Methods to improve the effectiveness 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 powerful and specific "living drugs." They have the potential to target tumor cells while preserving normal tissue, and several clinical observations suggest they may possess significant anti-cancer activity. Therefore, 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) is inversely related to leukemia relapse. 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) generated major clinical responses in patients with either solid tumors or leukemia. Finally, infusion of genetically modified autologous T lymphocytes induced complete and sustained remission 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 immunotherapy by expanding its applicability and enhancing its effectiveness. [Overview of the project]

[0003] The present invention relates to engineered immune cells having 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 activation receptor and a nucleic acid comprising a nucleotide sequence encoding a target-binding molecule linked to a localization domain.

[0004] In another embodiment, the present invention provides the use of engineered immune cells for treating cancer, comprising a gene encoding an immune-activating receptor and a gene encoding a target-binding molecule linked to a localization domain, the use comprising administering a therapeutic dose of the engineered immune cells to a subject in need.

[0005] In various embodiments, the present invention also provides a method for generating engineered immune cells, comprising introducing into immune cells 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 generating engineered immune cells.

[0006] In some embodiments, manipulated immune cells have improved therapeutic efficacy as a result of one or more of the following: reduction of graft-versus-host disease (GvHD) in the host, reduction or elimination of host rejection, extension of host survival, reduction of tumor suppression in the host, reduction of host autolysis, reduction of inflammatory cascades in the host, or sustained intrinsic / artificial receptor-mediated (e.g., CAR-mediated) signal transduction in the host.

[0007] The foregoing will become clear from the following more specific description of exemplary embodiments of the invention, as illustrated by the attached drawings (where similar reference letters refer to the same parts from different perspectives). The drawings are not necessarily to scale, and instead the focus is on illustrating embodiments of the invention. [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram of the design used in the present invention. Figure 1A shows the overall mechanism of CAR-mediated killing of cancer cells. Figure 1B shows the combined expression of CARs by different forms of compartment-oriented scFv (an example of a target-binding molecule linked to a localization domain) and examples of possible targets. The CAR may be replaced by other receptors that can enhance the immune cell capacity. [Figure 2]This is a schematic diagram of a construct containing scFv, along with a domain that localizes scFv to a specific cellular compartment. 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 diagram 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 constructs that do not have a signaling peptide in the CD8 transmembrane domain and have various numbers of amino acids. 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 Domains" and, as shown, describe the linker in several examples. 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 illustrate the downregulation of CD3 / TCR in T cells by scFv targeting of CD3ε. Figure 3A shows the expression of surface CD3ε in Jurcut cells transduced with either a retroviral vector containing only green fluorescent protein (GFP) ("fake") or a vector containing a different construct shown as GFP plus. One week after transduction, surface CD3ε expression in cell membranes was compared to that in fake-transduced cells using an anti-CD3 antibody coupled to allophycocyanin (BD Biosciences). All comparisons were performed after gating of GFP-positive cells. Figure 3B depicts a similar experiment performed on peripheral blood T lymphocytes grown with anti-CD3 / CD28 beads (Lifesciences). Staining was performed one week after transduction. [Figure 3C] Figure 3C shows flow cytometry plots illustrating the downregulation of membrane CD3ε in Jurcut cells after transduction in the indicated construct. The dashed rectangle in the upper right quadrant of each plot encloses GFP+CD3+ cells. [Figure 4] This shows the downregulation of CD3ε and TCRαβ on the cell membrane in Jarcut T cells during transduction with anti-CD3ε scFv-KDEL or -PEST, or -mb EEKKMP. Membrane marker expression was measured one week after transduction using anti-CD3 antibody conjugated to allophycocyanin (BD Biosciences) or anti-TCRαβ conjugated to phycoerythrin (Biolegend). The line labeled "control" represents the labeling of pseudotransduced cells. The vertical dashed line represents the upper limit of staining obtained with isotype-matched non-reactive antibodies. [Figure 5]This demonstrates that anti-scFv and CAR can be expressed simultaneously. Flow cytometry dot plots show staining of Jurcut cells (top row) or peripheral blood lymphocytes (bottom row) with anti-CD3 allophycocyanin antibody and streptavidin coupled with goat anti-mouse Fab2 biotin plus 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 of GFP-positive cells, those transduced with anti-CD3scFv-myc KDEL downregulated CD3 (left column, bottom left quadrant), while those transduced with the anti-CD19-4-1BB-CD3ζ construct expressed CAR (middle column, top right quadrant). A significant proportion of cells transduced with both constructs were CD3-negative and CAR-positive (right column, top left quadrant). [Figure 6] This study demonstrates that anti-CD19CAR induces T cell activation and degranulation regardless of CD3 / TCR downregulation. Jarcut 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 pseudotransduced cells. Cells were cultured alone or co-cultured with the CD19+ leukemia cell line OP-1 in a 1:1 ratio. After 18 hours, CD69 and CD25 expression was tested by flow cytometry using specific antibodies (from BD Biosciences), and after 6 hours, CD107a expression was tested (using antibodies from BD Biosciences). In the presence of OP-1 cells, CD69 and CD25 expression occurred in CAR-expressing cells regardless of whether the cells were also transduced with anti-CD3scFv-KDEL. Activation did not occur in pseudo- or anti-CD3scFv-myc KDEL-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 study demonstrates that anti-CD19CAR 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. Transduced T lymphocytes were co-cultured with Streck (Omaha, NE) treated OP-1 cells to suppress their proliferation for the indicated time. The proliferation of CD3-positive and CD3-negative T lymphocytes expressing anti-CD19CAR was compared with the proliferation of pseudotransduced T cells. Each symbol indicates the average cell number of the two parallel cultures. CAR T cells proliferated similarly well, regardless of CD3 / TCR expression. [Figure 8] This shows the expression of CD7 on the membrane of peripheral blood T lymphocytes transduced with either a retroviral vector containing only GFP ("fake") 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 fake-transduced cells using an anti-CD7 antibody conjugated to phycoerythrin (BD Biosciences). The dashed rectangle in the upper right quadrant of each plot encloses the GFP+CD7+ cells. [Figure 9] This study describes the downregulation of HLA class I in T cells by scFv targeting of β2-microglobulin. Jarcut 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 pseudotransduced cells using phycoerythrin (BD Biosciences)-coupled anti-HLA-ABC antibody. Staining with isotype-matched control antibodies is also shown. Analysis was performed after gating of GFP-positive cells. [Figure 10]This study describes the downregulation of killer immunoglobulin-like receptors (KIR) 2DL1 and KIR2DL2 / DL3 in human NK cells by scFv targeting of KIR2DL1 and KIR2DL2 / DL3. NK cells were grown in vitro and selected for KIR2DL1 expression, then transduced with anti-KIR2DL1-KIR2DL2 / DL3 scFv-linker(20)AEKDEL or -EEKKMP. Eight days after transduction, the expression of the corresponding KIR on the cell membrane was compared to that of pseudotransduced cells using anti-KIR2DL1 antibody coupled to allophycocyanin (R&D Systems) or anti-KIR2DL2 / DL3 antibody coupled to phycoerythrin (BD Biosciences). Staining with isotype-matched control antibodies is also shown. Analysis was performed after gating of GFP-positive cells. [Figure 11] This study describes the downregulation of NKG2A in human NK cells by scFv targeting. NK cells grown in vitro were transduced with anti-NKG2A scFv-EEKKMP. Eight days after transduction, NKG2A expression on the cell membrane was compared to that of pseudotransduced cells using phycoerythrin (Beckman-Coulter) coupled NKG2A antibody. Staining with isotype-matched control antibodies is also shown. Analysis was performed after gating of GFP-positive cells. [Modes for carrying out the invention]

[0009] The following is a description of exemplary embodiments of the present invention.

[0010] In recent years, the acquisition of knowledge about molecular pathways that regulate immune cells has been matched by remarkable advances in the ability to manipulate them in vitro (including their proliferation and genetic engineering). Currently, it is 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 amplified through 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 previously described artificial multimolecular proteins (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 extracellular domains, transmembrane domains, and cytoplasmic domains that bind to a specific target. For example, as described in U.S. Patent No. 8,399,645 (which is incorporated herein by reference in its entirety), the extracellular and transmembrane domains may originate from any desired source of such domains. In short, CARs can be designed to contain a single-chain variable region (scFv) of an antibody that specifically binds to a target. scFv can be ligated to T cell receptor (TCR)-related signaling molecules such as CD3ζ via its transmembrane and hinge domains. Ligation of scFv to congener antigens induces signal transduction. Therefore, CARs can instantaneously reorient cytotoxic T lymphocytes toward cancer cells, inducing 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). 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), so co-stimulating molecules such as CD28 and 4-1BB (or CD137) are incorporated into CAR constructs to support signal transduction.This dual signaling design ("second-generation CAR") is useful for extracting 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 specific CAR containing both 4-1BB and CD3ζ, anti-CD19CAR, is described in U.S. Patent No. 8,399,645. Infusion of autologous T cells expressing anti-CD19-4-1BB-CD3ζCAR resulted in dramatic clinical responses in patients with chronic lymphoid 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, as well as studies using 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 strong evidence of the clinical potential of this technology and of general immunotherapy.

[0013] The methods described herein enable the rapid removal or deactivation of specific proteins in immune cells that are reoriented by natural or artificial receptors (e.g., CARs), thereby expanding the application potential of engineered cells and significantly improving their function. The methods partially rely on single or multiple constructs containing an immunoactivating receptor, e.g., a CAR (including an extracellular domain (e.g., scFv), a transmembrane domain, and a cytoplasmic domain that binds to a specific target), along with a target-binding molecule that binds to the target to be removed or neutralized (e.g., a protein). The target-binding molecule is ligated to a domain (i.e., a localization domain) that, depending on the application, directs it to a specific cellular compartment (e.g., the Golgi or endoplasmic reticulum), a proteasome, or the cell membrane. For simplicity, a target-binding molecule ligated to a localization domain (LD) may also be referred to herein as an "LD-ligated target-binding molecule."

[0014] As will become apparent from the teachings herein, various immunoactivating receptors may be suitable for the method of the present invention. That is, any receptor containing a molecule that can activate an immune response upon binding (ligation) to a ligand (e.g., a peptide or antigen) expressed on cancer cells can be used according to this method. For example, as described above, the immunoactivating receptor may be a chimeric antigen receptor (CAR), and methods for designing and manipulating 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 ability 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 CAR but in which the scFv is replaced with an antibody-binding molecule (e.g., CD16, CD64, CD32). Furthermore, in the context of tumor cell HLA, T cell receptors containing alpha and beta chains that bind to peptides expressed on tumor cells can also be used according to this method. In addition, other receptors carrying molecules that activate the immune response by binding to ligands expressed on cancer cells (e.g., NKG2D-DAP10-CD3 zeta receptor that binds to the NKG2D ligand expressed on tumor cells) can also be used (see, for example, 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 the immune response upon binding to cancer cell ligands.” Therefore, immune-activating receptors having molecules activated by cancer cell ligands can be expressed together with LD-linked target-binding molecules according to this method.

[0015] This method significantly expands the potential uses of immunotherapy based on the injection of immune cells reoriented by artificial receptors. The methods described are 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, for example, by inserting an internal ribosome entry site (IRES) or a 2A peptide-coding region site between two cDNAs encoding the CAR and the LD-linked target-binding molecule. The design of a tricistronic delivery system for deleting two or more targets could also be feasible. Alternatively, separate transduction of two genes (simultaneously or sequentially) may be performed. In the context of cancer cell therapy, the CAR may be replaced by an antibody-binding signaling receptor (Kudo K, et al., Cancer Res. 2014;74(1):93-103), a T cell receptor oriented 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 ability of the CAR was not impaired.

[0016] Both anti-CD3ε scFv-KDEL (and -PEST) tested herein stably downregulate CD3 and TCR expression. Residual CD3+ T cells can be removed using CD3 beads, and this approach is also available in clinical-grade form. The ability to produce CD3 / TCR-negative cells that respond to CAR signaling represents a significant advance. Clinical studies with CAR T cells are generally performed using autologous T cells. Therefore, the quality of cell products varies from patient to patient, and responses are heterogeneous. Allogeneic T cell injection is currently not possible due to the unacceptably high potential risk of fatal GvHD caused by stimulation of the endogenous TCR by the recipient's tissue antigens. Since the absence of endogenous TCR eliminates GvHD capability, downregulation of CD3 / TCR opens up the possibility of injecting allogeneic T cells. Allogeneic products can be prepared with optimal cell compositions (e.g., those rich in highly cytotoxic T cells, those depleted of regulatory T cells, etc.) and selected so that the injected cells have high CAR expression and functional efficacy. Furthermore, fully standardized products can be cryopreserved and made available for use regardless of the patient's immune cell status and their fitness to undergo depletion therapy or extensive blood collection. Depletion 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 is difficult to implement in a clinical setting because it requires several cell selections and proliferations through extended culture. The method described herein has many practical advantages.

[0017] Furthermore, an LD-linked target-binding molecule (e.g., scFv-myc KDEL, scFv-EEKKMP, or scFv-PEST, where scFv targets a specific protein / molecule) can be used according to the present invention to delete HLA class I molecules and reduce the likelihood of allogeneic cell rejection. While the injection of allogeneic T cells is a future goal of CAR T cell therapy, the injection of allogeneic natural killer (NK) cells has already been used to treat patients with cancer. An important factor determining the success of NK cell-based therapies is that the NK cells must remain in a sufficient number to achieve an effector:target ratio that has the potential to generate tumor cell elimination (Miller JS. Hematology Am Soc Hematol Educ Program. 2013;2013:247-253). However, when allogeneic cells are injected, their persistence is limited. The immunosuppressive chemotherapy given to the patient allows for transient engraftment of the injected NK cells, but these are rejected within 2 to 4 weeks of injection (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, immunosuppressive agents also suppress NK cell function, so continuous immunosuppression is not an option. Since rejection is mainly mediated by the recognition of HLA class I molecules by the recipient's CD8+ T lymphocytes, removing HLA class I molecules from the injected NK cells (or T cells) will reduce or arrest the rejection rate and extend the survival of allogeneic cells and, therefore, their anti-tumor capacity.

[0018] Furthermore, inhibitory receptors can be targeted using LD-linked target-binding molecules according to the present invention. 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 oriented towards solid tumors, may be suppressed by a similar mechanism. Therefore, the expression of target-binding molecules (e.g., scFv or ligands) to PD1, CTLA-4, Tim3, or other inhibitory receptors will prevent the 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 (when linked to the transmembrane domain) prevent the receptor from binding to their ligands, thus perpetuating CAR-mediated signal transduction. Examples of inhibitory receptors in NK cells include killer immunoglobulin-like receptors (KIRs) and NKG2A (Vivier E, et al., Science, 2011;331(6013):44-49).

[0019] The method of the present invention also enables the targeting of a greater number of targets to which CAR-oriented T-cell therapy can be accepted. One of the main limitations of CAR-oriented 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 may lead to the elimination of the immune cells themselves through a "sibling killer" mechanism, potentially rendering their anti-cancer capabilities ineffective. If the target molecule can be removed from immune cells without functional adverse effects, a CAR with corresponding specificity may 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, 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, it has been shown that cytokine secretion by activated immune cells can cause cytokine release syndrome and macrophage activation syndrome, leading to serious adverse effects of immunotherapy (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 may contribute to such inflammatory cascades.

[0021] Accordingly, in one embodiment, the present invention relates to an engineered immune cell 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.

[0022] As used herein, “engineered” immune cells include immune cells that have been genetically modified compared to naturally occurring immune cells. For example, engineered T cells produced according to this method possess nucleic acids containing nucleotide sequences that are not naturally present in the T cells from which they originate. In some embodiments, the engineered immune cells of the present invention include a chimeric antigen receptor (CAR) and a target-binding molecule (LD-linked target-binding molecule) linked to the localization domain. In a particular embodiment, the engineered immune cells of the present invention include an anti-CD19-4-1BB-CD3ζCAR and an anti-CD3scFv linked to the 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, “immunoactivating receptor” refers to a receptor that activates an immune response upon binding to a cancer cell ligand. In some embodiments, the immunoactivating receptor comprises a molecule that can activate an immune response upon binding (ligation) to a ligand (e.g., a peptide or antigen) expressed on cancer cells. In one embodiment, the immunoactivating receptor is a chimeric antigen receptor (CAR), and methods for designing and manipulating CARs are known in the art. In other embodiments, the immunoactivating receptor is an antibody-binding receptor, which is similar to a CAR but in which the scFv is substituted 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, in the context of tumor cell HLA, T cell receptors comprising T cell receptor alpha and beta chains that bind to peptides expressed on tumor cells can also be used according to this method. In certain embodiments, other receptors carrying molecules that activate the immune response by binding to ligands expressed on cancer cells (e.g., the NKG2D-DAP10-CD3 zeta receptor that binds to the NKG2D ligand expressed on tumor cells) can also be used (see, e.g., Chang YH, et al., Cancer Res. 2013;73(6):1777-1786). All such suitable receptors that can activate the immune response upon binding (ligation) to ligands (e.g., peptides or antigens) expressed on cancer cells are collectively called “immunoactivating receptors.” As recognized by those skilled in the art, immunoactivating receptors do not need to contain an antibody or antigen-binding fragment (e.g., scFv); rather, the portion of the immunoactivating receptor that binds to the target molecule may originate, for example, from the receptor in a receptor-ligand pair or from the ligand in a receptor-ligand pair.

[0025] In certain embodiments, the immune-activating receptor binds 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, mesoserine, 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-CD3ζCAR). In certain embodiments, the immune-activating receptor includes, but is not limited to, antibodies or antigen-binding fragments (e.g., scFv) that 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, mesoserine, 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. For example, antibodies against CD3 and CD7 are commercially available and known in the art. As illustrated 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 common in the art.

[0026] The transmembrane domains of immunoactivating receptors (e.g., CARs) according to the present invention include, but are 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, and may be derived from single-pass transmembrane proteins. In some examples, the membrane protein is not CD8α. The transmembrane domain may also be a hydrophobic protein segment that does not exist naturally.

[0027] The hinge domain of an immune-activating receptor (e.g., CAR) may originate 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-natural peptide such as a polypeptide consisting of hydrophilic residues of varying lengths, or (GGGGS) n (Sequence ID 8) A polypeptide may also be (where n is, for example, an integer between 3 and 12 (including these)).

[0028] The signaling domains of immune-activating receptors (e.g., CARs) may originate from CD3ζ, FcεRIγ, DAP10, DAP12, or other molecules known to deliver activation signals in immune cells. At least one co-stimulating signaling domain of the receptor may be 4-1BB (also known as CD137), CD28, CD28 JPEG0007869384000001.jpg13

[0029] The co-stimulating molecules may include variants, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. Such molecules are readily available and known in the art.

[0030] As will be recognized by those skilled in the art, the components of an immunoactivating receptor can be manipulated to include several functional combinations, as described herein, in order to produce the desired results. Using a particular CAR anti-CD19-4-1BB-CD3ζ as an example, the antibody that binds to the molecule (e.g., or its antigen-binding fragment (e.g., scFv)) may be substituted with an antibody that binds to a different molecule (e.g., anti-CD20, anti-CD33, anti-CD123 instead of anti-CD19), as described herein. In other embodiments, the co-stimulating molecule (4-1BB in this specific example) may also be different by a different co-stimulating molecule (e.g., CD28). In some embodiments, the stimulating molecule (CD3ζ in this specific example) may be substituted with another known stimulating molecule. In various embodiments, the transmembrane domain of the receptor may also be different, if desired. The design, production, and testing of the functionality of such immunoactivating receptors can be readily determined by those skilled in the art. Similarly, the design, delivery to cells, and expression of nucleic acids encoding such immunoactivating 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 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. Furthermore, 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 double-stranded polymers, “nucleic acid” may refer to either or both strands of the molecule.

[0032] In relation to nucleic acids, the term "nucleotide sequence" refers to a series of adjacent nucleotides joined by covalent bonds such as phosphorus bonds (e.g., phosphate diester bonds, alkyl and aryl phosphonate bonds, phosphorothioate bonds, phosphotriester bonds) and / or non-phosphorus bonds (e.g., peptide bonds and / or sulfamate bonds). In certain embodiments, for example, a nucleotide sequence encoding 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 ribonucleotides or deoxyribonucleotide monomers, as well as their derivatives and analogues that do not exist in nature. Therefore, nucleotides may include, for example, nucleotides containing naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine), and nucleotides containing modified bases known in the art.

[0034] As will be recognized by those skilled in the art, in some embodiments, the nucleic acid further comprises plasmid sequences. The plasmid sequence may comprise, for example, one or more sequences selected from the group consisting of promoter sequences, selection marker sequences, and gene coordinate sequences.

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

[0036] In certain embodiments, the target-binding molecule is an antibody or its antigen-binding fragment. As used herein, “antibody” means an intact antibody or an antigen-binding fragment of an antibody, including an intact antibody or an antigen-binding fragment that is modified or engineered, or is a human antibody. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, multiple paratopic 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] "Fab fragment" is a single light chain, and C H It includes 1 and a variable region of one heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0038] The "Fc" region contains two heavy chain fragments, each containing the CH2 and CH3 domains of a given antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

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

[0040] The "F(ab′)2 fragment" consists of two light chains, as well as C, such that an interchain disulfide bond is formed between the two heavy chains. H 1 domain and C H 2 It contains two heavy chains, each containing a portion of the constant region between the domains. Therefore, the F(ab′)2 fragment consists of two Fab′ fragments, which are held together by a disulfide bond between the two heavy chains.

[0041] The "Fv region" includes variable regions derived from both heavy and light chains, but lacks a steady-state region.

[0042] In a particular embodiment, the target-binding molecule is a single-chain Fv antibody ("scFv antibody"). scFv refers to an antibody fragment containing the VH and VL domains of a given antibody, where these domains are located within a single polypeptide chain. Generally, an Fv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form a structure desired for antigen binding. For an overview of scFv, 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 WO88 / 01649, and U.S. Patents 4,946,778 and 5,260,203. As an example, linkers between the VH domain and VL domain of scFv disclosed herein include, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGSGGGGS (SEQ ID NO: 43). As will be recognized by those skilled in the art, various suitable linkers may be designed and tested for optimal function, as provided in the art and disclosed herein.

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

[0044] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a target binding molecule (e.g., 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 with 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 by a program such as 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 acts as a reference sequence (e.g., parent sequence), and the test sequence is compared against it. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence(s) relative to the reference sequence based on the specified program parameters.

[0046] The optimal sorting 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 sorting algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by 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 Ausubel et al., Current Protocols in Molecular Biology in general). An example of a suitable algorithm for determining sequence identity percentage and sequence similarity is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (publicly accessible through the NCBI Internet Server of the National Institutes of Health). Typically, sequence comparison can be performed using default program parameters, but customized parameters may also be used. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff & Henikoff, Proc.Natl.Acad.Sci. USA89:10915 (1989)) as defaults.

[0047] In certain embodiments, the antibody (e.g., scFv) comprises VH and VL having amino acid sequences defined 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) includes VH and VL sequences 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 with respect to the VH and VL sequences defined 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. 28 and 29, SEQ ID NOs. 32 and 33, or SEQ ID NOs. 36 and 37, respectively.

[0048] A "diabody" is a small antibody fragment having two antigen-binding sites. The fragment contains a heavy chain variable region (VH) (VH-VL or VL-VH) attached to a light chain variable region (VL) within the same polypeptide chain. 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 a complementary domain on another chain and create two antigen-binding sites. Diabodies are described, for example, in patent documents EP404,097, WO93 / 11161, and Holliger et al., (1993) Proc. Natl.Acad.Sci.USA90:6444-6448.

[0049] 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.Methods248(1-2):47-66, 2001.

[0050] A "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing 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 by a peptide linker to create a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same antigen or different antigens.

[0051] In some embodiments, antibodies are modified or manipulated. Examples of modified or manipulated antibodies include chimeric antibodies, multiple paratopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies).

[0052] As used herein, “multiple paratopic antibody” means an antibody comprising at least two single-domain antibodies, of which at least one single-domain antibody is oriented to a first antigenic determinant on an antigen, and at least one other single-domain antibody is oriented to a second antigenic determinant on the same antigen. Thus, for example, a “double paratopic” antibody comprises at least one single-domain antibody oriented to a first antigenic determinant on an antigen, and at least one further single-domain antibody oriented to a second antigenic determinant on the same antigen.

[0053] As used herein, “multispecific antibody” means an antibody comprising at least two single-domain antibodies, of which at least one single-domain antibody is oriented toward a first antigen and at least one other single-domain antibody is oriented toward a second antigen (different from the first antigen). Thus, for example, a “bispecific” antibody is an antibody comprising at least one single-domain antibody oriented toward a first antigen and at least one further single-domain antibody oriented toward, 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 producing 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 an LD-linked target-binding molecule is a receptor or ligand that binds to a 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 recognized by those skilled in the art, the target-binding molecule may be an antibody or a ligand / receptor that binds to a 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 within a frame adjacent to one or more genes encoding one or more localization domains (e.g., without a linker). Alternatively, the gene encoding the target-binding molecule may be linked to one or more genes encoding one or more localization domains via 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 domains. For example, non-natural peptides such as polypeptides consisting of hydrophilic residues of varying lengths, or (GGGGS) n A polypeptide (SEQ ID NO: 8) (where n is, for example, an integer between 3 and 12 (including these)) may be used in accordance with the present invention. In certain embodiments, the linker includes, for example, GGGGSGGGGS (SEQ ID NO: 62). In some embodiments, the linker includes, for example, GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41). In various embodiments, peptide linkers having an amino acid length of about 5 to about 100 (including these) can be used in the present invention. In certain embodiments, peptide linkers having an amino acid length of about 20 to about 40 (including these) can be used in the present invention. In some embodiments, peptide linkers having an amino acid length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 amino acids can be used in the present invention. As will be recognized by those skilled in the art, such linker sequences and variants of such linker sequences are known in the art. Methods for designing constructs that incorporate linker arrays, and for evaluating their functionality, are readily available to those skilled in the art.

[0057] In certain embodiments, LD-linked target-binding molecules bind to targets expressed on the surface of immune cells. In some embodiments, LD-linked target-binding molecules suppress the activity or function of target molecules. As an example, as disclosed herein, LD-linked target-binding molecules may bind to, for example, CD3, CD7, CD45, hB2MG, KIR2DL1, KIR2DL2 / DL3, or NKG2A, thereby downregulating the 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, LD-linked target-binding molecules inactivate the target (for example, the target can no longer interact with and / or bind to its homologous ligand or receptor).

[0058] In some embodiments, the manipulated immune cells of the present invention have improved therapeutic efficacy. As used herein, “improved therapeutic efficacy” means one or more of the following: reduction of graft-versus-host disease (GvHD) in the host, reduction or elimination of host rejection, extension of host survival, reduction of tumor suppression in the host, reduction of host autolysis, reduction of inflammatory cascade in the host, or sustained CAR-mediated signal transduction in the host.

[0059] In certain embodiments of the present invention, the target-binding molecule in the context of an LD-linked target-binding molecule binds to molecules in the CD3 / T cell receptor (TCR) complex, cytokines, human leukocyte antigen (HLA) class I molecules, or receptors that downregulate the immune response.

[0060] In certain embodiments, the molecules in the CD3 / TCR complex may 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, α1-microglobulin, α2-microglobulin, or α3-microglobulin.

[0062] In other embodiments, receptors that downregulate the immune response are selected from, for example, PD-1, CTLA-4, Tim3, killer immunoglobulin-like receptors (KIR-e.g., KIR2DL1 (also known as CD158a), KIR2DL2 / DL3 (also known as CD158b)), CD94 or NKG2A (also known as CD159a), and protein tyrosine phosphatases (such as Src homology domain 2-domain phosphatases (SHP)-1 and SHP-2). Thus, such receptors can be targeted by partially LD-linked target-binding molecules as described herein.

[0063] Examples of cytokines that can be targeted by partially LD-linked target-binding molecules in various embodiments 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, for example, CD2, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD52, or CD127.

[0065] Methods for generating antibodies against any target protein and their antibody fragments are well known and common in the art. Furthermore, as illustrated herein, LD-linked target-binding molecules can be constructed using commercially available antibodies against various targets (e.g., CD3 and CD7), as illustrated herein. As illustrated 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 may be an endoplasmic reticulum (ER) retained sequence KDEL (SEQ ID NO: 4), or KKXX (SEQ ID NO: 9), KXD / E (SEQ ID NO: 10) (wherein X may 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 Other ER or Golgi-retaining sequences such as Immunother 46:55-60, 1998; for example, proteosome-targeting sequences containing the "PEST" motif - SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7); and / or sequences that target target-binding molecules to the cell membrane, such as the CD8α transmembrane domain or other single-pass transmembrane proteins 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). Figure 2 shows an example of a specific localization domain (sequence) illustrated herein. Various other localization sequences are known and available in the art.

[0067] As shown in Figure 2, the LD-linked target-binding molecule of the present invention may contain one or more localization domains. For example, the LD-linked target-binding molecule 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 jointly linked localization domains. When two or more localization domains are 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, a single LD-linked target-binding molecule may use the localization domains CD8™, the PEST motif, and EEKKMP. This particular construct exhibits localization domains without any intervening linkers, while 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 recognized by those skilled in the art, immunoactivating receptors and / or LD-linked target-binding molecules may be designed to bind to the targets disclosed herein and variants of the targets disclosed herein. For example, immunoactivating receptors and / or LD-linked target-binding molecules may be designed to bind to molecules in the CD3 / TCR complex or to 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, the variant may have altered function compared to the wild-type form of the molecule (e.g., conferring a disease state).

[0069] As will be recognized 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., containing different linkers, different localization 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 realm of those skilled in the art, as disclosed herein.

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

[0071] In another embodiment, the present invention relates to the use of engineered immune cells for treating cancer, comprising nucleic acids comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and nucleic acids comprising a nucleotide sequence encoding a single-chain variable fragment (scFv) linked to a localization domain, the use comprising administering a therapeutic dose of the engineered immune cells to a subject in need thereof.

[0072] In another embodiment, the present invention relates to the use of engineered immune cells for treating autoimmune disorders, comprising nucleic acids comprising a nucleotide sequence encoding an immune-activating receptor and nucleic acids comprising a nucleotide sequence encoding a target-binding molecule (e.g., scFv) linked to a localization domain, the use comprising administering a therapeutic dose of the engineered immune cells to a subject in need.

[0073] In other embodiments, the present invention also relates to the use of engineered immune cells for treating infectious diseases, comprising nucleic acids comprising a nucleotide sequence encoding an immune-activating receptor and nucleic acids comprising a nucleotide sequence encoding a target-binding molecule (e.g., scFv) linked to a localization domain, the use comprising administering a therapeutic dose 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-CD3ζCAR).

[0075] In other embodiments, the single-chain variable fragment (scFv) linked to the localization domain is selected from one or more constructs shown in Figure 2.

[0076] In some embodiments, the engineered immune cells are administered by injection into a subject. Methods of 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 the symptoms of the disease. Typically, a dosage of 10 7 -10 10 cells, e.g., a dosage of 10 9 cells, is injected in a single setting. The injection is administered as a single dose of 10 9 cells or is divided into several doses of 10 9 cells. The injection frequency can be once every 3 - 30 days or even at longer intervals if desired or indicated. The injection volume is generally at least one injection per subject and preferably at least three injections per subject if tolerated, or until the disease symptoms are ameliorated. The cells can be injected intravenously at a rate of 50 - 250 ml / hr. Other suitable modes of administration include intraarterial injection, direct injection into the tumor and / or perfusion of the tumor bed 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, myelodysplastic syndromes, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin and non-Hodgkin 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 tumors.

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

[0079] In certain embodiments, nucleic acids containing nucleotide sequences are introduced into immune cells in vitro. In other embodiments, nucleic acids containing nucleotide sequences are introduced into immune cells 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 introduced nucleotide sequence may be a single bicistronic construct containing an immunoactivating receptor as 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 the immunoactivating receptor (e.g., CAR) and the target-binding molecule (e.g., scFv) as described herein. Designing a tricistronic delivery system for deleting two or more targets would also be feasible. Alternatively, separate transduction (simultaneous or sequential) of the individual constructs (e.g., CAR and LD-linked target-binding molecule) may be performed. Methods for introducing exogenous nucleic acids are illustrated herein and are well known in the art.

[0082] Where used herein, unless explicitly stated otherwise, the indefinite articles “a” and “an” should be understood to mean “at least one.” [Examples]

[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 M-MLV reverse transcriptase (Promega, Madison, WI) and oligothymidine were used. 15cDNA was synthesized using primers (Promega). The variable regions of the heavy chain (VH) and light chain (VL) were amplified using the IgG Library Primer Set Mouse BioGenomics (US Biological, Salem, MA), and the PCR products were cloned into the TOPO TA cloning kit (Life Technologies) for sequencing. The VH and VL genes were assembled into scFv using splicing by overlapping extension PCR with a mobile linker sequence encoding (Gly4Ser)4. The signal peptide domain of CD8α was subcloned by PCR using cDNA derived from healthy donor human activated T cells and annexed to the 5' end of the VL fragment. The 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). A synthesized gene consisting of a CD8 signal peptide, scFv for 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 created, as listed in Figure 2.

[0084] The "PEST" sequence-SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7) motif, corresponding to amino acids 422-461 of mouse ornithine decarboxylase, was obtained from GenBank (acceptance number NM_013614.2). Codon optimization and gene synthesis were performed using GenScript (Piscataway, NJ), and the motif was subcloned to the 3' end of VH by PCR. The construct was then subcloned to the EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

[0085] Cloning of scFv against human CD7 The scFv sequence derived from mouse TH69 (anti-CD7) antibody was obtained from the literature (Peipp et al., Cancer Res 2002(62):2848-2855). After codon optimization, a synthesized gene consisting of a CD8 signal peptide, scFv for human CD7, 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 substituted with other sequences were also prepared, 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, a synthesized 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.

[0087] Cloning of scFv into human KIR2DL1 and KIR2DL2 / DL3 The amino acid sequences of the human monoclonal antibodies I-7F9 (anti-KIR2DL1, KIR2DL2, and KIR2DL3) were derived from the published international patent application WO2006003179A2 by Moretta et al. After codon optimization, the scFv sequence was designed by linking the variable light (VL) and variable heavy (VH) regions with a linker sequence. The synthesized gene, consisting of the CD8 signal peptide, scFv (KIR2DL1, KIR2DL2, and KIR2DL3) against human KIR, the CD8 hinge and transmembrane domain, and the KKMP sequence, was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP is substituted with other sequences were also prepared, 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 (EP2247619A1) by Spee et al. After codon optimization, the scFv sequence was designed by linking the variable light (VL) and variable heavy (VH) regions with a linker sequence. The synthesized gene, consisting of the CD8 signal peptide, scFv against human NKG2A, CD8 hinge and transmembrane, and KKMP sequence, was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP is substituted with other sequences were also prepared, as listed in Figure 2. The sequence information of the scFvs prepared herein is shown in Table 1. The sequence information of the various components depicted in Figure 2 is also shown in Table 2.

[0089] Anti-CD19-4-1BB-CD3ζCAR This CAR was constructed 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. Arrangement information of the 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] Genetic transduction, cell proliferation, flow cytometry analysis, and functional research These were carried out as previously described (Kudo, K et al., Cancer Res. 2014; 74(1): 93-103).

[0104] result Creating scFv structures An overview of this technology is illustrated in Figure 1. A schematic diagram of the repressive construct we have created is shown in Figure 2. The scFv portion can be derived from the cloning of cDNA encoding variable light (VL) and variable heavy (VH) immunoglobulin chain regions of antibody-producing hybridoma cell lines or corresponding publicly available sequences. VL and VH are linked by a short peptide sequence ("linker") according to standard techniques for constructing complete scFv. To be expressed, the scFv is linked to a signal peptide at its N-terminus, and as confirmed in preliminary experiments, the signal peptide is required for scFv expression. Proteins containing scFv plus the signal peptide are generally released into the cellular environment. For example, in preliminary experiments (not shown), the anti-CD3ε scFv plus signal peptide expressed in Jurcat T cells was detected in the cell culture supernatant. By orienting scFv to a specific compartment and preventing its secretion, potential effects on other cells are prevented. To orient it towards the endoplasmic reticulum (ER), we utilized the KDEL (SEQ ID NO: 4) motif (which holds proteins in the ER) (Strebe N. et al., J Immunol Methods. 2009; 341(1-2): 30-40). To promote the degradation of the targeted protein, we ligated it to the proteasome-targeting PEST motif (Joshi, SN et al., MAbs. 2012; 4(6): 686-693). scFv may also be oriented towards the cell membrane by ligating it to the transmembrane domain and the 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 scheme could be applied to the creation of immune cells expressing CARs and lacking one or more markers, T cell receptor (TCR) expression was downregulated in anti-CD19CAR 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 even one component prevents CD3 / TCR expression and therefore antigen recognition. In preliminary studies, we cloned scFv of an anti-CD3ε hybridoma (purchased from Creative Diagnostics, Shirley, NY) and constructed constructs containing KDEL (SEQ ID NO: 4), PEST, CD8α transmembrane domain, or others as shown in Figure 2.

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

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

[0109] Next, we determined whether anti-CD3scFv-myc KDEL could be expressed co-expressed with anti-CD19-4-1BB-CD3ζCAR. As shown in Figure 5, this resulted in T cells expressing anti-CD19CAR but lacking CD3 expression. TCRs were also absent on these cells (not shown).

[0110] To evaluate whether CAR can signal in Jurcutt cells with a downregulated CD3 / TCR, the expression of activation markers CD69 and CD25 was tested, and exocytosis of lytic granules was measured by CD107a expression in Jurcutt cells co-cultured with the CD19+ leukemia cell line OP-1. As shown in Figure 6, downregulation of the CD3 / TCR by the anti-CD3scFv-myc KDEL construct did not reduce the ability of anti-CD19-4-1BB-CD3ζCAR to activate Jurcutt cells. To further explore the effect of CD3 / TCR deletion on CAR signaling, it was determined whether CD3-negative T lymphocytes expressing CAR could be stimulated by their ligation. As shown in Figure 7, co-culturing T lymphocytes expressing anti-CD19CAR with CD19+ leukemia cells resulted in T cell proliferation regardless of whether CD3 was downregulated or not, indicating that CD3 / TCR downregulation did not reduce the stimulation of CAR proliferation.

[0111] Therefore, 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 Downward Control We determined whether the successful design for regulating CD3 / TCR expression could be applied to other surface molecules. For this purpose, we regulated CD7 expression. The scFv sequence was derived from that published by Peipp et al. (Cancer Res 2002 (62): 2848-2855), and this was ligated to the CD8 signal peptide and myc-KDEL sequence described in Figure 2. Using an MSCV retroviral vector, the anti-CD7-myc KDEL construct was transduced into peripheral blood lymphocytes exhibiting high CD7 expression, as detected by an anti-CD7 antibody coupled to phycoerythrin (BD Bioscience). As shown in Figure 8, CD7 in T lymphocytes transduced with this construct was substantially suppressed.

[0113] HLA-Class I Downward Control Subsequently, this plan was applied to downcontrol 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 leads to suppression of HLA (MHC in mice) class I expression (Koller, BH et al., Science. 1990;248(4960):1227-1230). Using scFv that reacts with β2-microglobulin, we suppressed HLA class I expression in immune cells.

[0115] The scFv sequence was derived from the one published by Grovender et al. (Kidney Int. 2004;65(1):310-322), and was ligated to the CD8 signal peptide and myc KDEL sequence, as described in Figure 2. The anti-β2M-myc KDEL construct was transduced into Jurcut cells exhibiting high HLA class I expression, as detected by phycoerythrin (BD Pharmingen)-coupled anti-HLA-ABC antibody, using an MSCV retroviral vector. As shown in Figure 9, Jurcut cells transduced with this construct exhibited substantial downregulation of HLA-ABC expression. The cells maintained their morphology and proliferative capacity.

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

[0117] To downregulate KIR receptors, we suppressed their expression in NK cells using scFv sequences that react with KIR2DL1 and KIR2DL2 / DL3. The scFv sequences were derived from those published by Moretta et al. (Patent WO2006 / 003179A2) and were ligated to the CD8 signal peptide and ER retention sequence described in Figure 2. Using an MSCV retroviral vector, this construct was transduced into NK cells selected for KIR2DL1 expression, which were grown from human peripheral blood. These cells also exhibited high KIR2DL1 expression, as detected by an anti-KIR2DL1 antibody coupled to allophycocyanin (R&D Systems), and high KIR2DL2 / DL3 expression, as detected by an anti-KIR2DL2 / DL3 antibody coupled to phycoerythrin (BD Bioscience). Figure 10 shows the results obtained with scFv-linker(20)AEKEDL and scFv-EEKKMP, along with substantial downregulation of the targeted KIR.

[0118] To downregulate NKG2A, its expression in NK cells was suppressed using scFv, which reacts with NKG2A. The scFv sequence derived from published European patent application EP2247619A1 by Spee et al. was ligated to the CD8 signal peptide and ER retention sequence described in Figure 2. This construct was transduced into NK cells grown from human peripheral blood with high NKG2A expression, as detected by phycoerythrin (Beckman Coulter)-coupled anti-NKG2A antibody, using an MSCV retroviral vector. Figure 11 shows the substantial downregulation of NKG2A achieved by scFv-EEKKMP.

[0119] All patents, published applications, and references cited herein are incorporated in their entirety by reference.

[0120] While the present invention is specifically shown and described with reference to its exemplary embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therein without departing from the scope of the invention as covered by the appended claims.

Claims

1. An in vitro method for generating manipulated immune cells, The method involves introducing into immune cells (i) a first nucleic acid comprising a first polynucleotide sequence encoding an immune activation receptor which includes a first binding domain which binds to a molecule expressed on the surface of cancer cells, and (ii) a second nucleic acid comprising a second polynucleotide sequence encoding a target-binding molecule linked to a localization domain, wherein the target-binding molecule linked to the localization domain comprises a second binding domain which binds to a target of the immune cell, and the target-binding molecule linked to the localization domain downregulates the expression of the target of the immune cell, thereby generating immune cells that have been manipulated. Compared to otherwise identical immune cells that contain the first nucleic acid but not the second, the engineered immune cells retain similar or higher levels of (i) expression of immune-activating receptors, (ii) expression of T-cell activation markers, (iii) T-cell proliferation, or (iv) any combination thereof. The second polynucleotide sequence comprises, from the 5' end to the 3' end, a first nucleotide sequence encoding a second binding domain, a second nucleotide sequence encoding a peptide linker, and a third nucleotide sequence encoding a localization domain, wherein the peptide linker comprises at least 5 amino acids. A method in which molecules expressed on the surface of cancer cells are identical to the targets of immune cells.

2. The in vitro method according to claim 1, wherein the localization domain includes an ER-retaining sequence, a Golgi-retaining sequence, or a PEST sequence.

3. The in vitro method according to claim 1, wherein the first polynucleotide sequence and the second polynucleotide sequence are located on the same nucleic acid molecule.

4. The in vitro method according to claim 3, further comprising an internal ribosome entry site (IRES) or a 2A peptide-coding region between a first polynucleotide sequence encoding an immune-activating receptor and a second polynucleotide sequence encoding a target-binding molecule linked to a localization domain.

5. The in vitro method according to claim 2, wherein the localization domain includes the ER-retaining sequence.

6. The in vitro method according to claim 5, wherein the ER retention sequence includes the KDEL amino acid sequence.

7. The in vitro method according to claim 5, wherein the ER retention sequence includes a KKXX amino acid sequence, where X represents any amino acid.

8. The in vitro method according to claim 2, wherein the localization domain includes the Golgi-retaining sequence.

9. The in vitro method according to claim 8, wherein the Golgi retention sequence includes the YQRL amino acid sequence.

10. The in vitro method according to claim 2, wherein the localization domain includes the PEST sequence.

11. The in vitro method according to claim 1, wherein the immune cell is a T cell.

12. The in vitro method according to claim 1, wherein the immune cells are natural killer (NK) cells.

13. The in vitro method according to claim 1, wherein the second binding domain is a single-chain binding domain.

14. The in vitro method according to claim 13, wherein the single-chain binding domain is a single-chain variable fragment (scFv).

15. The in vitro method according to claim 1, wherein the target binding molecule linked to the localization domain further comprises a transmembrane domain.

16. The in vitro method according to claim 15, wherein the transmembrane domain is a CD8 transmembrane domain.

17. The in vitro method according to claim 1, wherein the immune-activating receptor is a chimeric antigen receptor (CAR) comprising a first binding domain, a signaling domain, and a co-stimulation domain.

18. The in vitro method according to claim 1, wherein the first binding domain and the second binding domain have the same sequence.

19. The in vitro method according to claim 1, wherein the second binding domain and the first binding domain are different.

20. The in vitro method according to claim 17, wherein the signal transduction domain comprises a CD3ζ, Fc, DAP10, or DAP12 domain.

21. The simultaneous stimulation domains are the 4-1BB domain, the CD28 domain, and the CD28 domain. LL->GG The in vitro method according to claim 17, comprising a variant domain, an OX40 domain, an ICOS domain, a CD27 domain, a GITR domain, an HVEM domain, a TIM1 domain, an LFA1 domain, or a CD2 domain.

22. The in vitro method according to claim 1, wherein the second polynucleotide sequence further comprises a nucleotide sequence encoding a signal peptide.

23. The in vitro method according to claim 1, wherein the first binding domain is a first single-chain variable fragment (scFv) and the second binding domain is a second scFv.

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