Method for improving 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 Art] Immune cells can be powerful and specific "living drugs". Immune cells have the potential to target tumor cells while sparing normal tissues, and several clinical observations have shown that they can 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. 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 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 that bridge them to tumor cells (Topp MS, et al. J. Clin. Oncol. 2011; 29(18):2493-2498) has generated major clinical responses in patients with either solid tumors or leukemia. Finally, infusion of genetically modified autologous T lymphocytes has 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 immunotherapy by expanding its applicability and improving its effectiveness. SUMMARY OF THE INVENTION

[0003] The present invention relates to engineered immune cells having, for example, improved therapeutic efficacy for cancer treatment. In certain embodiments, the present invention provides engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoreceptor 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 for the use of engineered immune cells comprising a gene encoding an immunoreceptor and a gene encoding a target binding molecule linked to a localization domain for treating cancer, the use comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof.

[0005] In various embodiments, the present invention also provides a method for generating engineered immune cells, the method comprising introducing into the immune cells a nucleic acid comprising a nucleotide sequence encoding an immunoreceptor 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, the engineered immune cells have improved therapeutic efficacy as a result of one or more of reduced graft-versus-host disease (GvHD) in a host, reduced or eliminated rejection by a host, extended survival in a host, reduced suppression by tumors in a host, reduced apoptosis in a host, reduced inflammatory cascade in a host, or sustained natural / artificial receptor-mediated (e.g., CAR-mediated) signal transduction in a host.

[0007] The foregoing will become apparent from the following more specific description of exemplary embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts through different views. The drawings are not necessarily to scale, and instead, emphasis is placed on explaining embodiments of the invention.

Brief Description of the Drawings

[0008]

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Mode for Carrying Out the Invention

[0009] The description of exemplary embodiments of the present invention continues.

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

[0011] CAR is an artificial multi - molecular protein described previously (Geiger TL, et al., J Immunol. 1999;162(10):5931 - 5939; Brentjens RJ, et al., Nat Med. 2003;9(3):279 - 286; Cooper LJ, et al., Blood. 2003;101(4):1637 - 1644). CAR contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain that binds 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 can be derived from any desired source of such domains. Briefly, CAR can be designed to contain a single - chain variable region (scFv) of an antibody that specifically binds to a target. The scFv can be linked to a T - cell receptor (TCR) - associated signaling molecule such as CD3ζ via a transmembrane domain and a hinge domain. Ligation of the scFv to its cognate antigen causes signal transduction. Thus, CAR can instantaneously re - orient cytotoxic T lymphocytes towards cancer cells and induce lysis of tumor cells (Eshhar, Z. et al., PNAS. 1993;90(2):720 - 724; Geiger TL, et al., J Immunol. 1999;162(10):5931 - 5939; Brentjens RJ, et al., Nat Med. 2003;9(3):279 - 286; Cooper LJ, et al., Blood. 2003;101(4):1637 - 1644; Imai C, et al., Leukemia. 2004;18:676 - 684). Since CD3ζ signaling alone is not sufficient to continuously 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), co - stimulatory molecules such as CD28 and 4 - 1BB (or CD137) are incorporated into the CAR construct to boost signal transduction.This dual signaling design (the "second-generation CAR") is useful for eliciting effective anti-tumor activity from T cells. (Imai C, et al., Leukemia. 2004;18:676-684; Campana D, et al., Cancer J. 2014;20(2):134-140).

[0012] A specific CAR containing both 4-1BB and CD3ζ, the anti-CD19 CAR, is described in U.S. Patent No. 8,399,645. Infusion of autologous T cells expressing the anti-CD19-4-1BB-CD3ζ CAR has led to 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 studies with CARs bearing 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 regarding the clinical potential of this technology and for immunotherapy in general.

[0013] The methods described herein enable the rapid removal or inactivation of specific proteins in immune cells that are reoriented by natural or artificial receptors (e.g., CARs), thus expanding the potential uses of the engineered cells and significantly improving their function. The method partially relies on a single construct or multiple constructs containing an immunoreceptor, such as a CAR (including an extracellular domain (e.g., scFv) that binds to a specific target, a transmembrane domain, and a cytoplasmic domain), together with a target-binding molecule that binds to the target (e.g., protein) to be removed or neutralized. The target-binding molecule is linked to a domain that directs it to a specific cellular compartment (such as the Golgi or endoplasmic reticulum), the proteasome, or the cell membrane (i.e., a localization domain) depending on the use. For the sake of purification, the target-binding molecule linked to the localization domain (LD) may also be referred to herein as the "LD-linked target-binding molecule".

[0014] As will be apparent from the teachings herein, various immunoreceptors may be suitable for the methods of the invention. That is, any receptor that can activate an immune response upon binding (ligation) to a ligand (e.g., peptide or antigen) expressed on a cancer cell can be used according to the method. For example, as described above, the immunoreceptor may be a chimeric antigen receptor (CAR), and methods for designing and engineering CARs are known in the art (Geiger TL, et al., J Immunol. 1999;162(10):5931-5939; Brentjens See RJ, et al., Nat Med. 2003;9(3):279-286; Cooper LJ, et al., Blood. 2003;101(4):1637-1644). Further, receptors having 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 CARs but with the scFv replaced by an antibody-binding molecule (e.g., CD16, CD64, CD32). Further, in the context of tumor cell HLA, T cell receptors comprising the alpha and beta chains of the T cell receptor that bind to peptides expressed on tumor cells can also be used according to the present method. Further, other receptors carrying molecules that activate an immune response 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 cancer cell ligands". Thus, immune-activating receptors having molecules activated by cancer cell ligands can be expressed according to the present method together with the LD-linked target-binding molecule.

[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-encoding 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 available in a clinical-grade format. The ability to generate CD3 / TCR-negative cells that respond to CAR signaling represents an important advance. Clinical studies with CAR T cells are generally performed using autologous T cells. Thus, the quality of the cell product varies by patient, and responses are heterogeneous. Infusion of allogeneic T cells is currently not possible because of the unacceptably high risk of potentially fatal GvHD caused by stimulation of the endogenous TCR by the recipient's tissue antigens. Downregulation of CD3 / TCR opens the possibility of infusing allogeneic T cells because it eliminates the GvHD potential. Allogeneic products can be prepared with an optimal cell composition (e.g., enriched in highly cytotoxic T cells, depleted of regulatory T cells, etc.) and selected such that the infused cells have high CAR expression and functional efficacy. Furthermore, a fully standardized product 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 draws. Removal of TCR expression has been addressed using gene editing tools such as nucleases (Torikai H, et al. Blood, 2012;119(24):5697-5705). While this is an effective approach, it is difficult to implement in a clinical setting because it requires several rounds of cell selection and expansion by extended culture. The methods described herein have several practical advantages.

[0017] Furthermore, LD-linked target-binding molecules (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 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. 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. Hematolog y Am Soc Hematol Educ Program. 2013;2013:247-253). However, when allogeneic cells are infused, their persistence is limited. The immunosuppressive chemotherapy given to patients allows for transient engraftment of the infused NK cells, but these are rejected within 2 to 4 weeks of infusion (Miller JS, et al. Blood. 2005;105:3051-3057; Rubnitz JE, et al., J Clin Oncol. 2010;28(6):955-959). In contrast to organ transplantation, 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 infused NK cells (or T cells) will reduce or prevent the rejection rate and extend the survival of allogeneic cells and, therefore, their antitumor capacity.

[0018] Furthermore, LD-linked target-binding molecules can be used according to 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 generated 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 similar mechanisms. Thus, expression of target-binding molecules (e.g., scFv or ligands) against PD1, CTLA-4, Tim3, or other inhibitory receptors will prevent expression of these molecules (when linked to, e.g., KDEL (SEQ ID NO: 4), EEKKMP (SEQ ID NO: 64), or the PEST motif SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7)) or prevent their binding to their ligands on the receptors (when linked to a transmembrane domain) and will sustain 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 greater number of targets that are 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 that are not expressed in non-hematopoietic cells may be potential targets, but since they are also expressed on T cells and / or NK cells, they cannot be used as CAR targets. Expression of such CARs on immune cells would result in the elimination of the immune cells themselves by the "kill-siblings" mechanism, which may render their anti-cancer ability ineffective. 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, 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 the secretion of cytokines by activated immune cells causes cytokine release syndrome and macrophage activation syndrome, presenting serious adverse effects of immune cell therapy (Lee DW, et al., Blood. 2014;124(2):188-195). Thus, 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 engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoreceptor and a nucleic acid comprising a nucleotide sequence encoding a target binding molecule linked to a localization domain.

[0022] As used herein, an “engineered” immune cell includes an immune cell that has been genetically modified as compared to a naturally occurring immune cell. For example, an engineered T cell generated according to the methods herein carries a nucleic acid comprising a nucleotide sequence that is not naturally present in the T cell from which it is derived. In some embodiments, the 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 particular embodiment, the 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, an "immune activation receptor" refers to a receptor that activates an immune response upon binding to a cancer cell ligand. In some embodiments, an immune activation receptor includes 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 activation receptor is a chimeric antigen receptor (CAR), and methods for designing and engineering CARs are known in the art. In other embodiments, the immune activation receptor is an antibody-binding receptor, which 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, in the context of tumor cell HLA, a T cell receptor comprising an alpha and a beta chain that binds to a peptide expressed on a tumor cell can also be used according to the methods described herein. In certain embodiments, other receptors that carry a molecule that activates an immune response by binding to a ligand expressed on a cancer cell (e.g., the NKG2D-DAP10-CD3 zeta receptor that binds to an NKG2D ligand expressed on a tumor cell) 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 an immune response upon binding (ligation) to a ligand (e.g., a peptide or antigen) expressed on a cancer cell are collectively referred to as "immune activation receptors." As will be appreciated by those skilled in the art, an immune activation receptor need not contain an antibody or antigen-binding fragment (e.g., scFv), rather, the portion of the immune activation receptor that binds to the target molecule can be derived, for example, from the receptor in a receptor-ligand pair or the ligand in a receptor-ligand pair.

[0025] In certain embodiments, the immune activation 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 activation receptor is a CAR (e.g., anti-CD19-4-1BB-CD3ζ CAR). In certain embodiments, the immune activation receptor is 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, and includes an antibody or antigen-binding fragment thereof (e.g., scFv) that binds to a molecule expressed on the surface of a tumor cell, including but not limited to these. 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 illustrated herein, such antibodies and antibody fragments derived therefrom (e.g., scFv) can be used in the present invention. Furthermore, methods for generating antibodies and antibody fragments against target proteins are well known and conventional in the art.

[0026] The transmembrane domain of the immune activation receptor (e.g., CAR) according to the present invention may be derived from a single-pass transmembrane 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 hydrophobic protein segment that does not occur naturally.

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

[0028] The signaling domain of an immunoreceptor (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 is 4-1BB (also known as CD137), CD28, CD28 LL→GG Variants, costimulatory molecules such as OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1, or CD2. Such molecules are readily available and known in the art.

[0029] As will be appreciated by those skilled in the art, the components of the immune activation receptor can be engineered to include several functional combinations as described herein to produce the desired results. Using a specific CAR anti-CD19-4-1BB-CD3ζ as an example, as described herein, the antibody (e.g., or its antigen-binding fragment such as scFv) that binds to the molecule may be replaced 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 co-stimulatory molecule (4-1BB in this specific example) may also differ by a different co-stimulatory molecule (e.g., CD28). In some embodiments, the stimulatory molecule (CD3ζ in this specific example) may be replaced with another known stimulatory molecule. In various embodiments, the transmembrane domain of the receptor may also differ if desired. The design, generation, and testing of the functionality of such immune activation receptors can be readily determined by those skilled in the art. Similarly, the design, delivery to cells, and expression of nucleic acids encoding such immune activation receptors are readily known and available in the art.

[0030] 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. Further, nucleic acid molecules may be single-stranded, double-stranded, or triple-stranded. In some embodiments, the nucleic acid molecule may be modified. In the case of a double-stranded polymer, "nucleic acid" may refer to either or both strands of the molecule.

[0031] In connection with nucleic acids, the term "nucleotide sequence" refers to a series of adjacent nucleotides joined by covalent bonds such as phosphorous linkages (e.g., phosphodiester bonds, alkyl and aryl-phosphonate bonds, phosphorothioate bonds, phosphotriester bonds) and / or non-phosphorous linkages (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 of a different species or cell type).

[0032] The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as derivatives and analogs thereof that do not occur naturally. Thus, nucleotides can 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.

[0033] As will be recognized by those skilled in the art, in some embodiments, a nucleic acid further includes a plasmid sequence. The plasmid sequence can include, for example, one or more sequences selected from the group consisting of a promoter sequence, a selectable marker sequence, and a locus targeting sequence.

[0034] 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".

[0035] In certain embodiments, the target-binding molecule is an antibody or an antigen-binding fragment thereof. As used herein, "antibody" means an intact antibody or an antigen-binding fragment of an antibody, which may be modified or engineered, or is a human antibody, including an intact antibody or an antigen-binding fragment thereof. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, multi-paratopic antibodies (e.g., bispecific 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.

[0036] A "Fab fragment" contains one light chain, and C H 1 and the variable region of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0037] The "Fc" region contains two heavy chain fragments that include 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.

[0038] A "Fab′ fragment" contains one light chain, as well as a portion of one heavy chain that also contains the VH domain and the CH1 domain and the region between the CH1 domain and the CH2 domain such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule.

[0039] An "F(ab′)2 fragment" contains two light chains, as well as two heavy chains that contain a portion of the constant region between the C H 1 domain and the C H 2 domain. Thus, an F(ab′)2 fragment is composed of two Fab′ fragments held together by an interchain disulfide bond between the two heavy chains.

[0040] The "Fv region" contains variable regions derived from both the heavy and light chains, but lacks constant regions.

[0041] In certain embodiments, the target-binding molecule is a single-chain Fv antibody ("scFv antibody"). An scFv refers to an antibody fragment that contains the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the structure desired for antigen binding. For an overview of scFv, see Pluckthun (1994) The Pharmacology Of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315. See also PCT Publication No. WO88 / 01649, as well as U.S. Patent Nos. 4,946,778 and 5,260,203. As an example, the linker between the VH domain and the VL domain of the scFv disclosed herein includes, 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 can be designed and tested for optimal function as provided in the art and as disclosed herein.

[0042] The scFv, which is part of the LD-linked target-binding molecule, is not necessarily the same as the scFv that occurs, for example, in the context of a chimeric antigen receptor (CAR) or a similar antibody-binding signaling receptor. In some embodiments, the scFv that is part of the LD-linked target-binding molecule is the same as the scFv that occurs, for example, in the context of a chimeric antigen receptor (CAR) or a similar antibody-binding signaling receptor.

[0043] 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.

[0044] The term "sequence identity" means that two nucleotide or amino acid sequences 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 when optimally aligned by a program such as GAP or BESTFIT that uses a default gap weight. For sequence comparison, typically one sequence serves as a reference sequence (e.g., a parental sequence) against which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, partial sequence coordinates are specified as necessary, and the sequence algorithm program parameters are specified. Thereafter, the sequence comparison algorithm calculates the percent sequence identity of the test sequence(s) to the reference sequence based on the specified program parameters.

[0045] Optimal alignment of arrays 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 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 (generally, see Ausubel et al., Current Protocols in Molecular Biology). An example of an algorithm 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 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 comparisons can be performed using the default program parameters, but customized parameters may also be used. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 score matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0046] In certain embodiments, the antibody (e.g., scFv) comprises a VH and a VL having amino acid sequences defined by SEQ ID NO: 12 and 13, respectively, SEQ ID NO: 16 and 17, respectively, SEQ ID NO: 20 and 21, respectively, SEQ ID NO: 24 and 25, respectively, SEQ ID NO: 28 and 29, respectively, SEQ ID NO: 32 and 33, respectively, or SEQ ID NO: 36 and 37, respectively. In some embodiments, the antibody (e.g., scFv) has 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 defined by SEQ ID NO: 12 and 13, respectively, SEQ ID NO: 16 and 17, respectively, SEQ ID NO: 20 and 21, respectively, SEQ ID NO: 24 and 25, respectively, SEQ ID NO: 28 and 29, respectively, SEQ ID NO: 32 and 33, respectively, or SEQ ID NO: 36 and 37, respectively.

[0047] 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) (VH-VL or VL-VH) in the same polypeptide chain. 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 on another chain and create two antigen-binding sites. Diabodies are described, for example, in the patent documents EP404,097, WO93 / 11161, and Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0048] In certain embodiments, the antibody is a tribody or a tetrabody. Methods for designing and generating tribodies and tetrabodies are known in the art. For example, Todorovska et al., J. Immunol. Methods 24 See 8(1-2):47-66,2001.

[0049] 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 examples, 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.

[0050] In some embodiments, the antibody is modified or engineered. Examples of modified or engineered antibodies include chimeric antibodies, multi-paratopic antibodies (e.g., bis-paratopic antibodies), and multi-specific antibodies (e.g., bispecific antibodies).

[0051] As used herein, a "multi-paratopic antibody" means an antibody comprising at least two single domain antibodies, wherein at least one of the single domain antibodies is oriented against a first antigenic determinant on an antigen and at least one other single domain antibody is oriented against a second antigenic determinant on the same antigen. Thus, for example, a "bis-paratopic" antibody comprises at least one single domain antibody oriented against a first antigenic determinant on an antigen and at least one further single domain antibody oriented against a second antigenic determinant on the same antigen.

[0052] As used herein, a "multi-specific antibody" means an antibody comprising at least two single domain antibodies, wherein at least one of the single domain antibodies is oriented against a first antigen and at least one other single domain antibody is oriented 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 oriented against a first antigen and at least one further single domain antibody oriented against, for example, a second antigen different from the first antigen.

[0053] In some embodiments, the antibodies disclosed herein are monoclonal antibodies, e.g., mouse monoclonal antibodies. Methods for generating monoclonal antibodies are known in the art. See, e.g., Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315.

[0054] 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 the target molecule.

[0055] As used herein, "linked" in the context of an LD-linked target binding molecule refers to a gene encoding a target binding molecule that is directly (e.g., without a linker) within the reading frame adjacent to 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. Various suitable linkers known in the art can be used to tether the target binding molecule to the localization domain. For example, unnatural peptides such as polypeptides consisting of hydrophilic residues of various lengths, or (GGGGS) n (SEQ ID NO: 8) polypeptide (n is an integer from, for example, 3 to 12 (including these)) may be used according to 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 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 at least 5 amino acid length, at least 10 amino acid length, at least 15 amino acid length, at least 20 amino acid length, at least 25 amino acid length, at least 30 amino acid length, at least 35 amino acid length, 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 incorporating linker sequences and methods for evaluating functionality are readily available to those skilled in the art.

[0056] 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 suppresses the activity or function of a target molecule. As an example, as disclosed herein, the LD-linked target binding molecule may be designed to 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 molecules 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).

[0057] In some embodiments, the engineered immune cells of the invention have improved therapeutic efficacy. As used herein, "improved therapeutic efficacy" refers to one or more of a reduction in graft-versus-host disease (GvHD) in a host, a reduction or elimination of rejection by the host, an extension of survival in the host, a reduction in suppression by a tumor in the host, a reduction in apoptosis in the host, a reduction in an inflammatory cascade in the host, or a persistence of CAR-mediated signal transduction in the host.

[0058] 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.

[0059] 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.

[0060] In another embodiment, the HLA class I molecule is beta-2 microglobulin, alpha1-microglobulin, alpha2-microglobulin, or alpha3-microglobulin.

[0061] In other embodiments, the receptor that downregulates the immune response is selected from, for example, PD-1, CTLA-4, Tim3, killer immunoglobulin-like receptor (KIR - e.g., KIR2DL1 (also known as CD158a), KIR2DL2 / DL3 (also known as CD158b)), CD94 or NKG2A (also known as CD159a), protein tyrosine phosphatase (such as Src homology region 2 domain-containing phosphatase (SHP)-1 and SHP-2). Thus, such receptors can be targeted by the partial LD-linked target binding molecule as described herein.

[0062] In various embodiments, examples of cytokines that can be targeted by a partial 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.

[0063] In a further aspect, 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.

[0064] Methods for generating antibodies and their antibody fragments against any target protein are well known and conventional in the art. Further, 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.

[0065] 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) (wherein X can be any amino acid - Gao C, et al., Trends (see, e.g., in Plant Science 19:508-515, 2014), and other ER or Golgi retention sequences such as YQRL (SEQ ID NO: 11) (see, e.g., Zhan J, et al., Cancer Immunol Immunother 46:55-60, 1998); for example, a proteasome targeting sequence containing the "PEST" motif - SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7); and / or a CD8α transmembrane domain, or a transmembrane targeting sequence of another single-pass transmembrane 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 FIG. 2. Various other localization sequences are known and available in the art.

[0066] As shown in FIG. 2, the LD-linked target binding molecule of the present invention may include one or more localization domains. For example, the LD-linked target binding molecule may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 localization domains linked together. In a given LD-linked target binding molecule, when two or more localization domains are used, each localization domain can be linked with or without any intervening linker. As an example, as shown in FIG. 2, in a single LD-linked target binding molecule, the localization domains CD8TM, the PEST motif, and EEKKMP can be used. This particular construct shows localization domains without any intervening linker, while various intervening linkers may be incorporated between some or all of the localization domains. Other examples are shown in FIG. 2.

[0067] As will be appreciated by those skilled in the art, the immunoreceptor and / or LD-linked target binding molecule may be designed to bind to the targets disclosed herein and variants of the targets disclosed herein. As an example, the immunoreceptor and / or LD-linked target binding molecule may be designed to bind to a molecule in the CD3 / TCR complex or a naturally occurring variant molecule thereof. Such a naturally occurring variant may have the same function as the wild-type form of the molecule. In other embodiments, the variant may have an altered function compared to the wild-type form of the molecule (e.g., confer a disease state).

[0068] As will be appreciated by those skilled in the art, the various components of the LD-linked target binding molecule construct 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 results in a functional LD-linked target binding molecule. Methods for assessing the functionality of a particular construct are within the purview of those skilled in the art disclosed herein.

[0069] In a further aspect, the invention relates to the use of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoreceptor for treating cancer 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 therapeutically effective amount of the engineered immune cells to a subject in need thereof.

[0070] In another aspect, the invention relates to the use of engineered immune cells comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) for treating cancer and a nucleic acid comprising a nucleotide sequence encoding a single-chain variable fragment (scFv) linked to a localization domain, the use comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof.

[0071] In other aspects, the present invention relates to the use of engineered immune cells comprising a nucleic acid encoding an immune activation receptor and a nucleic acid encoding a target binding molecule (e.g., scFv) linked to a localization domain for treating autoimmune disorders, the use comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof.

[0072] In other aspects, the present invention also relates to the use of engineered immune cells comprising a nucleic acid encoding an immune activation receptor and a nucleic acid encoding a target binding molecule (e.g., scFv) linked to a localization domain for treating infectious diseases, the use comprising administering a therapeutically effective amount of the engineered immune cells to a subject in need thereof.

[0073] In various embodiments, the immune activation receptor is a CAR (e.g., anti-CD19-4-1BB-CD3ζ CAR).

[0074] 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 Figure 2.

[0075] In some aspects, the engineered immune cells are administered by injection into the 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 alleviate the symptoms of the disease. Typically, a drug dose of 10 7 ~10 10 cells, e.g., a drug dose of 10 9 cells, is injected in a single setting. The injection is administered as a single dose of 10 9 cells or as several doses of 10 9It is divided into dosages for individual cells. The injection frequency can be once every 3 to 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 if permitted, or until the disease symptoms are alleviated. The cells can be intravenously injected at a rate of 50 to 250 ml / hour. Other suitable modes of administration include intra-arterial injection, direct injection into the tumor and / or perfusion of the tumor bed after surgery, transplantation 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. The speed can be intravenously injected. Other suitable modes of administration include intra-arterial injection, direct injection into the tumor and / or perfusion of the tumor bed after surgery, transplantation 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.

[0076] 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 syndrome, 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.

[0077] In another embodiment, the present invention relates to a method for generating engineered immune cells of the present invention, which comprises introducing into immune cells a nucleic acid comprising a nucleotide sequence encoding an immunoreceptor and a nucleic acid comprising a nucleotide sequence encoding a target-binding molecule linked to a localization domain, thereby generating engineered immune cells.

[0078] In certain embodiments, the nucleic acid comprising the nucleotide sequence is introduced into immune cells in vitro. In other embodiments, the nucleic acid comprising the nucleotide sequence is introduced into immune cells in vivo.

[0079] In some embodiments, "immune cells" include, for example, T cells, natural killer (NK) cells, NK / T cells, monocytes, macrophages, or dendritic cells.

[0080] The nucleic acid containing the nucleotide sequence to be introduced may be a single bicistronic construct containing an immunoreceptor 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 site between two cDNAs encoding an immunoreceptor (e.g., CAR) and a target-binding molecule (e.g., scFv) described herein. The design of a tricistronic delivery system for eliminating two or more targets may also be feasible. Alternatively, separate transduction of individual constructs (e.g., CAR and LD-linked target-binding molecule) (simultaneously or sequentially) may be performed. Methods for introducing foreign nucleic acids are exemplified herein and are well known in the art.

[0081] As used herein, unless expressly stated to the contrary, the indefinite articles "a" and "an" are to be understood to mean "at least one".

Examples

[0082] Illustration Method

[0083] Cloning of scFv from a mouse anti-human CD3 hybridoma

[0084] PLU4 hybridoma cells (IgG2a isotype, Creative Diagnostics, Shirley, NY) secreting an anti-human CD3 monoclonal antibody were cultured in IMDM plus GlutaMAX medium (Life Technologies, Carlsbad, CA) with 20% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and antibiotics. TRIzol Total RNA was extracted using the reagent (Life Technologies), and cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI) and oligo thymidine 15 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 for Sequencing (Life Technologies). The VH and VL genes were assembled into scFv using splicing by overlap extension PCR with a flexible linker sequence encoding (Gly4Ser)4. The signal peptide domain of CD8α was subcloned by PCR using cDNA from healthy donor human activated T cells and connected 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 by PCR using the sense primer: 5′-ATATATGAATTCGGCTTCCACCATGGCCTTACCAGTGACC-3′ (SEQ ID NO: 5) and the reverse primer: 5′-TATATACTCGAGTTACAACTCGTCCTTCAGATCTTCTTCAGAAATAAG-3′ (SEQ ID NO: 6). The synthetic 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 generated, as listed in Figure 2.

[0085] The "PEST" sequence - SHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 7) motif corresponding to amino acids 422 - 461 of mouse ornithine decarboxylase was obtained from GenBank (accession number NM_013614.2). It was codon-optimized and gene-synthesized by GenScript (Piscataway, NJ), and subcloned to the 3' end of VH by PCR. The construct was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector.

[0086] Cloning of scFv against human CD7

[0087] The sequence scFv derived from the mouse TH69 (anti-CD7) antibody was obtained from the literature (Peipp et al., Cancer Res 2002(62):2848 - 2855). After codon optimization, the synthesized gene consisting of the CD8 signal peptide, scFv against human CD7, Myc tag, and KDEL (SEQ ID NO: 4) sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. As listed in Figure 2, constructs in which myc-KDEL was replaced by other sequences were also prepared.

[0088] Cloning of scFv against human beta-2 microglobulin (hB2MG)

[0089] The sequence scFv derived from the mouse BBM.1 (anti-hB2MG) IgG2b antibody was obtained from the literature (Grovender, E.A. et al., Kidney Int. 2004;65(1):310 - 322). After codon optimization, the synthesized gene consisting of the CD8 signal peptide, scFv against 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.

[0090] Cloning of scFv against human KIR2DL1 and KIR2DL2 / DL3

[0091] The amino acid sequence of the human monoclonal antibody I-7F9 (against KIR2DL1, KIR2DL2, and KIR2DL3) was derived from the published international patent application No. WO2006003179A2 by Moretta et al. After codon optimization, the sequence of the single-chain variable fragment (scFv) was designed by connecting the variable light (VL) region and the variable heavy (VH) region with a linker sequence. A synthetic gene consisting of a CD8 signal peptide, the scFv against human KIR (KIR2DL1, KIR2DL2, and KIR2DL3), 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 was replaced by other sequences were also generated, as listed in Figure 2.

[0092] Cloning of the scFv against human NKG2A

[0093] The sequence of the mouse antibody Z199 (against NKG2A) was derived from the published patent (EP2247619A1) by Spee et al. After codon optimization, the sequence of the scFv was designed by connecting the VL region and the VH region with a linker sequence. A synthetic gene consisting of a CD8 signal peptide, the scFv against human NKG2A, the CD8 hinge and transmembrane, and the KKMP sequence was subcloned into the EcoRI and XhoI sites of the MSCV-IRES-GFP vector. Constructs in which KKMP was replaced 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.

[0094] Anti-CD19-4-1BB-CD3ζ CAR

[0095] 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).

[0096] Table 1. scFv Array Information

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

[0097] Table 2. Array Information of the Components Depicted in Figure 2

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

[0098] Gene transfection, cell proliferation, flow cytometry analysis, and functional studies

[0099] These were performed as previously described (Kudo, K et al., Cancer Res. 2014;74(1):93-103).

[0100] Results

[0101] Generation of scFv constructs

[0102] The outline of this technique is described in Figure 1. A schematic diagram of the inhibitory constructs we generated is shown in Figure 2 Yes. The scFv portion can be derived from the cloning of cDNA encoding the variable light (VL) and variable heavy (VH) immunoglobulin chain regions of an antibody-producing hybridoma cell line or the corresponding published sequence. The VL and VH are linked by a short peptide sequence ("linker") according to standard techniques for generating a full scFv. For expression, the scFv is linked to a signal peptide at the N-terminus, and as confirmed in preliminary experiments, the signal peptide is required for the scFv to be expressed. Proteins containing the scFv plus the signal peptide are generally released into the cellular environment. For example, in preliminary experiments (not shown), an anti-CD3ε scFv plus signal peptide expressed in Jurkat T cells was detected in the cell culture supernatant. By orienting the scFv to a specific compartment and preventing its secretion, potential effects on other cells are prevented. To orient it to the endoplasmic reticulum (ER), the KDEL (SEQ ID NO: 4) motif (which retains proteins in the ER) was utilized (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, S.N. et al., MAbs. 2012;4(6):686-693). The scFv may also be oriented to the cell membrane by linking it to a transmembrane domain and the hinge of CD8α or another transmembrane protein.

[0103] Downregulation of the T cell receptor in T lymphocytes expressing the anti-CD19-BB-ζ CAR

[0104] To determine whether the proposed protocol can be applied to generate 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.

[0105] For expression on the cell membrane, the CD3 / TCR complex requires the assembly of all its components (TCRα, TCRβ, CD3δ, CD3ε, CD3γ, CD3ζ). The absence of a single component prevents CD3 / TCR expression and thus antigen recognition. In preliminary studies, the scFv of an anti-CD3ε hybridoma (purchased from Creative Diagnostics, Shirley, NY) was cloned, and constructs containing KDEL (SEQ ID NO: 4), PEST, the CD8α transmembrane domain, or others shown in Figure 2 were generated.

[0106] Using a murine stem cell virus (MSCV) retroviral vector containing green fluorescent protein (GFP), the constructs disclosed herein were transduced in CD3 / TCR+ Jurkat cell lines. 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 measured by flow cytometry. Antibody staining of CD3ε decreased to varying degrees in cells transduced with the listed constructs. Similar downregulation of CD3ε was obtained with human peripheral blood T lymphocytes (Figure 3B). Figure 3C shows an illustrative flow cytometry dot plot of CD3ε expression in GFP-positive Jurkat 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 Jurkat cells or the expression of all other cell markers tested (including CD2, CD4, CD8, CD45, CD25, CD69). Suppression of CD3 expression persisted for over 3 months. Further enrichment of CD3-negative cells can be achieved by depletion of CD3+ T cells with anti-CD3 magnetic beads (Dynal, Life Technologies, Carlsbad, CA).

[0107] 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 (Figure 4).

[0108] Next, it was determined whether anti-CD3scFv-myc KDEL could be co-expressed with anti-CD19-4-1BB-CD3ζ CAR. As shown in Figure 5, this resulted in T cells that expressed anti-CD19 CAR while lacking CD3 expression. On these cells, the TCR was also absent (not shown).

[0109] To evaluate whether the CAR could signal in Jurkat cells with downregulated CD3 / TCR, the expression of activation markers CD69 and CD25 was tested, and exocytosis of lytic granules was measured by CD107a expression in Jurkat cells co-cultured with the CD19+ leukemia cell line OP-1. As shown in Figure 6, downregulation of CD3 / TCR by the anti-CD3scFv-myc KDEL construct did not reduce the ability of the anti-CD19-4-1BB-CD3ζ CAR to activate Jurkat cells. To further explore the effect of CD3 / TCR ablation on CAR signaling, it was determined whether CD3-negative T lymphocytes expressing the CAR could be stimulated by its ligation. As shown in Figure 7, co-culturing T lymphocytes expressing anti-CD19 CAR with CD19+ leukemia cells resulted in T cell proliferation regardless of whether CD3 was downregulated, indicating that CD3 / TCR downregulation did not reduce CAR proliferation stimulation.

[0110] Therefore, the anti-CD3scFv-myc KDEL construct can effectively downregulate CD3 / TCR in CAR-T cells without affecting CAR-driven T cell activation, degranulation, and proliferation.

[0111] Downregulation of CD7

[0112] We determined whether the plan that had succeeded in 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 the MSCV retroviral vector, the anti - CD7 - myc KDEL construct was transduced into peripheral blood lymphocytes with high CD7 expression, which was detected by an anti - CD7 antibody conjugated to phycoerythrin (BD Bioscience). As shown in Figure 8, CD7 in T lymphocytes transduced with this construct was substantially suppressed.

[0113] Down - regulation of HLA - class I

[0114] Subsequently, we applied this plan to down - regulate another surface molecule, HLA class I.

[0115] 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 an scFv that reacts with β2 - microglobulin, we suppressed the expression of HLA class I in immune cells.

[0116] The scFv sequence was derived from that published by Grovender et al. (Kidney Int. 2004;65(1):310 - 322), and this was ligated to the CD8 signal peptide and myc KDEL sequence described in Figure 2. Using the MSCV retroviral vector, the anti - β2M - myc KDEL construct was transduced into Jurkat cells with high HLA class I expression, which was detected by an anti - HLA - ABC antibody conjugated to phycoerythrin (BD Pharmingen). As shown in Figure 9, this construct... The jackknife cells transfected in the building had substantial downregulation of HLA-ABC expression. The cells maintained their morphology and proliferative capacity.

[0117] Downregulation of inhibitory receptors in NK cells

[0118] To determine whether the scheme described above also applies to surface molecules expressed in other immune cells, the downregulation of the functions of the inhibitory receptors KIR2DL1, KIR2DL2 / DL3, and NKG2A was tested in NK cells.

[0119] To downregulate the KIR receptor, their expression in NK cells was suppressed using scFv that reacts with KIR2DL1 and KIR2DL2 / DL3. The scFv sequences were derived from those published by Moretta et al. (Patent WO2006 / 003179A2), and this was ligated to the CD8 signal peptide and ER retention sequence described in Figure 2. Using the MSCV retroviral vector, this construct was transfected into NK cells grown from human peripheral blood and selected for KIR2DL1 expression. These cells also had high KIR2DL1 expression detected by an anti-KIR2DL1 antibody conjugated to allophycocyanin (R&D Systems) and high KIR2DL2 / DL3 expression detected by an anti-KIR2DL2 / DL3 antibody conjugated to phycoerythrin (BD Bioscience). Figure 10 shows the results obtained with scFv-linker (20)AEKEDL and scFv-EEKKMP with substantial downregulation of the targeted KIR.

[0120] To downregulate NKG2A, its expression in NK cells was suppressed using an scFv that reacts with NKG2A. The scFv sequence derived from the 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 expanded from human peripheral blood with high NKG2A expression, detected by an anti-NKG2A antibody conjugated to phycoerythrin (Beckman Coulter), using an MSCV retroviral vector. Figure 11 shows the substantial downregulation of NKG2A obtained by scFv-EEKKMP.

[0121] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0122] While the invention has been specifically shown and described with reference to its exemplary embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. An isolated and engineered immune cell that expresses a chimeric antigen receptor (CAR) on the cell surface, wherein the engineered immune cell comprises (i) a polynucleotide encoding a CAR comprising a CD7 binding domain, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain, and (ii) a polynucleotide encoding an engineered polypeptide comprising a second CD7 binding domain linked to a localization domain that downregulates surface expression of endogenous CD7, wherein the CD7 binding domain is a single-chain variable region (scFv) that specifically binds to CD7, a) a variable heavy chain sequence having at least 95% sequence identity with SEQ ID NO: 16, wherein the heavy chain (HC) complementarity-determining region (CDR) 1, HC CDR2, and HC CDR3 are identical to the HC CDR1, HC CDR2, and HC CDR3 of the amino acid sequence of SEQ ID NO: 16, a variable heavy chain sequence, and a variable light chain sequence having at least 95% sequence identity with SEQ ID NO: 17, wherein the light chain (LC) CDR1, LC CDR2, and LC CDR3 are identical to the LC CDR1, LC CDR2, and LC CDR3 of the amino acid sequence of SEQ ID NO: 17, a variable light chain sequence, or b) a variable heavy chain sequence having at least 95% sequence identity with SEQ ID NO: 20, wherein the HC CDR1, HC CDR2, and HC CDR3 are identical to the HC CDR1, HC CDR2, and HC CDR3 of the amino acid sequence of SEQ ID NO: 20, a variable heavy chain sequence, and a variable light chain sequence having at least 95% sequence identity with SEQ ID NO: 21, wherein the LC CDR1, LC CDR2, and LC CDR3 are identical to the LC CDR1, LC CDR2, and LC CDR3 of the amino acid sequence of SEQ ID NO: 21, a variable light chain sequence, comprising an scFv containing the same, wherein the signaling domain is a CD3ζ signaling domain, and wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain or a CD28 co-stimulatory domain, wherein the second CD7 binding domain comprises a second scFv that specifically binds to CD7, wherein the localization domain comprises an ER retention sequence, a Golgi retention sequence, or a proteasome localization sequence, When the ER retention sequence contains the amino acid sequence KDEL, the isolated and engineered immune cell in which the polypeptide that downregulates the surface expression of endogenous CD7 further includes a linker sequence between the second CD7 binding domain and the localization domain. **Claim 2** The isolated and engineered immune cell according to claim 1, wherein the transmembrane domain is selected from the group consisting of CD8α, CD8β, 4-1BB, CD28, CD34, CD4, FcεRIγ, OX40, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, and FGFR2B. **Claim 3** The isolated and engineered immune cell according to claim 1 or 2, wherein the engineered immune cell is an engineered T cell or an engineered natural killer (NK) cell. **Claim 4** The isolated and engineered immune cell according to any one of claims 1 to 3, wherein the second CD7 binding domain includes the HC CDR1, the HC CDR2, the HC CDR3, the LC CDR1, the LC CDR2, and the LC CDR3 of the CD7 binding domain of the CAR. **Claim 5** The isolated and engineered immune cell according to any one of claims 1 to 4, wherein the ER retention sequence contains the amino acid sequence KDEL (SEQ ID NO: 4), KKXX (SEQ ID NO: 9), or KXD / E (SEQ ID NO: 10), wherein X is any amino acid. **Claim 6** The isolated and engineered immune cell according to any one of claims 1 to 5, wherein the Golgi retention sequence contains the amino acid sequence YQRL (SEQ ID NO: 11), or the proteasome localization sequence contains a PEST motif. **Claim 7** An isolated and engineered immune cell comprising a polynucleotide encoding an engineered polypeptide comprising a CD7 binding domain linked to a localization domain that downregulates the surface expression of endogenous CD7, wherein the CD7 binding domain includes a single-chain variable region (scFv) that specifically binds to CD7, the localization domain includes an ER retention sequence, a Golgi retention sequence, or a proteasome localization sequence, When the ER retention sequence contains the amino acid sequence KDEL, the isolated and engineered immune cell in which the polypeptide that downregulates the surface expression of endogenous CD7 further includes a linker sequence between the CD7 binding domain and the localization domain. **Claim 8** The CD7 binding domain is a) A variable heavy chain sequence having at least 95% identity with SEQ ID NO: 16, wherein the heavy chain (HC) complementarity-determining region (CDR) 1, HC CDR2, and HC CDR3 are identical to the HC CDR1, HC CDR2, and HC CDR3 of the amino acid sequence of SEQ ID NO: 16, and a variable heavy chain sequence, a variable light chain sequence having at least 95% identity with SEQ ID NO: 17, wherein the light chain (LC) complementarity-determining region (CDR) 1, LC CDR2, and LC CDR3 are identical to the LC CDR1, LC CDR2, and LC CDR3 of the amino acid sequence of SEQ ID NO: 17, and a variable light chain sequence, or b) A variable heavy chain sequence having at least 95% identity with SEQ ID NO: 20, wherein the heavy chain (HC) complementarity-determining region (CDR) 1, HC CDR2, and HC CDR3 are identical to the HC CDR1, HC CDR2, and HC CDR3 of the amino acid sequence of SEQ ID NO: 20, and a variable heavy chain sequence, a variable light chain sequence having at least 95% identity with SEQ ID NO: 21, wherein the light chain (LC) complementarity-determining region (CDR) 1, LC CDR2, and LC CDR3 are identical to the LC CDR1, LC CDR2, and LC CDR3 of the amino acid sequence of SEQ ID NO: 21, and a variable light chain sequence, the isolated and engineered immune cell according to claim 7.

9. The isolated and engineered immune cell according to claim 7 or 8, wherein the engineered immune cell is an engineered T cell or an engineered natural killer (NK) cell.

10. The isolated and engineered immune cell according to any one of claims 7 to 9, wherein the ER retention sequence comprises the amino acid sequence KDEL (SEQ ID NO: 4).

11. The isolated and engineered immune cell according to any one of claims 7 to 9, wherein the ER retention sequence comprises KKXX (SEQ ID NO: 9), or KXD / E (SEQ ID NO: 10), wherein X is any amino acid.

12. The isolated and engineered immune cell according to any one of claims 7 to 11, wherein the Golgi retention sequence comprises the amino acid sequence YQRL (SEQ ID NO: 11), or the proteasome localization sequence comprises a PEST motif.

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