerythrin (BD Biosciences). The dashed rectangle in the upper right quadrant of each plot encloses GFP+CD7+ cells. [Figure 9] This figure depicts downregulation of HLA class I in T cells by scFv targeting of β2-microglobulin. Jurkat T cells were transduced with anti-β2M scFv-myc KDEL. One week after transduction, HLA-ABC expression on the cell membrane was compared to that of mock-transduced cells using an 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. [Figure 10]This figure depicts downregulation of killer immunoglobulin-like receptor (KIR) 2DL1 and KIR2DL2 / DL3 in human NK cells by scFv targeting of KIR2DL1 and KIR2DL2 / DL3. NK cells expanded in vitro and selected for KIR2DL1 expression were transduced with anti-KIR2DL1-KIR2DL2 / DL3 scFv-linker (20)AEKDEL or -EEKKMP. Eight days after transduction, expression of the corresponding KIR on the cell membrane was compared with that of mock-transduced cells using anti-KIR2DL1 antibody conjugated to allophycocyanin (R&D Systems) or anti-KIR2DL2 / DL3 antibody conjugated to phycoerythrin (BD Biosciences). Staining with isotype-matched control antibodies is also shown. Analysis was performed after gating on GFP-positive cells. [Figure 11] Figure 1 depicts downregulation of NKG2A in human NK cells by scFv targeting. Ex vivo expanded NK cells were transduced with anti-NKG2A scFv-EEKKMP. Eight days after transduction, NKG2A expression on the cell membrane was compared to that of mock-transduced cells using an NKG2A antibody conjugated to phycoerythrin (Beckman Coulter). Staining with an isotype-matched control antibody is also shown. Analysis was performed after gating on GFP-positive cells. DETAILED DESCRIPTION OF THE INVENTION

[0009] A description of exemplary embodiments of the present invention follows.

[0010] In recent years, the gain in knowledge about the molecular pathways that control immune cells has been matched by significant advances in our ability to manipulate them in vitro, including their proliferation and genetic manipulation. It is now possible to reliably prepare highly complex clinical-grade immune cell products in a timely manner. A prime example of how the anti-cancer activity of immune cells can be directed and expanded by in vitro cell manipulation is the development of chimeric antigen receptor (CAR) T cells (Eshhar, Z. et al., PNAS. 1993; 90(2): 720-724).

[0011] CARs are artificial multimolecular proteins that have been previously described (Geiger TL, et al., J Immunol. 1999; 162(10):5931-5939; Brentjens RJ, et al., NatMed. 2003; 9(3):279-286; Cooper LJ, et al., Blood. 2003; 101(4):1637-1644). CARs contain an extracellular domain that binds to a specific target, a transmembrane domain, and a cytoplasmic domain. For example, as described in U.S. Pat. No. 8,399,645 (incorporated herein by reference in its entirety), the extracellular domain and transmembrane domain can be derived from any desired source of such domains. Briefly, CARs can be designed to contain the single-chain variable region (scFv) of an antibody that specifically binds to a target. The scFv can be linked via the transmembrane and hinge domains to T cell receptor (TCR)-associated signaling molecules such as CD3ζ. Ligation of the scFv to its cognate antigen triggers signal transduction. Thus, CARs can instantly redirect cytotoxic T lymphocytes toward cancer cells and induce 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., NatMed. 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 sufficient to sustainably activate T cells (Schwartz RH. Annu Rev Immunol. 2003;21:305-334; Zang X and Allison JP. Clin Cancer Res. 2007;13(18Pt1):5271-5279), costimulatory molecules such as CD28 and 4-1BB (or CD137) have been incorporated into CAR constructs to boost signal transduction.This dual signaling design ("second-generation CAR") is useful for eliciting effective antitumor activity from T cells. (Imai C, et al., Leukemia. 2004; 18:676-684; Campana D, et al., Cancer J. 2014; 20(2):134-140).

[0012] A particular CAR containing both 4-1BB and CD3ζ, an anti-CD19 CAR, is described in US Pat. No. 8,399,645. Infusion of autologous T cells expressing anti-CD19-4-1BB-CD3ζ CAR resulted in dramatic clinical responses in patients with chronic lymphocytic leukemia (CLL) (Porter DL, et al., Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia;2011:N Engl J Med.2011;365(8):725-733; Kalos M, et al., SciTranslMed.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 those with CARs carrying 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 compelling evidence for the clinical potential of this technology, and for immunotherapy in general.

[0013] The methods described herein enable the rapid removal or deactivation of specific proteins in immune cells that are redirected by natural or artificial receptors (e.g., CARs), thus broadening the potential applications of engineered cells and significantly improving their function. The methods rely in part on a single construct or multiple constructs containing an immune activation receptor, e.g., a CAR (comprising an extracellular domain (e.g., an scFv) that binds to a specific target, a transmembrane domain, and a cytoplasmic domain) along with a target-binding molecule that binds to the target (e.g., protein) to be removed or neutralized, where the target-binding molecule is linked to a domain (i.e., a localization domain) that directs it to a specific cellular compartment (such as the Golgi or endoplasmic reticulum), the proteasome, or the plasma membrane, depending on the application. For simplicity, a target-binding molecule linked to a localization domain (LD) is sometimes referred to herein as an "LD-linked target-binding molecule."

[0014] As will be clear from the teachings of this specification, various immune activating receptors can be suitable for the method of the present invention. That is, any receptor, including a molecule that can activate immune response when binding (ligating) to a ligand (e.g., peptide or antigen) expressed on cancer cells, can be used according to this method. For example, as mentioned above, immune activating receptors can be chimeric antigen receptors (CARs), and methods for designing and engineering CARs are known in the art (see Geiger TL, et al., J Immunol. 1999;162(10):5931-5939; Brentjens RJ, et al., NatMed. 2003;9(3):279-286; Cooper LJ, et al., Blood. 2003;101(4):1637-1644). Furthermore, receptors with antibody binding capabilities may be used (e.g., CD16-4-1BB-CD3 zeta receptor—Kudo K, et al. Cancer Res. 2014; 74(1):93-103), which are similar to CARs, but in which the scFv is replaced with an antibody-binding molecule (e.g., CD16, CD64, CD32). Furthermore, T cell receptors comprising T cell receptor alpha and beta chains that bind to peptides expressed on tumor cells in the context of tumor cell HLA can also be used according to the present method. Furthermore, other receptors carrying molecules that activate immune responses by binding to ligands expressed on cancer cells (e.g., NKG2D-DAP10-CD3 zeta receptor that binds to NKG2D ligands expressed on tumor cells) can also be used (see, e.g., Chang YH, et al., Cancer Res. 2013; 73(6):1777-1786). As used herein, all such suitable receptors are collectively referred to as "immune activating receptors" or "receptors that activate an immune response upon binding to a cancer cell ligand." Thus, an immune activating receptor having a molecule activated by a cancer cell ligand can be expressed with an LD-linked target binding molecule according to the present method.

[0015] This method significantly expands the potential applications of immunotherapy based on the injection of immune cells redirected by artificial receptors. The described method is practical and can be easily incorporated into clinical-grade cell processing. For example, a single bicistronic construct containing a CAR and an LD-linked target-binding molecule (e.g., scFv-myc KDEL (or PEST or transmembrane)) can be prepared by inserting an internal ribosome entry site (IRES) or a 2A peptide-coding region site between the two cDNAs encoding the CAR and the LD-linked target-binding molecule. Designing a tricistronic delivery system to delete two or more targets would also be feasible. Alternatively, separate transduction of two genes (simultaneous or sequential) may be performed. In the context of cancer cell therapy, a CAR can be used to target an antibody-binding signaling receptor (Kudo K, et al., Cancer Res. 2014;74(1):93-103), a T cell receptor directed against a specific HLA-peptide combination, or any receptor activated by contact with cancer cells (Chang YH, et al., Cancer Res. 2013;73(6):1777-1786). The results of the studies described herein with simultaneous anti-CD19-4-1BB-CD3ζ CAR and anti-CD3ε scFv-KDEL demonstrate that the signaling capacity of the CAR was not impaired.

[0016] Both anti-CD3ε scFvs tested herein, KDEL (and PEST), stably down-regulate CD3 and TCR expression. Residual CD3+ T cells can be removed using CD3 beads, an approach that is also available in clinical-grade formats. The ability to generate CD3 / TCR-negative cells that respond to CAR signaling represents an important advance. Clinical studies with CAR T cells have generally been performed using autologous T cells. Therefore, the quality of the cell product varies between patients, and responses are heterogeneous. Infusion of allogeneic T cells is currently impossible due to the unacceptably high and potentially fatal risk of GvHD caused by stimulation of endogenous TCRs by recipient tissue antigens. Down-regulation of CD3 / TCR opens the possibility of infusing allogeneic T cells, as the lack of endogenous TCRs eliminates GvHD potential. Allogeneic products can be prepared with optimal cell composition (e.g., highly enriched in cytotoxic T cells, depleted in regulatory T cells, etc.), and infused cells can be selected to have high CAR expression and functional potency. Furthermore, fully standardized products can be cryopreserved and available for use regardless of the patient's immune cell status and their suitability for apheresis or extensive blood sampling. Removal of TCR expression has been addressed using gene editing tools such as nucleases (Torikai H, et al. Blood, 2012;119(24):5697-5705). Although this is an effective approach, it requires several rounds of cell selection and expansion through extended culture, making it difficult to implement in a clinical setting. The method described herein has many practical advantages.

[0017] Furthermore, LD-linked target binding molecules (e.g., scFv-myc KDEL, scFv-EEKKMP, or scFv-PEST, where the scFv targets a specific protein / molecule) can be used in accordance with the present invention to delete HLA class I molecules and reduce the likelihood of allogeneic cell rejection. While the infusion of allogeneic T cells is a future goal of CAR T cell therapy, the infusion of allogeneic natural killer (NK) cells has already been used to treat patients with cancer. A key factor determining the success of NK cell-based therapy is that NK cells must persist in sufficient numbers to achieve an effector:target ratio that is likely to produce tumor cell ablation (Miller JS. Hematology Am Soc Hematol Educ Program. 2013; 2013: 247-253). However, when allogeneic cells are infused, their persistence is limited. Although immunosuppressive chemotherapy administered to patients allows transient engraftment of infused NK cells, 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). Contrary to organ transplantation, continuous immunosuppression is not an option because immunosuppressive drugs also suppress NK cell function. Because rejection is primarily mediated by recognition of HLA class I molecules by recipient CD8+ T lymphocytes, removing HLA class I molecules from infused NK cells (or T cells) would reduce or abrogate the rejection rate and prolong the survival, and therefore antitumor, of allogeneic cells.

[0018] Furthermore, LD-linked target binding molecules can be used in accordance with the present invention to target inhibitory receptors. Specifically, administration of antibodies that release T cells from inhibitory signals, such as anti-PD1 or anti-CTLA-4, has produced dramatic 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, particularly those directed against solid tumors, can be inhibited by a similar mechanism. Thus, expression of target-binding molecules (e.g., scFvs or ligands) against PD1, CTLA-4, Tim3, or other inhibitory receptors will prevent expression of these molecules (e.g., when linked to KDEL (SEQ ID NO: 4), EEKKMP (SEQ ID NO: 64), or the PEST motif SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7)), or will prevent receptor binding to their ligands (when linked to transmembrane domains), sustaining CAR-mediated signal transduction. In NK cells, examples of inhibitory receptors include killer immunoglobulin-like receptors (KIR) and NKG2A (Vivier E, et al., Science, 2011;331(6013):44-49).

[0019] The method of the present invention also enables targeting of a larger number of targets amenable to CAR-directed T cell therapy. One of the major limitations of CAR-directed therapy is the lack of specific antigens expressed by tumor cells. In the case of hematological malignancies such as leukemia and lymphoma, molecules not expressed in non-hematopoietic cells may be potential targets, but they cannot be used as CAR targets because they are also expressed on T cells and / or NK cells. Expression of such CARs on immune cells would potentially result in the elimination of the immune cells themselves through a "brother-killing" mechanism, potentially nullifying their anti-cancer capabilities. If the target molecule can be removed from immune cells without functional adverse effects, a CAR with the corresponding specificity can be expressed. This opens up many new opportunities for targeting hematological malignancies. Examples of potential 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, and CD45 and CD52 expressed in all hematological malignancies. These molecules are also expressed in a significant proportion of T cells and NK cells.

[0020] Furthermore, secretion of cytokines by activated immune cells has been shown to cause cytokine release syndrome and macrophage activation syndrome, which are serious adverse effects of immune cell therapy (Lee DW, et al., Blood. 2014;124(2):188-195). Therefore, LD-linked target binding molecules can be used in accordance with the present invention to block cytokines that may contribute to such inflammatory cascades, 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.

[0021] Thus, 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.

[0022] As used herein, "engineered" immune cells include immune cells that are genetically modified compared to naturally occurring immune cells. For example, engineered T cells generated according to the present methods harbor a nucleic acid comprising a nucleotide sequence that does not naturally occur in the T cell from which it was derived. In some embodiments, engineered immune cells of the present invention comprise a chimeric antigen receptor (CAR) and a target binding molecule linked to a localization domain (LD-linked target binding molecule). In one specific embodiment, engineered immune cells of the present invention comprise an anti-CD19-4-1BB-CD3ζ CAR and an anti-CD3 scFv linked to a localization domain.

[0023] In certain embodiments, the engineered immune cells are engineered T cells, engineered natural killer (NK) cells, engineered NK / T cells, engineered monocytes, engineered macrophages, or engineered dendritic cells.

[0024] In certain embodiments, as used herein, "immune activating receptor" refers to a receptor that activates an immune response upon binding to a cancer cell ligand. In some embodiments, the immune activating receptor comprises a molecule that can activate an immune response upon binding (ligation) to a ligand (e.g., a peptide or antigen) expressed on a cancer cell. In one embodiment, the immune activating receptor is a chimeric antigen receptor (CAR), and methods for designing and engineering CARs are known in the art. In other embodiments, the immune activating receptor is an antibody-binding receptor that is similar to a CAR, but in which the scFv is replaced with an antibody-binding molecule (e.g., CD16, CD64, CD32) (e.g., CD16-4-1BB-CD3 zeta receptor—Kudo K, et al. Cancer Res. 2014;74(1):93-103). In various embodiments, T cell receptors comprising T cell receptor alpha and beta chains that bind to peptides expressed on tumor cells in the context of tumor cell HLA can also be used according to the present methods. In certain embodiments, other receptors (e.g., NKG2D-DAP10-CD3 zeta receptors that bind to NKG2D ligands expressed on tumor cells) carrying molecules that activate immune responses by binding to ligands expressed on cancer cells can also be used (see, for example, Chang YH, et al., Cancer Res. 2013; 73(6):1777-1786). All such suitable receptors that can activate immune responses upon binding (ligation) to a ligand (e.g., a peptide or antigen) expressed on cancer cells are collectively referred to as "immune activating receptors." As will be recognized by those skilled in the art, immune activating receptors do not necessarily contain antibodies or antigen-binding fragments (e.g., scFvs); rather, the portion of the immune activating receptor that binds to the target molecule can be derived, for example, from the receptor in a receptor-ligand pair or from the ligand in a receptor-ligand pair.

[0025] In certain aspects, the immune activating receptor binds to a molecule expressed on the surface of a tumor cell, 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, the immune activating receptor is a CAR (e.g., anti-CD19-4-1BB-CD3ζ CAR). In certain embodiments, the immune activating receptor comprises an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to a molecule 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 against such molecules expressed on the surface of tumor cells are known and available in the art. As an example, antibodies against CD3 and CD7 are commercially available and known in the art. As exemplified herein, such antibodies and antibody fragments (e.g., scFv) derived therefrom can be used in the present invention. Furthermore, methods for generating antibodies and antibody fragments against target proteins are well known and routine in the art.

[0026] The transmembrane domain of an immune activating receptor (e.g., CAR) according to the present invention may be derived from a single-pass membrane protein, including, but not limited to, 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, and FGFR2B. In some examples, the membrane protein is not CD8α. The transmembrane domain may also be a non-naturally occurring hydrophobic protein segment.

[0027] The hinge domain of an immune activating receptor (e.g., CAR) can be derived from a protein such as CD8α or IgG. The hinge domain can be a non-naturally occurring peptide, such as a fragment of the transmembrane or hinge domain of CD8α, or a polypeptide consisting of hydrophilic residues of various lengths, or (GGGGS). n (SEQ ID NO: 8) polypeptide (where n is an integer of, for example, 3 to 12, inclusive).

[0028] The signaling domain of an immune activating receptor (e.g., CAR) can be derived from CD3ζ, FcεRIγ, DAP10, DAP12, or other molecules known to deliver activation signals in immune cells. At least one costimulatory signaling domain of the receptor may be derived from 4-1BB (also known as CD137), CD28, CD28 JPEG2025169264000002.jpg13

[0029] The variant may be a costimulatory molecule such as OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. Such molecules are readily available and known in the art.

[0030] As will be appreciated by those skilled in the art, components of an immune activating receptor can be engineered to include several functional combinations as described herein to produce a desired result. Using the specific CAR anti-CD19-4-1BB-CD3ζ as an example, as described herein, the antibody (e.g., or antigen-binding fragment thereof (such as an scFv)) that binds to a molecule can be substituted with an antibody that binds to a different molecule (e.g., anti-CD20, anti-CD33, anti-CD123, etc., instead of anti-CD19). In other embodiments, the costimulatory molecule (in this specific example, 4-1BB) can also be replaced with a different costimulatory molecule (e.g., CD28). In some embodiments, the stimulatory molecule (in this specific example, CD3ζ) can be replaced with another known stimulatory molecule. In various embodiments, the transmembrane domains of the receptor can also be different, if desired. The design, production, and functionality testing of such immune activating receptors can be readily determined by those skilled in the art. Similarly, the design, delivery into cells, and expression of nucleic acids encoding such immune activating receptors are readily known and available in the art.

[0031] As used herein, the term "nucleic acid" refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide or deoxyribonucleotide monomers). "Nucleic acid" includes, for example, genomic DNA, cDNA, RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. Furthermore, nucleic acid molecules can be single-stranded, double-stranded, or triple-stranded. In some embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, "nucleic acid" can refer to either or both strands of the molecule.

[0032] In the context of nucleic acids, the term "nucleotide sequence" refers to a series of contiguous nucleotides joined by covalent bonds, such as phosphorus bonds (e.g., phosphodiester, alkyl and aryl-phosphonate, phosphorothioate, phosphotriester) and / or non-phosphorus bonds (e.g., peptide and / or sulfamate). In certain embodiments, the nucleotide sequence encoding, for example, a target binding molecule linked to a localization domain is a heterologous sequence (e.g., a gene originating from a different species or cell type).

[0033] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Thus, nucleotides can include, for example, nucleotides containing naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine), as well as nucleotides containing modified bases known in the art.

[0034] As will be appreciated by those skilled in the art, in some embodiments, the nucleic acid further comprises a plasmid sequence, which may include, for example, one or more sequences selected from the group consisting of a promoter sequence, a selectable marker sequence, and a gene targeting sequence.

[0035] As used herein, a gene encoding a target binding molecule linked to a localization domain may also be referred to as an "LD-linked target binding molecule."

[0036] 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 thereof, including an intact antibody or antigen-binding fragment that has been modified or engineered, or is a human antibody. Examples of modified or engineered antibodies are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies.

[0037] A "Fab fragment" is a fragment that contains one light chain and one C H 1 and one heavy chain variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0038] The "Fc" region contains two heavy chain fragments containing 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.

[0039] A "Fab' fragment" contains one light chain and a portion of one heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0040] A "F(ab')2 fragment" is a fragment of two light chains and a C fragment of two heavy chains separated by an interchain disulfide bond. H 1 Domain and C H 2 The F(ab')2 fragment contains two heavy chains containing a portion of the constant region between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0041] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0042] 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 further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also PCT Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. As an example, a linker between the VH and VL domains of an scFv disclosed herein comprises, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGSGGGGGS (SEQ ID NO: 43). As will be recognized by those skilled in the art, a variety of suitable linkers can be designed and tested for optimal function as provided in the art and as disclosed herein.

[0043] The scFv that is part of an LD-linked target binding molecule is not necessarily identical to the scFv that occurs 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 an LD-linked target binding molecule is identical to the scFv that occurs in the context of, for example, a chimeric antigen receptor (CAR) or similar antibody-binding signaling receptor.

[0044] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., an 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 NOs: 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, or 39.

[0045] The term "sequence identity" means that two nucleotide or amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least, for example, 70% sequence identity, or at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% or more sequence identity. For sequence comparison, typically, one sequence serves as a reference sequence (e.g., parent sequence) to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0046] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. 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). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (publicly accessible through the NCBI internet server of the National Institutes of Health). Typically, default program parameters can be used to perform the sequence comparison, although customized parameters may also be used. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0047] In certain embodiments, the antibody (e.g., scFv) comprises a VH and a VL having the amino acid sequences set forth in SEQ ID NOs: 12 and 13, respectively, SEQ ID NOs: 16 and 17, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 24 and 25, respectively, SEQ ID NOs: 28 and 29, respectively, SEQ ID NOs: 32 and 33, respectively, 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 have at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the VH and VL sequences set forth in SEQ ID NOs: 12 and 13, respectively, SEQ ID NOs: 16 and 17, respectively, SEQ ID NOs: 20 and 21, respectively, SEQ ID NOs: 24 and 25, respectively, SEQ ID NOs: 28 and 29, respectively, SEQ ID NOs: 32 and 33, respectively, or SEQ ID NOs: 36 and 37, respectively.

[0048] A "diabody" is a small antibody fragment having two antigen-binding sites. The fragment comprises 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 the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described, for example, in patent documents EP 404,097, WO 93 / 11161, and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0049] In certain embodiments, the antibody is a triabody or tetrabody. Methods for designing and generating triabodies and tetrabodies are known in the art. See, e.g., Todorovska et al., J. Immunol. Methods 248(1-2):47-66, 2001.

[0050] A "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more VH regions are covalently linked with a peptide linker to create a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment may target the same antigen or different antigens.

[0051] In some embodiments, the antibody is modified or engineered. Examples of modified or engineered antibodies include chimeric antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies).

[0052] As used herein, a "multi-paratopic antibody" refers to an antibody comprising at least two single domain antibodies, of which 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 "double paratopic" 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.

[0053] As used herein, a "multispecific antibody" means an antibody comprising at least two single domain antibodies, of which 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 an antibody comprising at least one single domain antibody directed against a first antigen and at least one further single domain antibody directed against, for example, a second antigen different from the first antigen.

[0054] In some embodiments, the antibodies disclosed herein are monoclonal antibodies, such as mouse monoclonal antibodies. Methods for generating monoclonal antibodies are known in the art. See, for example, Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

[0055] 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). Thus, as will be appreciated by those skilled in the art, the target-binding molecule may be an antibody or a ligand / receptor that binds to the target molecule.

[0056] As used herein, "linked" in the context of LD-linked target binding molecules refers to a gene encoding a target binding molecule that is directly adjacent to (e.g., without a linker) one or more genes encoding one or more localization domains. Alternatively, the gene encoding the target binding molecule may be connected to one or more genes encoding one or more localization domains through a linker sequence, as described herein. A variety of suitable linkers known in the art can be used to tether the target binding molecule to the localization domain. For example, a non-naturally occurring peptide, such as a polypeptide consisting of hydrophilic residues of various lengths, or (GGGGS) n (SEQ ID NO: 8) polypeptides (n is an integer, e.g., 3 to 12, inclusive) may be used in accordance with the present invention. In certain embodiments, the linker comprises, for example, GGGGSGGGGS (SEQ ID NO: 62). In some embodiments, the linker comprises, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41). In various embodiments, peptide linkers having a length of about 5 to about 100, inclusive, amino acids can be used in the present invention. In certain embodiments, peptide linkers having a length of about 20 to about 40, inclusive, amino acids can be used in the present invention. In some embodiments, peptide linkers having 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 recognized by those of skill in the art, such linker sequences and variants of such linker sequences are known in the art. Methods for designing constructs incorporating linker sequences and for assessing functionality are readily available to those of skill in the art.

[0057] In certain embodiments, the LD-linked target binding molecule binds to a target expressed on the surface of an immune cell. In some embodiments, the LD-linked target binding molecule inhibits the activity or function of the target molecule. By way of example, as disclosed herein, the LD-linked target binding molecule may be designed to bind, for example, CD3, CD7, CD45, hB2MG, KIR2DL1, KIR2DL2 / DL3, or NKG2A, thereby downregulating cell surface expression of such molecules. Downregulation of such molecules can be achieved, for example, through localization / targeting of the molecule for degradation and / or internalization. In other embodiments, the LD-linked target binding molecule inactivates the target (e.g., the target can no longer interact and / or bind to its cognate ligand or receptor).

[0058] In some embodiments, the engineered immune cells of the present invention have improved therapeutic efficacy. As used herein, "improved therapeutic efficacy" refers to one or more of the following: reduced graft-versus-host disease (GvHD) in the host, reduced or eliminated rejection by the host, prolonged survival in the host, reduced tumor suppression in the host, reduced self-killing in the host, reduced inflammatory cascade in the host, or sustained CAR-mediated signal transduction in the host.

[0059] In certain embodiments of the invention, the target binding molecule in the context of the 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 the immune response.

[0060] In certain embodiments, the molecule in the CD3 / TCR complex can be CD3ε, TCRα, TCRβ, TCRγ, TCRδ, CD3δ, CD3γ, or CD3ζ. In one particular embodiment, the molecule is CD3ε.

[0061] In another embodiment, the HLA class I molecule is beta-2 microglobulin, alpha 1-microglobulin, alpha 2-microglobulin, or alpha 3-microglobulin.

[0062] In other embodiments, the receptor that downregulates the immune response is selected from, for example, PD-1, CTLA-4, Tim3, killer 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). Accordingly, such receptors can be targeted by partial LD-linked target binding molecules as described herein.

[0063] In various embodiments, examples of cytokines that can be targeted by a moiety LD-linked target binding molecule 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 transforming growth factor (TGF)-β.

[0064] In a further embodiment, the LD-linked target binding molecule binds to a molecule selected from, e.g., CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127.

[0065] Methods for generating antibodies and antibody fragments thereof against any target protein are well known and routine in the art. Furthermore, as exemplified herein, commercially available antibodies against various targets (e.g., CD3 and CD7) can be used to generate LD-linked target binding molecules, as exemplified herein. As exemplified herein, antibodies known in the art and antibody fragments derived therefrom (e.g., scFv) can be used in the present invention.

[0066] In other embodiments, the localization domain of the LD-linked target binding molecule is the endoplasmic reticulum (ER) retention sequence KDEL (SEQ ID NO: 4), or KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10) (where 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) (Zhan J, et al., Cancer Immunol Immunother 46:55-60, 1998); a proteosome targeting sequence comprising, for example, a "PEST" motif -SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7); and / or a sequence that targets a target binding molecule to the cell membrane, such as the CD8α transmembrane domain, or the transmembrane domain of another single-pass membrane protein described herein (e.g., CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16 (such as CD16A or CD16B), OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32 (such as CD32A or CD32B), CD64 (such as CD64A, CD64B, or CD64C), VEGFR2, FAS, or FGFR2B). Examples of specific localization domains (sequences) exemplified herein are shown in Figure 2. A variety of other localization sequences are known and available in the art.

[0067] As shown in Figure 2, LD-linked target binding molecules of the invention may contain one or more localization domains. For example, 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 linker. As an example, as shown in Figure 2, the localization domains CD8™, PEST motif, and EEKKMP can be used in a single LD-linked target binding molecule. While this particular construct shows localization domains without any intervening linkers, various intervening linkers may be incorporated between some or all of the localization domains. Other examples are shown in Figure 2.

[0068] As will be appreciated by those skilled in the art, immune activating receptors and / or LD-linked target binding molecules may be designed to bind to the targets disclosed herein, as well as variants of the targets disclosed herein. As an example, immune activating receptors and / or LD-linked target binding molecules may be designed to bind to a molecule in the CD3 / TCR complex, or a naturally occurring variant molecule thereof. Such naturally occurring variants may have the same function as the wild-type form of the molecule. In other embodiments, variants may have an altered function (e.g., confer a disease state) compared to the wild-type form of the molecule.

[0069] As will be appreciated by those skilled in the art, the various components of the LD-linked target binding molecule constructs shown in Figure 2 may be substituted in different combinations (e.g., to contain different linkers, different localization sequences, different scFvs, etc.) so long as the combination produces a functional LD-linked target binding molecule. Methods for assessing the functionality of a particular construct are within the realm of one skilled in the art as disclosed herein.

[0070] In a further aspect, the present invention relates to the use of engineered immune cells 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 (e.g., an scFv) linked to a localization domain, for treating cancer, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0071] In another aspect, the present invention relates to the use of engineered immune cells comprising 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) linked to a localization domain, for treating cancer, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0072] In another aspect, the present invention relates to the use of engineered immune cells 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 (e.g., an scFv) linked to a localization domain, for treating an autoimmune disorder, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0073] In other aspects, the present invention also relates to the use of engineered immune cells 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 (e.g., an scFv) linked to a localization domain, for treating an infectious disease, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

[0074] In various embodiments, the immune activating receptor is a CAR (e.g., anti-CD19-4-1BB-CD3ζ CAR).

[0075] In other embodiments, the single chain variable fragment (scFv) linked to the localization domain is selected from any one or more of the constructs shown in FIG.

[0076] In some embodiments, the engineered immune cells are administered by injection into a subject. Methods for injecting immune cells (e.g., allogeneic or autologous immune cells) are known in the art. A sufficient number of cells are administered to the recipient to ameliorate disease symptoms. Typically, 10 7 ~10 10 A dose of 10 cells, e.g., 9 A dose of cells is injected in a single setting. The injection is a single 10 9 administered as a dose of several 10 cells 9 The cells are divided into individual cell doses. The frequency of injections can be once every 3 to 30 days, or even longer intervals if desired or indicated. The injection volume is generally at least one injection per subject, and preferably at least three injections if tolerated, or until disease symptoms are remitted. Cells can be infused intravenously at a rate of 50 to 250 ml / hour. Other suitable modes of administration include intra-arterial infusion, direct injection into the tumor and / or perfusion of the tumor matrix after surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration, and intraocular administration. Methods for adapting the present invention to such delivery modes are readily available to those skilled in the art.

[0077] In certain embodiments, 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 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, and brain tumor.

[0078] In another embodiment, the present invention relates to a method for generating an engineered immune cell of the present invention, comprising introducing into an immune cell 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, thereby generating the engineered immune cell.

[0079] In certain embodiments, a nucleic acid comprising the nucleotide sequence is introduced into an immune cell ex vivo, while in other embodiments, a nucleic acid comprising the nucleotide sequence is introduced into an immune cell in vivo.

[0080] In some embodiments, "immune cells" include, for example, T cells, natural killer (NK) cells, NK / T cells, monocytes, macrophages, or dendritic cells.

[0081] The nucleic acid containing the nucleotide sequence to be introduced may be a single bicistronic construct containing an immune activating receptor described herein and a target-binding molecule (e.g., scFv) linked to a localization domain. As described herein, a single bicistronic construct can be prepared by inserting an internal ribosome entry site (IRES) or a 2A peptide coding region between two cDNAs encoding an immune activating receptor (e.g., CAR) and a target-binding molecule (e.g., scFv) described herein. Designing a tricistronic delivery system to delete two or more targets may also be feasible. Alternatively, separate transduction (simultaneous or sequential) of each construct (e.g., a CAR and an LD-linked target-binding molecule) may be performed. Methods for introducing exogenous nucleic acids are exemplified herein and are well known in the art.

[0082] As used herein, unless expressly indicated to the contrary, the indefinite articles "a" and "an" should be understood to mean "at least one." [Example]

[0083] Example method Cloning of scFv from mouse anti-human CD3 hybridoma PLU4 hybridoma cells (IgG2a isotype, Creative Diagnostics, Shirley, NY), secreting anti-human CD3 monoclonal antibodies, 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 purified with M-MLV reverse transcriptase (Promega, Madison, WI) and oligothymidine dinucleotides. 15cDNA was synthesized using primers (Promega). The variable regions of the heavy (VH) and light (VL) chains were amplified using the IgG Library Primer Set Mouse BioGenomics (US Biological, Salem, MA), and the PCR products were cloned into a TOPO TA cloning kit (Life Technologies) for sequencing. Splicing by overlap extension PCR was used to assemble the VH and VL genes into scFvs with a flexible linker sequence encoding (Gly4Ser)4. The signal peptide domain of CD8α was subcloned and attached to the 5' end of the VL fragment by PCR using cDNA derived from human activated T cells from a healthy donor. A Myc tag (EQKLISEEDL, SEQ ID NO: 1) was added to the C-terminus of VH by PCR using the sense primer 5'-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3' (SEQ ID NO: 2) and the reverse primer 5'-CAGATCTTCTTCAGAAATAAGTTTTTGTTCGGCTGAGGAGACTGTGAGAG-3' (SEQ ID NO: 3). After the Myc tag, the KDEL (SEQ ID NO: 4) coding sequence was also generated using the sense primer 5'-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3' (SEQ ID NO: 5) and the reverse primer 5'-TATATACTCGAGTTACAACTCGTCCTTCAGATCTTCTTCAGAAATAAG-3' (SEQ ID NO: 6). The synthesized gene consisting of the CD8 signal peptide, scFv against human CD3, Myc tag, and 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 by other sequences were also made, as listed in Figure 2.

[0084] The "PEST" sequence, corresponding to amino acids 422-461 of mouse ornithine decarboxylase, -SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7), was obtained from GenBank (accession number NM_013614.2). Codon optimization and gene synthesis were performed using 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.

[0085] Cloning of scFv against human CD7 The sequence of the 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 synthesized gene consisting of the CD8 signal peptide, the scFv against 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 by other sequences were also generated, as listed in Figure 2.

[0086] Cloning of scFv against human beta-2 microglobulin (hB2MG) The scFv sequence 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 synthesized gene consisting of the CD8 signal peptide, the scFv against human B2MG, a Myc tag, and the KDEL (SEQ ID NO: 4) sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

[0087] Cloning of scFv against human KIR2DL1 and KIR2DL2 / DL3 The amino acid sequence of human monoclonal antibody I-7F9 (anti-KIR2DL1, KIR2DL2, and KIR2DL3) was derived from published International Patent Application WO2006003179A2 by Moretta et al. After codon optimization, the scFv sequence was designed by connecting the variable light (VL) and variable heavy (VH) regions with a linker sequence. The synthesized gene consisting of the CD8 signal peptide, scFvs against human KIRs (KIR2DL1, KIR2DL2, and KIR2DL3), CD8 hinge and transmembrane domains, and KKMP sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP was replaced by other sequences were also generated, as listed in Figure 2.

[0088] Cloning of scFv against human NKG2A The sequence of the mouse antibody Z199 (anti-NKG2A) was derived from the published patent by Spee et al. (EP2247619A1). After codon optimization, the sequence of the scFv was designed by connecting the variable light (VL) and variable heavy (VH) regions with a linker sequence. The synthesized gene consisting of the CD8 signal peptide, the scFv against human NKG2A, the CD8 hinge and transmembrane domains, and the KKMP sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP was replaced by other sequences were also generated, as listed in Figure 2. The sequence information of the scFvs generated herein is shown in Table 1. The sequence information of the various components depicted in Figure 2 is shown in Table 2.

[0089] Anti-CD19-4-1BB-CD3ζCAR 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).

[0090] Table 1. scFv sequence information

[0091] [Table 1-1]

[0092] [Table 1-2]

[0093] [Table 1-3]

[0094] [Table 1-4]

[0095] [Table 1-5]

[0096] [Table 1-6]

[0097] [Table 1-7] Table 2. Sequence information of components depicted in Figure 2

[0098] [Table 2-1]

[0099] [Table 2-2]

[0100] [Table 2-3]

[0101] [Table 2-4]

[0102] [Table 2-5]

[0103] [Table 2-6] Gene transduction, cell proliferation, flow cytometry analysis, and functional studies These were performed as previously described (Kudo, K et al., Cancer Res. 2014;74(1):93-103).

[0104] result Generation of scFv constructs An overview of this technology is illustrated in Figure 1. A schematic diagram of the suppression constructs we generated is shown in Figure 2. The scFv portion can be derived from cloning cDNAs encoding the variable light (VL) and variable heavy (VH) immunoglobulin chain regions of an antibody-producing hybridoma cell line or from the corresponding published sequence. The VL and VH are linked by a short peptide sequence ("linker"), according to standard techniques for generating complete scFvs. To be expressed, the scFv is linked to a signal peptide at its N-terminus; as confirmed in preliminary experiments, the signal peptide is required for scFv expression. Proteins containing the scFv plus the signal peptide are generally released into the cellular environment. For example, in preliminary experiments (not shown), anti-CD3ε scFv plus the 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, potential effects on other cells are prevented. To target it to the endoplasmic reticulum (ER), we utilized the KDEL (SEQ ID NO: 4) motif (which retains proteins 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 a proteasome-targeting PEST motif (Joshi, SN et al., MAbs. 2012;4(6):686-693). The scFv can also be targeted to the cell membrane by linking it to the transmembrane domain and hinge of CD8α or another transmembrane protein.

[0105] Downregulation of T cell receptors in T lymphocytes expressing anti-CD19-BB-ζCAR To determine whether the proposed strategy could be applied to generating immune cells that express a CAR and lack one or more markers, T cell receptor (TCR) expression was downregulated in anti-CD19 CAR T cells.

[0106] 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 absence 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 to generate constructs containing KDEL (SEQ ID NO: 4), PEST, the CD8α transmembrane domain, or others shown in Figure 2.

[0107] A murine stem cell virus (MSCV) retroviral vector containing green fluorescent protein (GFP) was used to transduce the constructs disclosed herein into a CD3 / TCR+ Jurkat cell line. The percentage of GFP+ cells after transduction was greater than 90% in all experiments. Figure 3A shows the results of staining with anti-CD3ε antibody among GFP+ cells, as measured by flow cytometry. Antibody staining for CD3ε was reduced to various degrees in cells transduced with the listed constructs. Similar downregulation of CD3ε was obtained with human peripheral blood T lymphocytes (Figure 3B). Figure 3C shows illustrative flow cytometry dot plots of CD3ε expression in GFP-positive Jurkat cells after transduction with different gene constructs compared to cells transduced with a vector containing GFP alone. CD3 downregulation did not affect Jurkat cell proliferation or the expression of all other cell markers tested, including CD2, CD4, CD8, CD45, CD25, and CD69. Suppression of CD3 expression remained for more than 3 months. Further enrichment of CD3-negative cells can be achieved by CD3+ T cell depletion with anti-CD3 magnetic beads (Dynal, Life Technologies, Carlsbad, CA).

[0108] Staining of Jurkat cells or human peripheral blood T lymphocytes with anti-TCRαβ antibody showed that downregulation of CD3ε expression was associated with downregulation of TCRαβ expression (Fig. 4 ).

[0109] Next, we determined whether anti-CD3 scFv-myc KDEL could be co-expressed with anti-CD19-4-1BB-CD3ζ CAR. As shown in Figure 5, this resulted in T cells expressing anti-CD19 CAR but lacking CD3 expression. TCR was also absent on these cells (not shown).

[0110] To assess whether CARs can signal in Jurkat cells with downregulated CD3 / TCRs, we examined the expression of activation markers CD69 and CD25 and measured exocytosis of lytic granules by CD107a expression in Jurkat cells cocultured with the CD19+ leukemia cell line OP-1. As shown in Figure 6, downregulation of CD3 / TCRs with the anti-CD3scFv-myc KDEL construct did not reduce the ability of anti-CD19-4-1BB-CD3ζ CAR to activate Jurkat cells. To further explore the effect of CD3 / TCR deletion on CAR signaling, we determined whether CD3-negative T lymphocytes expressing CARs could be stimulated by their ligation. As shown in Figure 7, co-culture of anti-CD19 CAR-expressing T lymphocytes with CD19+ leukemia cells resulted in T cell proliferation whether CD3 was downregulated or not, indicating that CD3 / TCR downregulation did not reduce CAR proliferation stimulation.

[0111] Thus, the anti-CD3scFv-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.

[0112] CD7 downregulation We determined whether the strategy that successfully regulated CD3 / TCR expression could be applied to other surface molecules. To this end, CD7 expression was modulated. The scFv sequence was derived from that published by Peipp et al. (Cancer Res 2002(62):2848-2855) and linked to the CD8 signal peptide and myc-KDEL sequence illustrated in Figure 2. Using an MSCV retroviral vector, we transduced an anti-CD7-myc KDEL construct into peripheral blood lymphocytes with high expression of CD7, as detected by anti-CD7 antibodies conjugated to phycoerythrin (BD Bioscience). As shown in Figure 8, CD7 expression in T lymphocytes transduced with this construct was substantially suppressed.

[0113] HLA-class I downregulation This strategy was then applied to downregulate another surface molecule, HLA class I.

[0114] HLA class I consists of a polymorphic α chain and a non-polymorphic chain called β2-microglobulin. Knockdown of the latter subunit results in suppression of HLA (MHC in mice) class I expression (Koller, BH et al., Science. 1990;248(4960):1227-1230). Using scFvs reactive with β2-microglobulin, we suppressed HLA class I expression in immune cells.

[0115] The scFv sequence was derived from that published by Grovender et al. (Kidney Int. 2004;65(1):310-322) and was linked to the CD8 signal peptide and myc KDEL sequence illustrated in Figure 2. An MSCV retroviral vector was used to transduce the anti-β2M-myc KDEL construct into Jurkat cells with high expression of HLA class I, as detected by anti-HLA-ABC antibodies conjugated to phycoerythrin (BD Pharmingen). As shown in Figure 9, Jurkat cells transduced with this construct had a substantial downregulation of HLA-ABC expression. The cells maintained their morphology and proliferation capacity.

[0116] Downregulation of inhibitory receptors in NK cells To determine whether the strategy described above also applies to surface molecules expressed in other immune cells, downregulation of the function of the inhibitory receptors KIR2DL1, KIR2DL2 / DL3, and NKG2A was examined in NK cells.

[0117] To downregulate KIR receptors, scFvs reacting with KIR2DL1 and KIR2DL2 / DL3 were used to suppress their expression in NK cells. The scFv sequence was derived from that published by Moretta et al. (Patent WO 2006 / 003179 A2) and was linked to a CD8 signal peptide and ER retention sequence as illustrated in Figure 2. Using an MSCV retroviral vector, this construct was transduced into NK cells grown from human peripheral blood and selected for KIR2DL1 expression. These cells also had high KIR2DL1 expression, as detected by anti-KIR2DL1 antibodies conjugated to allophycocyanin (R&D Systems), and high KIR2DL2 / DL3 expression, as detected by anti-KIR2DL2 / DL3 antibodies conjugated to phycoerythrin (BD Bioscience). FIG. 10 shows the results obtained with scFv-linker(20)AEKEDL and scFv-EEKKMP, with substantial downregulation of the targeted KIR.

[0118] To downregulate NKG2A, an scFv reactive with NKG2A was used to suppress its expression in NK cells. The scFv sequence derived from published European Patent Application No. EP2247619A1 by Spee et al. was linked to the CD8 signal peptide and ER retention sequence illustrated in Figure 2. An MSCV retroviral vector was used to transduce this construct into NK cells expanded from human peripheral blood with high NKG2A expression, as detected by anti-NKG2A antibodies conjugated to phycoerythrin (Beckman Coulter). Figure 11 shows the substantial downregulation of NKG2A obtained by scFv-EEKKMP.

[0119] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0120] While the present 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 appended claims.

Claims

1. 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.

2. 2. The engineered immune cell of claim 1, wherein 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.

3. 2. The engineered immune cell of claim 1, wherein the receptor is a chimeric antigen receptor (CAR).

4. The engineered immune cell of claim 1, wherein the CAR is an anti-CD19-4-1BB-CD3ζ CAR.

5. The engineered immune cell of claim 1 , wherein the target binding molecule is an antibody.

6. 6. The engineered immune cell of claim 5, wherein the antibody is a single-chain variable fragment (scFv).

7. 6. The engineered immune cell of claim 5, wherein the antibody binds to a factor 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.

8. 8. The engineered immune cell of claim 7, wherein the factor in the CD3 / TCR complex is CD3ε, TCRα, TCRβ, TCRγ, TCRδ, CD3δ, CD3γ, or CD3ζ.

9. 8. The engineered immune cell of claim 7, wherein the HLA class I molecule is β2-microglobulin, α1-microglobulin, α2-microglobulin, or α3-microglobulin.

10. 8. The engineered immune cell of claim 7, wherein the receptor that downregulates an immune response is selected from programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing 3 (Tim3), killer immunoglobulin-like receptor (KIR), CD94, NKG2A, or protein tyrosine phosphatase.

11. 8. The engineered immune cell of claim 7, wherein the cytokine is 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 transforming growth factor (TGF)-β.

12. 2. The engineered immune cell of claim 1, wherein the target binding molecule binds to CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127.

13. 8. The engineered immune cell of claim 7, wherein the localization domain comprises an endoplasmic reticulum (ER) or Golgi retention sequence; a proteasome localization sequence; a transmembrane domain sequence derived from CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, CD16, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, or FGFR2B.

14. 14. The engineered immune cell of claim 13, wherein the ER or Golgi retention sequence comprises the amino acid sequence KDEL (SEQ ID NO: 4), KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10), or YQRL (SEQ ID NO: 11), where X is any amino acid.

15. The engineered immune cell of claim 13 , wherein the proteasome localization sequence comprises a PEST motif.

16. 10. The engineered immune cell of claim 1, wherein the immune activation receptor is a non-naturally occurring molecule.

17. 2. The engineered immune cell of claim 1, wherein the target binding molecule linked to a localization domain is a non-naturally occurring molecule.

18. 10. Use of the engineered immune cells of claim 1 for treating cancer, comprising administering a therapeutic amount of the engineered immune cells to a subject in need thereof.

19. 19. The use of claim 18, wherein the engineered immune cells are administered to the subject by intravenous infusion, intra-arterial infusion, direct injection into the tumor and / or perfusion of the tumor matrix after surgery, implantation at the tumor site in an artificial scaffold, intrathecal administration, and intraocular administration.

20. 19. The use of claim 18, wherein the cancer is a solid tumor or a hematological malignancy.

21. 10. A method for generating the engineered immune cells of claim 1, comprising: A method comprising introducing into an immune cell 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, thereby producing an engineered immune cell.

22. 22. The method of claim 21, wherein the engineered immune cells are generated in vitro.

Images