Immunotoxin-armored immune effector cells and uses thereof

Abstract

Provided herein are engineered effector cells that include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin. In some embodiments, the engineered effector cell further includes a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen.

Description

[0001] IMMUNOTOXIN-ARMORED IMMUNE EFFECTOR CELLS AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 637, 191 , filed on April 22, 2024, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 44807-0486WOl_SL_ST26.xml. The XML file, created on April 4, 2025, is 64,587 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to the field of biotechnology7, and more specifically, to engineered effector cells.

[0008] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0009] This invention was made with government support under grant CA006973 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] BACKGROUND

[0011] Immunotoxins, recombinant proteins comprising a targeting domain (e.g., an antibody or ligand) fused with a bacterial or plant toxin, can be powerful cancer therapeutics. Currently, a limited number of fusion immunotoxins are approved for clinical use, showing efficacy in cutaneous T-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm, and hairy cell leukemia. Despite these successes, the widespread use of immunotoxins has been restrained by their toxicities with systemic administration and the development of anti-drug antibodies that limit their efficacy with intermittent administration. Cellular therapies, such as chimeric antigen receptor (CAR)-T cells, have also exhibited only limited efficacy against solid tumors. The heterogeneity of antigen expression and antigen escape in tumors are primary factors restricting CAR-T cell effectiveness. Tumor cell resistance to perforin / granzyme, tumor necrosis factor-a (TNFa) and interferon-g (IFNg) mediated cytotoxicity can be another factor. Engineering T cells to express a CAR and also secrete a toxin could overcome limitations of these two therapeutic modalities by providing synergistic bystander killing. Local secretion of the toxin by CAR-T cells infiltrating the tumor could moreover reduce or abrogate systemic toxicities and toxin neutralization. The toxin could also kill tumor cells via an orthogonal mechanism to T cell cytotoxicity pathways.

[0012] Furthermore, toxin activity could induce an inflammatory tumor microenvironment that would enhance CAR-T cell activity.

[0013] SUMMARY

[0014] The present disclosure is based on the discovery’ that engineered immune cells (e.g., T cells) as described herein, can more selectively target a cancer cell, rather than a corresponding non-cancerous cell. The presently provided immune cells can provide for selective treatment of a cancer in a subject and / or a reduction in on-target off-tumor cytotoxicity in non-cancer cells.

[0015] Provided herein are engineered effector cells that include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin. In some embodiments, the engineered effector cell further includes a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen. In some embodiments, an engineered effector cell includes (i) a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen; and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. In some embodiments, the toxin comprises an ADP-ribosylating toxin (ADPRT). In some embodiments, the toxin comprises a Pseudomonas aeruginosa exotoxin A (PE), Corynebacterium diphtheria toxin (DT), or Vibrio cholerae cholix toxin (ChxA) and parts thereof.

[0016] In some embodiments, an engineered effector cell further includes a mutation in a gene in the diphthamide biosynthesis pathway. In some embodiments, the gene in the diphthamide biosynthesis pathway comprises a DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, or DPH7 gene. In some embodiments, an engineered effector cell further includes a mutation in an EEF2 gene. In some embodiments, the mutation comprises a p.G717R mutation in the EEF2 gene. Also provided herein are methods of generating any one of the engineered effector cells described herein, the method comprising delivering a gene-editing agent into an effector cell, wherein the gene-editing agent alters a target gene of the effector cell, thereby generating the engineered effector cell that is resistant to the toxin.

[0017] In some embodiments, the altering comprises inactivating the target gene. In some embodiments, the gene-editing agent comprises CRISPR / Cas9 components. In some embodiments, the gene-editing agent comprises a guide RNA (gRNA). wherein the gRNA is targeted to the target gene of the effector cell. In some embodiments, the target gene comprises a gene in the diphthamide biosynthesis pathway. In some embodiments, the target gene comprises a DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, or DPH7 gene. In some embodiments, the gRNA comprises a SEQ ID NO: 1-28.

[0018] In some embodiments, the altering comprises introducing a mutation into the target gene. In some embodiments, the target gene comprises a EEF2 gene. In some embodiments, the mutation comprises a p.G717R mutation in the EEF2 gene. In some embodiments, the gRNA comprises a SEQ ID NO: 29-34.

[0019] Also provided herein are pharmaceutical compositions that include any one of the engineered effector cells described herein and a pharmaceutically acceptable carrier.

[0020] Also provided herein are methods of treating a subj ect having a disease that include administering to the subject any one of the engineered effector cells, or any one of the pharmaceutical compositions described herein. In some embodiments, the disease is a cancer. In some embodiments, the cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0022] BRIEF DESCRIPTION OF DRAWINGS

[0023] FIGs. 1A-1D show ADPRT resistance in mammalian cells through genetic engineering of DPH and EEF2. (FIG. 1A) Grow th of 293FT cells cultured in the presence of vary ing concentrations of DT or PE was quantified over time using CellTiter-Glo luciferase activity. (FIG. IB) 293FT cells that were wild-type (WT). knockouts for DPH1-7 (DPH1.K0- DPH7.K0) or EEF2 mutant (EEF2 p.G717R) w ere cultured in the presence of DT. Cell confluence over time was quantified using live cell microscopy. Shown are the results for the single best gRNA from multiple tested for each gene (FIG. 7). (FIG. 1C) Jurkat cells and (FIG. ID) primary human T cells that w ere wild-type (WT) or DPH1 knockout (DPH 1. KO) were cultured in the absence or presence of 2 pg / ml DT (+DT). Relative cell area over time was quantified using live-cell microscopy. Points represent mean ± SEM of n>3 replicates. DT: diphtheria toxin. PE: pseudomonas exotoxin.

[0024] FIGs. 2A-2D show evaluation of effector cell function with DPH and EEF2 mutation. (FIGs. 2A-2C) CRTSPR / Cas9 was used to knockout DPH1, DPH2, DPH3, or DPH4 or to introduce an EEF2 p.G717R mutation in primary7human T cells. After selection in DT, T cells were cocultured with CD19+ NALM6 target cells and a 0, 0.05, 0.5, or 50 ng / ml of a CD19xCD3 bispecific antibody. (FIG. 2A) T cell expansion index, (FIG. 2B) supernatant IFNy concentration, and (FIG. 2C) NALM6 cell count were quantified by flow cytometry on day 7 of co-culture. (FIG. 2D) 293FT cells edited with CRISPR / Cas9 for DPH1 or DPH2 knockout or EEF2 p.G717R mutation were selected in DT and then cultured in the presence of exogenous TNFa. Cell confluence over time was quantified by live cell microscopy. DT: diphtheria toxin. IFNy: interferon- / . TNFa: tumor necrosis factor-a. WT: wild-type.

[0025] FIGs. 3A-3D show7activity' of immunotoxins expressed by engineered 293FT cells. (FIG. 3A) 293FT cells engineered for resistance with knockout of DPH genes (DPH.KO) or EEF2 p.G717R mutation w ere transfected for expression of immunotoxin or control constructs. The percentage of live cells expressing the constructs was quantified by intracellular flow7cytometry7staining using a FLAG-tag on the constructs. DTdl80 and PE38d578 are catalytically inactive forms of the DT and PE toxins. Mock: 293FT cells subjected to mock transfection protocol w7ith no DNA. (FIG. 3B) Conditioned media supernatant from transfected 293FT cells was applied to NCI-H358 target cells. NCI-H358 cell viability was measured by SteadyGlo luciferase activity after 4 days of supernatant exposure. (FIG. 3C) 293FT cells engineered for resistance with DPH2 knockout or EEF2 p.G717R mutation were transfected with immunotoxin constructs and then cultured across a transwell insert from NCI-H358 cells. Confluence ofNCI-H358 cells was quantified over time using live cell microscopy. (FIG. 3D) 293FT cells engineered for resistance were transfected with immunotoxin constructs and then co-cultured with NCI-H358 cells. GFP-labeled NCI-H358 cells were quantified longitudinally using live cell microscopy. NT: not transfected.

[0026] FIGs. 4A-4E show selective killing of cancer cell lines by immunotoxin constructs expressed in resistance engineered Jurkat cells. Target cells were co-cultured across a transwell barrier insert with media alone, exogenous DT, Jurkat DPH1.KO effector cells transfected to express GFP, or Jurkat DPH1.K0 cells transfected to express tEGF-PE38. Target cell area was quantified across time using live-cell microscopy. (FIG. 4A) Target cells were engineered for resistance with DPH1.K0; (FIG. 4B) did not express EGFR; (FIG. 4C) expressed EGFRvIII; (FIG. 4D) expressed EGFR. (FIG. 4E) Jurkat WT cells without resistance or Jurkat DPH1.K0 cells with resistance were transfected to express tEGF-PE38 and co- cultured across transwell barrier inserts from target cells. Target cell area across time was quantified using live-cell microscopy. Points represent mean ± SEM of n>3 replicates. DT: diphtheria toxin. WT: wild-type.

[0027] FIG. 5 shows an exemplary schematic of engineered immune effector cells secreting immunotoxins as a cancer therapeutic. Immune effector cells are engineered via knockout of diphthamide biosynthesis genes or EEF2 mutation to be resistant to immunotoxins. The effector cells locally secrete immunotoxins in tumors, leading to inhibition of translation and subsequently cancer cell death.

[0028] FIG. 6 shows a schematic of diphthamide biosynthesis and mutations conferring resistance to ADPRT. Diphthamide synthesis is a multistep reaction catalyzed by a group of seven enzy mes (DPH1-7), resulting in the posttranslational modification of histidine residue 715 of human EEF2. ADP-ribosylation of the diphthamide residue on EEF2 by bacterial ADPRT results in abrogation of protein synthesis and secondary cell death. Mutation of EEF2 at residue 717 (p.G717R) prevents posttranslational modification of H71 by diphthamide, rendering EEF2 resistant to the action of ADPRTs in generating ADP-ribosyl-diphthamide and thereby halting protein synthesis. ADP: adenosine diphosphate. ADPRT: adenosine diphosphate-ribosylating toxin. EEF2: eukaryotic elongation factor 2. FIG. 7 shows resistance to DT of CRISPR / Cas-engineered diphthamide biosynthesis protein deficient and EEF2 p.G717R mutant 293FT cells. Human 293FT cells were edited via CRISPR / Cas9 to knockout genes of the diphthamide biosynthesis pathway, namely DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, and DPH7 using up to 5 different guide RNAs per gene. Alternatively, CRISPR / Cas9-mediated homology-directed repair was used to introduce a p.G717R mutation into EEF2. 293FT mutant cells were then cultured in the presence of exogenous DT and their proliferation was quantified by live-cell imaging over time. Mock cells were subjected to the CRISPR / Cas9 editing protocol with no guide RNA. Points represent mean ± SEM of n=4 replicates. Mock: DT: diphtheria toxin. EEF2: eukaryotic elongation factor 2. WT: wild-type.

[0029] FIGs. 8A-8B show proliferation in the presence of DT of CRISPR / Cas-engineered diphthamide biosynthesis protein deficient and EEF2 p.G717R mutant T cells. Primary human T cells were edited via CRISPR / Cas9 to knockout DPH1, DPH2, DPH3, or DPH4 (DPH.KO) or to introduce an EEF2 p.G717R mutation, testing multiple guide RNAs per gene. T cell expansion in the presence of 1 pg / ml DT was tracked over time using the CellTiter-Glo luciferase activity (FIG. 8A) or live cell microscopy (FIG. 8B). Mock cells were subjected to the CRISPR / Cas9 editing protocol with no guide RNA. Untreated cells were cultured in the absence of DT. Points represent mean ± SEM of n=2 replicates. DT: diphtheria toxin. EEF2: eukaryotic elongation factor 2.

[0030] FIG. 9 shows replication after activation of T cells engineered with DPH and EEF2 mutations. CRISPR / Cas9 was used to knockout DPH1, DPH2, DPH3, or DPH4 (DPH.KO) or to introduce an EEF2 p.G717R mutation in primary human T cells. After selection in DT, T cells were co-cultured with CD19+ NALM6 target cells and 0, 0.05, 0.5, or 50 ng / ml of a CD19xCD3 bispecific antibody. Percentages of CellTrace Violet labeled T cells in each replication generation as quantified by flow cytometry are shown. DT: diphtheria toxin. EEF2: eukaryotic elongation factor 2. WT: wild-type.

[0031] FIG. 10 shows sensitivity to TNFa of cells engineered with DPH or EEF2 mutations. 293FT cells edited with CRISPR / Cas9 for DPH1, DPH2, or DPH4 knockout (DPH.KO) or EEF2 p.G717R mutation were selected in DT and then cultured in the presence of varying concentrations of exogenous TNFa. Cell death over time was quantified by live cell microscopy using the Cytotox Red marker. DT: diphtheria toxin. EEF2: eukaryotic elongation factor 2. TNFa: tumor necrosis factor-a. WT: wild-type. FIG. 11 shows immunotoxin and control constructs tested in experiments. Full sequences are described in Table 3. Cetux: cetuximab.

[0032] FIG. 12 shows expression of GFP transfection control in 293FT. 293FT cells engineered with resistance by CRISPR / Cas9 editing of DPH genes (DPH.KO) or introduction of EEF2 p.G717R mutation were transfected for expression of immunotoxin or control constructs. The percentage of live cells expressing GFP was quantified by flow cytometry. EEF2: eukaryotic elongfation factor 2. WT: wild-type.

[0033] FIG. 13 shows inhibition of NCI-H358 target cell growth by conditioned media supernatant from 293FT cells transfected with immunotoxin constructs. 293FT cells engineered with resistance by CRISPR / Cas9 editing of DPH genes (DPH.KO) or introduction of EEF2 p.G717R mutation were transfected for expression of immunotoxin or control constructs. Conditioned media supernatant from transfected 293FT cells was applied to NCI-H358 target cells. NCI-H358 cell confluence w as measured by microscopy after 4 days of supernatant exposure. EEF2: eukaryotic elongation factor 2. NT: non-transfected. WT: wild-type.

[0034] FIGs. 14A-14B show morphology of NCI-H358 target cells following exposure to conditioned media supernatant from 293FT cells transfected for expression of immunotoxin constructs. 293FT cells engineered for resistance with DPH1 knockout were not transfected or transfected with the tEGF-PE38 immunotoxin construct. Media supernatant from nontransfected control 293FT cells (FIG. 14A) or IEGF-PE38 293FT cells (FIG. 14B) was applied to NCI-H358 target cells. NCI-H358 cell morphology was examined by microscopy after 4 days of supernatant exposure. Cells are highlighted with yellow mask.

[0035] FIG. 15 shows NCI-H358 target cell viability following exposure to immunotoxin producing 293FT cells. 293FT cells engineered for resistance with DPH2 knockout or EEF2 p.G717R mutation were transfected with immunotoxin constructs and then cultured across a transwell barrier insert from NCI-H358 target cells. NCI-H358 cells were quantified using Steady-Gio luciferase activity after 9 days of culture. NT: non-transfected.

[0036] FIG. 16 shows immunotoxin constructs with additional targeting and catalytic domains. Full sequences are described in Table 3.

[0037] FIGs. 17A-17B show NCI-H358 target cell growth following exposure to 293FT cells expressing alternative immunotoxin constructs. 293FT cells engineered for resistance were transfected with immunotoxin constructs and (FIG. 17A) cultured across a transwell barrier insert fromNCI-H358 target cells or (FIG. 17B) co-cultured with NCI-H358 cells. GFP- labeled NCI-H358 cells were quantified longitudinally using live cell microscopy.

[0038] FIG. 18 shows flow cytometry gating strategy for NALM6 and T cell co-culture. FIG. 19 shows flow cytometry gating strategy for analysis of immunotoxin construct expression in transfected 293FT cells.

[0039] DETAILED DESCRIPTION

[0040] Immunotoxins can be potent targeted therapies against cancer but their clinical util i ty is limited by adverse events with systemic administration and neutralizing immune responses. Engineered T cells expressing T cell receptors or chimeric antigen receptors (CARs) against solid tumors have exhibited low efficacy in part due to heterogeneous expression of targeted cancer cell antigens. The present disclosure is based on the discovery that therapeutic immune effector cells engineered to express both a CAR and a secreted toxin could overcome these limitations because trafficking of the effector cell into the tumor could enable local delivery' of the toxin that targets and eliminates by stander cancer cells resistant to receptor- mediated cytotoxicity. The presently provided human effector cells that are engineered for resistance against adenosine diphosphate-ribosylating transferases (ADPRTs) by knockout of genes in the diphthamide biosynthesis pathway or selective mutation of eukaryotic elongation factor 2 (EEF2) can produce toxins that kill cancer cells. In some embodiments, genetically resistant cells (e.g., T cells) are more potent effectors than cells not engineered for resistance, wherein disruption of diphthamide biosynthesis does not prevent primary T cell proliferation and effector function.

[0041] Provided herein are engineered effector cells that include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and w herein the engineered effector cell is resistant to the toxin. In some embodiments, an engineered effector cell further comprises a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen. In some embodiments, provided herein are engineered effector cells comprising (i) a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. Also provided herein are methods of generating any one of the engineered effector cells described herein, that include delivering a gene-editing agent into an effector cell, wherein the gene-editing agent alters a target gene of the effector cell, thereby generating the engineered effector cell that is resistant to the toxin.

[0042] Also provided herein are pharmaceutical compositions that include any one of the engineered effector cells described herein and a pharmaceutically acceptable carrier. Also provided herein are methods of treating a subject having a disease that include administering to the subject any one of the engineered effector cells, or any one of the pharmaceutical compositions described herein.

[0043] Various non-limiting aspects of these effector cells are described herein, and can be used in any combination without limitation. Additional aspects of various components of effector cells are known in the art.

[0044] As used in the specification and the appended claims, the singular forms ‘"a,” "‘an7’ and “the” include plural referents unless the context clearly dictates otherwise.

[0045] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0046] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is. or is included in. the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. In some particular embodiments, administration may be intramuscular, intraventricular, intravenous, or subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0047] As used herein, the term “antibody” refers to an immunoglobulin (human, mammalian, non-mammalian, or synthetic) molecule that includes one or more antigenbinding domains that specifically bind to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin (Ig) structural elements sufficient to confer specific binding. Exemplary antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody may include one or more sequence elements that are humanized, primatized, chimeric, etc., as is known in the art. In some embodiments, the term “antibody” is used to refer to one or more of the art-know n or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with the present disclosure can be in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain variable fragments (scFvs); polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camelid antibodies or fragments thereof (e.g., VHHs); masked antibodies (e g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ”); single chain or Tandem diabodies (TandAb®); Anticalins®; Nanobodies®; minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Transbodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent is or comprises at least a portion of a chimeric antigen receptor (CAR).

[0048] As used herein, “delivering” or “gene delivery” can refer to the introduction of an exogenous polynucleotide into a host cell, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector- mediated gene transfer (e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (e.g., electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide maybe stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0049] In some embodiments, a polynucleotide can be inserted into a host cell by a gene delivery molecule. Examples of gene delivery molecules can include, but are not limited to, liposomes, micelles biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles ty pically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0050] As used herein, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when the polypeptide sequence is altered or manipulated. For example, in some embodiments, an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, deletions and / or insertions that have been introduced by the hand of man into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (i.e., covalently linked) to one or more additional polypeptides by the hand of man, to form a fusion polypeptide that would not naturally occur in vivo. Comparably, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, derivatives and / or progeny of an engineered polypeptide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0051] As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0052] As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0053] As used herein, the terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma” refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and nonHodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, precancerous pathology such as myelodysplastic syndromes, acquired aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria (PNH) and 5q- syndrome and the like.

[0054] Engineered Effector Cells

[0055] In some embodiments, provided herein are engineered effector cells that include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin. In some embodiments, the engineered effector cell further comprises a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen. In some embodiments, provided herein are engineered effector cells that include (i) a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. As used herein, an “effector cell"’ refers to a cell that performs a specific function in response to a stimulus. In some embodiments, an effector cell can provide a specific response against a stimulation and / or pathogen. In the immune system, effector cells are relatively short-lived activated cells that defend the body in an immune response. For example, effector B cells are called plasma cells and secrete antibodies, and activated T cells include cytotoxic T cells and helper T cells, which carry out cell-mediated responses. In some embodiments, an effector cell can refer to specific cells in the immune system (e.g., a T cell. aNK cell, a NKT cell, a B cell, a macrophage, a neutrophil). In some embodiments, an engineered effector cell provided herein comprises an engineered T cell.

[0056] In some embodiments, an effector cell can be modified in one or more than one manner. In some embodiments, an effector cell is an engineered effector cell, which means the effector cell has been genetically modified to express a non-naturally occurring protein (e.g., CAR, immunotoxin), or modified to include a non-coded amino acid, or modified to include posttranslational modifications, or modified to include an exogenous nucleic acid. In some embodiments, an engineered effector cell can include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin. In some embodiments, an engineered effector cells can include (i) a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. In some embodiments, an engineered effector cells can include (i) a T cell receptor (TCR), wherein the TCR binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. In some embodiments, an engineered effector cells can include (i) a HLA-independent T cell (HIT) receptor, wherein the HIT receptor binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin, wherein the engineered effector cell is resistant to the toxin. In some embodiments, an engineered effector cell can include an immunotoxin expressing module, wherein the immunotoxin expressing module expresses a toxin coupled to an antigen binding domain (e.g.. an antibody, a scFv, a growth factor), and wherein the engineered effector cell is resistant to the toxin.

[0057] Chimeric Antigen Receptors (CARs) In some embodiments, an engineered effector cell provided herein has been engineered to express at least one chimeric antigen receptor (CAR), including a CAR that targets a specific tumor antigen. As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably, and refer to engineered immune receptors capable of triggering or inhibiting the activation of an immune cell. With a CAR, a receptor can be programmed to recognize an antigen (e.g.. tumor antigen), which when bound, activates immune cells (e.g.. effector cells) to kill the cell expressing that antigen (e.g., tumor antigen). Therefore, immune cells expressing C AR(s) for a tumor antigen expressed on a tumor cell can target and kill the tumor cell. For example, recent clinical trials of a CD19-targeted CAR- transduced T cell (CD19-CAR T cell) against hematologic malignancies showed a strong effect of CAR T technology. (Kochenderfer, J. N. et al. (2010) Blood 116: 4099-4102; Porter, D. L„ et al. (2011) N. Engl. J. Med. 365: 725-733; Grupp, S. A. et al. (2013) N. Engl. J. Med. 368: 1509-1518; Kochenderfer, J. N. et al. (2015) J. Clin. Oncol. 33: 540-549; Brown, C. E. et al. (2016) N. Engl. J. Med. 375: 2561-2569). The clinical success of CAR T is attributed, at least in part, to the fusion structure of the CAR, which is made by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus. M. V. et al. (2014) Blood 123: 2625-2635; van der Stegen, S. J. et al. (2015) Nat. Rev. Drug Discov. 14: 499-509).

[0058] In some embodiments, a CAR comprises an extracellular antigen binding domain (e.g.. a ligand / anti gen-binding domain), a transmembrane domain and one or more intracellular signaling domains. As is known in the art, a CAR can typically comprise at least an extracellular antigen-binding domain, a transmembrane domain, a hinge region, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) that is capable of recognizing a tumor-associated antigen. In some embodiments, a CAR comprises an extracellular antigenbinding domain, a hinge domain or a spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR further comprises a co-stimulatory domain. In some embodiments, a CAR further comprises two co-stimulatory domains. In some embodiments, a CAR further comprises two or more co-stimulatory domains. In some embodiments, the transmembrane domain includes a CD8-alpha transmembrane domain. In some embodiments, the hinge region includes a CD28 hinge region. In some embodiments, the intracellular signaling domain includes a CD28 intracellular signaling domain or a CD3- zeta intracellular signaling domain. See. e.g., Abate-Daga et al., Molecular Therapy Oncolytics (2016) 3, 16014, the disclosure of which is incorporated herein by reference in its entirety.

[0059] In some embodiments, the transmembrane domain for use in a CAR provided herein includes a transmembrane domain from a endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D). CD103, CD137, CD160 (BY55), CD18, CD19, CD 19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29. CD3delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CDl la,CDl lb, CDl lc, CDlld, CDS, CEACAM1, CTLA-4, CRT AM, cytokine receptor, DAP-10,DNAM1 (CD226), Fc gamma receptor, GADS. GITR, HVEM (LIGHTR). IA4, ICAM-l,Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocy te function-associated antigen- 1 (LFA-1), an MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44. NKp46, NKp80 (KLRF1). OX-40. PAG / Cbp. programmed death-1 (PD-1), PSGL1, SELPLG (CD162), a Signaling Lymphocytic Activation Molecule (a SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, a TNF receptor protein, TNFR2. TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0060] In some embodiments, a transmembrane domain for use in a CAR provided herein comprises portions of transmembrane domains present in two or more endogenous proteins, such that the chimeric transmembrane domain retains the ability to fold correctly and span the cell membrane. In some embodiments, a CAR provided herein can include a transmembrane domain that differs from a transmembrane domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein can include a transmembrane domain that shares a degree of amino acid sequence identity to a transmembrane domain present in an endogenous protein. For example, a transmembrane domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity' with a transmembrane domain present in an endogenous protein.

[0061] In some embodiments, the intracellular signaling domain for use in a CAR provided herein includes an intracellular signaling domain from an endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D). CD103, CD137, CD160 (BY55), CD18, CD19. CD19a,CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8,CD8alpha, CD8beta, CD96 (Tactile), CDlla, CDl lb, CDl lc, CDlld, CDS, CEACAM1, CTLA-4, CRT AM. a cytokine receptor, DAP- 10, DNAM1 (CD226), Fc gamma receptor. GADS. GITR, HVEM (LIGHTR). IA4, ICAM-1, Ig alpha (CD79a), IL-2Rbeta, IL- 2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen- 1(LF A- 1), a MHC class 1 molecule, NKG2C, NKG2D, NKp30. NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), a Signaling Lymphocytic Activation Molecules (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL. VLA1, and VLA-6, or any combination thereof.

[0062] In some embodiments, an intracellular signaling domain for use in a CAR provided herein comprises portions of intracellular signaling domains present in two or more endogenous proteins, such that the chimeric intracellular signaling domain retains the ability to fold correctly and mediate signaling. In some embodiments, a CAR provided herein include an intracellular signaling domain that differs from an intracellular signaling domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein include an intracellular signaling domain that shares a degree of amino acid sequence identity to an intracellular signaling domain present in an endogenous protein. For example, an intracellular signaling domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a an intracellular signaling domain present in an endogenous protein.

[0063] In some embodiments, a CAR provided herein can include a co-stimulatory domain. In some embodiments, a CAR provided herein can include two or more co-stimulatory domains. For example, a CAR can include a co-stimulatory domain that is present in an endogenous polypeptide. Non-limiting examples of polypeptides having co-stimulatory domains that are suitable to include in engineered immune receptors provided herein include 4-1BB (CD137), CD28, CD2, CD4, 0X40, ICOS, BTLA, CD27, CD30, GITR, and HVEM, and CD8. In some embodiments, a co-stimulatory domain for use in a CAR provided herein comprises portions of co-stimulatory domains present in two or more endogenous proteins, such that the chimeric co-stimulatory domain retains the ability to fold correctly and enhance signaling. In some embodiments, a CAR provided herein include a co-stimulatory domain that differs from a co-stimulatory domain present in an endogenous protein by one or more amino acids, e.g., 1, 2. 3. 4, 5, 6. 7, 8, 9. 10. or more amino acids. In some embodiments, a CAR provided herein include a co-stimulatory domain that shares a degree of amino acid sequence identity' to a co-stimulatory domain present in an endogenous protein. For example, a co-stimulatory domain for use in a CAR provided herein can share at least 80%, at least 81%. at least 82%. at least 83%, 84%, 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a co-stimulatory domain present in an endogenous protein.

[0064] Exemplary CARs, exemplary domains within CARs, and derivatives thereof (e g., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ral 72; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2): 141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(l):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0065] In some embodiments, an engineered effector cell includes a CAR that binds specifically to a target antigen. In some embodiments, an engineered effector cell includes a CAR that binds specifically to a target antigen, wherein the target antigen comprises a tumor antigen.

[0066] As used herein, an '‘antigen” or ‘'target antigen” refers to a molecule or molecular structure that binds to a specific antibody, B-cell receptor, or T-cell receptor and induces a specific immune response in a subject. The term “tumor antigen” includes any antigenic substance produced in a tumor cell, which triggers an immune response in a subject. In some embodiments, a tumor antigen can include, e g., tumor-specific antigens (TSA), tumor- associated antigens (TAA), neoantigens, tissue differentiation antigens, mutant protein antigens, oncogenic viral antigens, cancer-testis antigens and vascular or stromal specific antigens. In some embodiments, a target antigen can include any tumor antigen (e.g.. neoantigens identified from a patient using genomic sequencing, human gplOO tumor antigen, transplantation antigens, cell surface proteins found on mammalian cells, cancerspecific proteins, or proteins associated with abnormal physiological responses), and combinations, derivatives, antigenic fragments of any the above target antigens. In some embodiments, a target antigen is a neoantigen or a tumor-associated antigen. In some embodiments, a target antigen is presented on a MHC class I molecule. In some embodiments, a target antigen is presented on a tumor cell.

[0067] In some embodiments, a tumor-associated antigen can include, but is not limited to, 5T4, AIM2, AKAP4 2, Art-4, Aura Al (AURKA), Aura Bl (AURKB), BAGE, BCAN, B- cyclin, BSG, CCND1, CD133, CDC45L, CDCA1 (TTK), CEA, CHI3L2 (Chitinase 3-like 2), CSPG4, EpCAM 4, Epha2, EPHX1, Ezh2, FABP7, Fosll (Fra-1), GAGE, Galt-3, G250 (CA9), gBK. glast. GnT-V, gplOO, HB-EGF, HER2, HNPRL, HO-1. hTERT, IGF2BP3. IL13-Ra2, IMP-3, IQGAP1, ITGAV, KIF1C, KIF20A, KIF21B, KIFC3, KK-LC-1, LAGE-1, Lek, LRRC8A, MAGE-1 (MAGEA1), MAGE-2 (MAGEA2B), MAGE-3, MAGE-4, MAGE- 6, MAGE-10, MAGE-12, MAGE-CI (CT7), MAGE-C2, MAGE-C3, Mart-1, MELK, MRP3, MUC1, NAPS A. NLGN4X, Nrcam. NY-ESO-1 (CTAG1B), NY-SAR-35, OFA / iLRP. PCNA. PIK3R1. Prame. PRKDC. PTH-rP, PTPRZ1, PTTG1 2. PRKDC. RAN, RGS1, RGS5, RHAMM (RHAMM-3R), RPL19, Sart-1, Sart-2, Sart-3, SEC61G, SGT-1, SOX2, SoxlO, Soxl l, SP17, SPANX-B, SQSTM1, SSX-2, STAT1, STAT3, Survivin, TARA, TNC, Trag-3. TRP-1, TRP2, Tyrosinase, URLC10 (LY6K), Ube2V, WT1, XAGE-lb (GAGED2a), YKL-40 (CHI3L1), ACRBP, SCP-1, SSX-I, SSX-4, NY-TLU-57, CAIX, Brachyury, NY- BR-1 , ErbB, Mesothelin, EGFRvIII, IL-13Ra2, MSLN, GPC3, FR, PSMA, GD2, LI -CAM, VEGFR1, VEGFR2, KOC1, OF A, SL-701, Mutant P53, DEPDC1, MPHOSPH1, ONT- 10, GD2L, GD3L, TF, PAP, BRCA1 DLC1, XPO1, HIF1A, ADAM2, CALR3, SAGE1. SCP-1. ppMAPkkk, WHSC. Mutant Ras, COX1, COX2, FOXP3, IDOL IDO2, TDO, PDL1, PDL2. and PGE2.

[0068] In some embodiments, neoantigens can include, but are not limited to, neoantigens associated with any tumor / cancer, e.g., lung cancer (MTFR2 D326Y, CHTF18 L769V, MY ADM R30W, HERC1 P3278S, FAM3C K193E, CSMD1 G3446E, SLC26A7 R117Q, PGAP1 Y903F, HELB P987S. ANKRD K603T); melanoma (TMEM48 F169L, TKT R438W, SEC24A P469L, AKAP13 Q285K, EXOC8 Q656P, PABPC1 R520Q, MRPS5 P59L, ABCC2 S1342F, SEC23A P52L, SYTL4 S363F, MAP3K9 E689K, AKAP6 M1482I, RPBM P42L, HCAPG2 P333L, H3F3C T41, GABPA E161K, SEPT2 Q125R, SRPX P55L, WDR46 T300I, PRDX3 P101L, HELZ2 D614N, GCN1L1 P769L, AFMID A52V. PLSCR4 R247C, CENPL P79L, TPX2 H458Y, SEC22C H218Y, POLA2 L420F, SLC24A5 mut); mesothelioma (NOTCH2 G703D, PDE4DIP L288M, BAP1 V523fs, ATP10B E210K, NSD1 K2482T); glioma / glioblastoma (IDH1 R132H, POLE L424V); breast cancer (mPALB2, mR0B03, mZDHHC16, mPTPRS, RBPJ H204L); cholangiocarcinoma (ERBB2IP E805G); and cervical cancer (MAPK1 E322K, PIK3CA E545K, PIK3CA E542K, EP300 D1399N, ERBB2 S310F, ERBB3 V104M, KRAS G12D).

[0069] Toxin Expressing Module

[0070] In some embodiments, an engineered effector cell provided herein has been engineered to include a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin. As used herein, an “toxin expressing module” refers to a module that expresses a toxin in a cell (e.g., an effector cell). A “toxin” refers to a toxic molecule that can be a small molecule, peptide, or protein that interacts with biological macromolecules (e.g., enzymes, cellular receptors).

[0071] In some embodiments, a toxin can be derived from bacteria, fungi, and plants, wherein most toxins function by inhibiting protein synthesis. For example, toxins such as diphtheria toxin (DT) and Pseudomonas exotoxin (PE) prevent protein synthesis by an effect on elongation factor 2 (EF-2). In some embodiments, bacterial toxins can include, but are not limited to, Diphtheria toxin (DT) and the toxin from Pseudomonas exotoxin (PE). Plant toxins can include the A chain of ricin (RTA), and the ribosome inactivating proteins (RIPs) gelonin, pokeweed antiviral protein, and dodecandron. In some embodiments, a toxin can include an ADP-ribosylating toxin (ADPRT). In some embodiments, a toxin comprises a Pseudomonas aeruginosa exotoxin A (PE) or Corynebacterium diphtheria toxin (DT). In some embodiments, a toxin can include a Vibrio cholerae cholix toxin-derived ADPRT. In some embodiments, a toxin can include a Vibrio cholerae cholix toxin (ChxA) and parts thereof.

[0072] In some embodiments, an engineered effector cell is engineered to be resistant to a toxin, which means the engineered effector cell has increased persistence and / or survival in the presence of, and / or after exposure to, toxins. In some embodiments, an engineered effector cell can further include a mutation in a gene in the diphthamide biosynthesis pathway, thereby allowing the effector cell to be resistant to a toxin. In some embodiments, the gene in the diphthamide biosynthesis pathway comprises a DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, or DPH7 gene. In some embodiments, an engineered effector cell can also include a mutation in a eukaryotic translation elongation factor 2 (EEF2) gene, thereby allowing the effector cell to be resistant to a toxin. In some embodiments, an engineered effector cell can comprise a mutation that includes a p.G717R mutation in the EEF2 gene.

[0073] Immunotoxin Expressing Module

[0074] In some embodiments, an engineering effector cell can include an immunotoxin expressing module, wherein the immunotoxin expressing module expresses an immunotoxin, and wherein the engineered effector cell is resistant to the immunotoxin. As used herein, an “immunotoxin expressing module" refers to a module that expresses an immunotoxin in a cell (e.g., an effector cell). The term “immunotoxin'’ refers to a protein that includes a toxin coupled to an antibody or growth factor that binds specifically to a target cell.

[0075] In some embodiments, toxins used in immunotoxin constructs can be derived from bacteria, fungi, and plants, wherein most toxins function by inhibiting protein synthesis. For example, toxins such as diphtheria toxin (DT) and Pseudomonas exotoxin (PE) prevent protein synthesis by an effect on elongation factor 2 (EF-2). In some embodiments, bacterial toxins that are commonly used in immunotoxins can include, but are not limited to, Diphtheria toxin (DT) and the toxin from Pseudomonas exotoxin (PE). Plant toxins utilized in immunotoxins can include the A chain of ricin (RTA), and the ribosome inactivating proteins (RIPs) gelonin. pokeweed antiviral protein, and dodecandron. In some embodiments, an immunotoxin can include an ADP-ribosylating toxin (ADPRT). In some embodiments, an immunotoxin comprises a Pseudomonas aeruginosa exotoxin A (PE), Corynebacterium diphtheria toxin (DT), or Vibrio cholerae cholix toxin (ChxA) and parts thereof.

[0076] In some embodiments, an engineered effector cell is engineered to be resistant to a toxin that is expressed by an immunotoxin expressing module, which means the engineered effector cell has increased persistence and / or survival in the presence of, and / or after exposure to, the toxin. In some embodiments, an engineered effector cell can further include a mutation in a gene in the diphthamide biosynthesis pathway, thereby allowing the effector cell to be resistant to an toxin. In some embodiments, the gene in the diphthamide biosynthesis pathway comprises a DPH1, DPH2, DPH3, DPH4, DPH5. DPH6. or DPH7 gene. In some embodiments, an engineered effector cell can also include a mutation in a eukaryotic translation elongation factor 2 (EEF2) gene, thereby allowing the effector cell to be resistant to a toxin. In some embodiments, an engineered effector cell can comprise a mutation that includes a p.G717R mutation in the EEF2 gene.

[0077] Method of Generating Engineered Effector Cells

[0078] Provided herein are methods of generating any one of the engineered effector cells described herein that include delivering a gene-editing agent into an effector cell, wherein the gene-editing agent alters a target gene of the effector cell, and thereby generating the engineered effector cell that is resistant to a toxin. In some embodiments, a method of generating any one of the engineered effector cells described herein includes genetically modifying an effector cell to express (i) a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen, and (ii) a toxin expressing module, wherein the toxin expressing module expresses a toxin.

[0079] In some embodiments, an engineered effector cell provided herein has been engineered to express at least one chimeric antigen receptor (CAR), including a CAR that targets a specific tumor antigen. In some embodiments, a CAR comprises an extracellular antigen binding domain (e.g., a ligand / antigen-binding domain), a transmembrane domain and one or more intracellular signaling domains. As is known in the art, a CAR can typically comprise at least an extracellular antigen-binding domain, a transmembrane domain, a hinge region, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) that is capable of recognizing a tumor-associated antigen. In some embodiments, a CAR comprises an extracellular antigen-binding domain, a hinge domain or a spacer, a transmembrane domain, and an intracellular signaling domain. In some embodiments, a CAR further comprises a costimulatory domain. In some embodiments, a CAR further comprises two co-stimulatory domains. In some embodiments, a CAR further comprises two or more co-stimulatory domains. In some embodiments, the transmembrane domain includes a CD8-alpha transmembrane domain. In some embodiments, the hinge region includes a CD28 hinge region. In some embodiments, the intracellular signaling domain includes a CD28 intracellular signaling domain or a CD3-zeta intracellular signaling domain. See, e g., Abate- Daga et al.. Molecular Therapy Oncolytics (2016) 3, 16014, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the transmembrane domain for use in a CAR provided herein includes a transmembrane domain from a endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD137, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84. CD8, CD8alpha. CD8beta. CD96 (Tactile), CDl la.CDl lb. CDl lc, CDl ld, CDS, CEACAM1, CTLA-4, CRT AM, cytokine receptor, DAP-10,DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l,Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin. ITGA4, ITGA6, ITGAD, ITGAE. ITGAL, ITGAM.ITGAX. ITGB2. ITGB7, ITGB1, KIRDS2. LAT. LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), an MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), a Signaling Lymphocytic Activation Molecule (a SLAM protein), SLAM (SLAMF1). SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7. SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0080] In some embodiments, a transmembrane domain for use in a CAR provided herein comprises portions of transmembrane domains present in two or more endogenous proteins, such that the chimeric transmembrane domain retains the ability to fold correctly and span the cell membrane. In some embodiments, a CAR provided herein can include a transmembrane domain that differs from a transmembrane domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5. 6, 7, 8. 9, 10, or more amino acids. In some embodiments, a CAR provided herein can include a transmembrane domain that shares a degree of amino acid sequence identity to a transmembrane domain present in an endogenous protein. For example, a transmembrane domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or more sequence identity with a transmembrane domain present in an endogenous protein.

[0081] In some embodiments, the intracellular signaling domain for use in a CAR provided herein includes an intracellular signaling domain from an endogenous polypeptide selected from an activating NK cell receptor, an immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD137, CD160 (BY55), CD18, CD19, CD19a,CD2, CD247, CD27, CD276 (B7-H3). CD28, CD29, CD3delta, CD3epsilon, CD3gamma, CD3zeta. CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8,CD8alpha, CD8beta, CD96 (Tactile), CD I la, CD l ib, CD 11c, CD l id, CDS, CEACAM1, CTLA-4, CRTAM, a cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2Rbeta, IL- 2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE. ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1. KIRDS2, LAT, ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen- 1(LF A- 1), a MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD 162), a Signaling Lymphocytic Activation Molecules (SLAM protein). SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0082] In some embodiments, an intracellular signaling domain for use in a CAR provided herein comprises portions of intracellular signaling domains present in two or more endogenous proteins, such that the chimeric intracellular signaling domain retains the ability to fold correctly and mediate signaling. In some embodiments, a CAR provided herein include an intracellular signaling domain that differs from an intracellular signaling domain present in an endogenous protein by one or more amino acids, e.g., 1. 2, 3, 4. 5, 6, 7. 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein include an intracellular signaling domain that shares a degree of amino acid sequence identity to an intracellular signaling domain present in an endogenous protein. For example, an intracellular signaling domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a an intracellular signaling domain present in an endogenous protein.

[0083] In some embodiments, a CAR provided herein can include a co-stimulatory domain. In some embodiments, a CAR provided herein can include two or more co-stimulatory domains. For example, a CAR can include a co-stimulatory domain that is present in an endogenous polypeptide. Non-limiting examples of polypeptides having co-stimulatory domains that are suitable to include in engineered immune receptors provided herein include 4-1BB (CD137). CD28, CD2, CD4, 0X40, ICOS, BTLA, CD27. CD30, GITR, and HVEM. and CD8. In some embodiments, a co-stimulatory domain for use in a CAR provided herein comprises portions of co-stimulatory domains present in two or more endogenous proteins, such that the chimeric co-stimulatory domain retains the ability to fold correctly and enhance signaling. In some embodiments, a CAR provided herein include a co-stimulatory domain that differs from a co-stimulatory domain present in an endogenous protein by one or more amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a CAR provided herein include a co-stimulatory domain that shares a degree of amino acid sequence identity to a co-sli mulatory domain present in an endogenous protein. For example, a co-stimulatory domain for use in a CAR provided herein can share at least 80%, at least 81%, at least 82%, at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a co-stimulatory domain present in an endogenous protein.

[0084] Exemplary CARs, exemplary domains within CARs, and derivatives thereof (e.g., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ral72; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2): 141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(l):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0085] In some embodiments, a method of generating any one of the engineered effector cells described herein includes delivering a gene-editing agent into an effector cell, wherein the gene-editing agent alters a target gene of the effector cell, and thereby generating the engineered effector cell that is resistant to the toxin.

[0086] In some embodiments, the gene-editing agent alters a target gene by inactivating the target gene of the effector cell, wherein the target gene is prevented from being expressed as a protein. In some embodiments, the altering of a target gene can include a knocking out of the target gene. In some embodiments, the altering of a target gene includes a knocking out of a target gene, wherein the target gene comprises a gene in the diphthamide biosynthesis pathway. In some embodiments, the altering of a target gene includes knocking out a DPH1. DPH2, DPH3, DPH4, DPH5, DPH6, or DPH7 gene. In some embodiments, the altering of a target gene can include introducing a mutation into the target gene. In some embodiments, the altering of a target gene includes introducing a mutation into a EEF2 gene. In some embodiments, the altering of a target gene includes introducing a p.G717R mutation in the EEF2 gene. As used herein, a “gene-editing agent’' can refer to an agent that can target and bind to a specific sequence in DNA. In some embodiments, a gene-editing agent comprises CRISPR / Cas9 components. As used herein, the term “CRISPR” refers to a technique of sequence specific genetic manipulation relying on the clustered regularly interspaced short palindromic repeats pathway, which unlike RNA interference regulates gene expression at a transcriptional level. As used herein, a “Cas effector” or “CRISPR-associated protein” can refer to an enzyme or protein that uses CRISPR sequences as a guide to recognize and cleave specific nucleic acid strands that are complementary to the CRISPR sequence. A gene-editing Cas effector can associate with a CRISPR RNA sequence to bind to, and alter DNA or RNA target sequences. In some embodiments, the gene-editing agent comprises a gene-editing Cas effector. In some embodiments, the gene-editing Cas effector comprises a Cas9 protein, a Casl3b protein, or a Casl3d protein. In some embodiments, a gene-editing Cas effector can be a Cas9 endonuclease that makes a double-stranded break in a target DNA sequence. In some embodiments, a gene-editing Cas effector can be a Cas 12a nuclease that also makes a double-stranded break in a target DNA sequence. In some embodiments, a gene-editing Cas effector can be a Cas 13 nuclease which targets RNA. In some embodiments, a gene-editing Cas effector comprises a Cas9 protein, a Cas 13b protein, or a Cas 13d protein. In some embodiments, the gene-editing Cas effector comprises a nuclease dead Cas9 (dCas9) protein. In some embodiments, the gene-editing Cas effector comprises a Cas 13b protein. In some embodiments, the gene-editing Cas effector comprises a Cas 13d protein.

[0087] In some embodiments, the gene-editing agent further comprises a guide RNA (gRNA), wherein the gRNA is targeted to an individual gene of a cell. The term “guide RNA” or “gRNA” is a specific ty pe of gRNA that combines tracrRNA (transactivating RNA), which binds to Cas9 to activate the complex to create the necessary strand breaks, and crRNA (CRISPR RNA), comprising complimentary' nucleotides to the tracrRNA, into a single RNA construct. Exemplary methods of employing the CRISPR technique are described in WO 2017 / 091630, which is incorporated by reference in its entirety.

[0088] In some embodiments, the guide RNA can recognize a target RNA, for example, by hybridizing to the target RNA. In some embodiments, the guide RNA comprises a sequence that is complementary to the target RNA. In some embodiments, the gRNA can include one or more modified nucleotides. In some embodiments, the gRNA has a length that is about 10 nt (e.g., about 20 nt, about 30 nt. about 40 nt, about 50 nt, about 60 nt, about 70 nt, about 80 nt, about 90 nt. about 100 nt. about 120 nt, about 140 nt, about 160 nt, about 180 nt, about 200 nt, about 300 nt, about 400 nt, about 500 nt. about 600 nt, about 700 nt, about 800 nt, about 900 nt. about 1000 nt, or about 2000 nt).

[0089] In some embodiments, the gene-editing agent comprises a guide RNA (gRNA), wherein the gRNA is targeted to a target gene of an effector cell. In some embodiments, a gRNA can be targeted to a gene in the diphthamide biosynthesis pathway. In some embodiments, a gRNA can be targeted to a DPH1, DPH2, DPH3. DPH4, DPH5, DPH6, or DPH7 gene. In some embodiments, a gRNA comprises a SEQ ID NO: 1-28.

[0090] [Table 1] - gRNAs targeting a diphthamide biosynthesis pathway gene In some embodiments, a gRNA can be targeted to an EEF2 gene. In some embodiments, a gRNA comprises a SEQ ID NO: 29-34

[0091] [Table 2] - gRNA targeting a EEF2 gene

[0092] Therapeutic Applications

[0093] Provided herein are pharmaceutical compositions that include any of the engineered effector cells described herein. In some embodiments, pharmaceutical compositions comprising an engineered effector cells also comprise a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition can include a buffer, a diluent, solubilizer, emulsifier, preservative, adjuvant, an excipient, or any combination thereof. In some embodiments, a composition, if desired, can also contain one or more additional therapeutically active substances.

[0094] In some embodiments, compositions are formulated for parenteral administration. For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g.. a form that is suitable for subcutaneous injection, intramuscular injection, or intravenous infusion). For example, in some embodiments, a pharmaceutical composition is provided in a liquid dosage form that is suitable for injection. In some embodiments, a pharmaceutical composition is provided as powders (e.g.. lyophilized and / or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g.. water, buffer, salt solution, etc.) prior to injection. In some embodiments, a pharmaceutical composition is diluted and / or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, a powder should be mixed gently with the aqueous diluent (e.g., not shaken).

[0095] In some embodiments, a pharmaceutical composition of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer’s solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. A vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e g., buffers and preservatives). In some embodiments, a formulation is sterilized by known or suitable techniques. A pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening, or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0096] Also provided herein are methods of treating a subject having a disease that include administering to the subject any one of the engineered effector cells, or any one of the pharmaceutical compositions descnbed herein. In some embodiments, the disease is a cancer.

[0097] Cancers suitable for treatment by a method of the present disclosure can include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer. Non-limiting examples of cancer include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embry onal tumor, endometrial cancer, ependy moma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy7cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer. Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary7, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and para-nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, orophary ngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary7syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms’ tumor. In some embodiments, a cancer suitable for treatment by a method of the present disclosure can include hairy cell leukemia. In some embodiments, a cancer suitable for treatment by a method of the present disclosure can include acute myelogenous leukemia. In some embodiments, a cancer suitable for treatment by a method of the present disclosure can include a lymphoma. In some embodiments, a cancer suitable for treatment by a method of the present disclosure can include a solid tumor.

[0098] In some embodiments, effective doses for treatment can vary depending on the risk and / or the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. An effective amount of any one of the engineered effector cells described herein can be any amount that treats a cancer present within the subject without producing significant toxicity to the subject. If a particular subject fails to respond to a particular amount, then the amount of any one of the engineered effector cells described herein can be increased (e.g., by twofold, three-fold, four-fold, or more). After receiving this higher amount, the subject can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the actual effective amount administered.

[0099] In some embodiments, the frequency of administration of any one of the engineered effector cells described herein can be any frequency that effectively treats a mammal having a cancer without producing significant toxicity to the mammal. For example, the frequency of administration of any one of the engineered effector cells described herein can be from about two to about three times a week to about two to about three times a year. In some cases, a subject having cancer can receive a single administration of any one of the engineered effector cells described herein. The frequency of administration of any one of the engineered effector cells described herein can remain constant or can be variable during the duration of treatment. A course of treatment with any one of the engineered effector cells described herein can include rest periods. For example, any one of the engineered effector cells described herein can be administered every other month over a two-year period followed by a six-month rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in administration frequency.

[0100] In some embodiments, an effective duration for administering any one of the engineered effector cells described herein can be any duration that effectively treats a cancer present within the subject without producing significant toxicity to the subject. In some cases, the effective duration can vary from several months to several years. In general, the effective duration for treating a subject having a cancer can range in duration from about one or two months to five or more years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

[0101] In certain instances, a cancer within a subject can be monitored to evaluate the effectiveness of the cancer treatment. Any appropriate method can be used to determine whether or not a subject having cancer is treated. For example, imaging techniques or laboratory assays can be used to assess the number of cancer cells and / or the size of a tumor present within a subject. For example, imaging techniques or laboratory assays can be used to assess the location of cancer cells and / or a tumor present within a subject.

[0102] In some embodiments, any one of the engineered effector cells described herein can be administered to a subject having a cancer as a combination therapy with one or more additional cancer treatments. A cancer treatment can include any appropriate cancer treatments. For example, a cancer treatment can include surgery7. For example, a cancer treatment can include radiation therapy. For example, a cancer treatment can include administration of one or more therapeutic agents (e.g., one or more anti-cancer agents). In some cases, an anti-cancer agent can be an immunotherapy (e.g., a checkpoint inhibitor). In some embodiments, the subject has previously been administered one or more additional anticancer therapies selected from the group consisting of ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, or a checkpoint inhibitor. In some embodiments, the subject will be administered one or more additional anticancer therapies selected from the group consisting of ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, or a checkpoint inhibitor.

[0103] EXAMPLES

[0104] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0105] Cell culture

[0106] 293FT (ThermoFisher #R70007) cells were cultured in DMEM high glucose (25 mM), GlutaMAX, sodium pyruvate (1 mM) (ThermoFisher #10569010) supplemented with lx MEM Non-Essential Amino Acids (ThermoFisher #1 1140050), Geneticin (ThermoFisher #10131027), 10% FBS (ThermoFisher #16000044), and penicillin / streptomycin (ThermoFisher #15140122) (293FT media). AsPCl (ATCC #CRL-1682), H9 (ATCC #HTB- 176), Jurkat, (ATCC #TIB-152). NALM6 (ATCC #CRL-3273), NCI-H358 (ATCC #CRL- 5807), and Ramos (ATCC #CRL-1596) cells were cultured in RPMI-1640 (ATCC #30-2001) supplemented with 10% FBS and penicillin / streptomycin. LN-229 (ATCC #CRL-2611) and U-l 18 (ATCC #HTB-15) cells were cultured in DMEM (ATCC #30-2002) supplemented with 10% FBS and penicillin / streptomycin. U-87 (ATCC #HTB-14) cells were cultured in EMEM (ATCC # 30-2003) supplemented with 10% FBS and penicillin / streptomycin. CFPAC-1 (ATCC #CRL-1918) cells were cultured in IMDM (ATCC #30-2005) supplemented with 10% FBS and penicillin / streptomycin. NCI-H358 and NALM6 cells were previously engineered to express GFP and luciferase. Primary human CD3+ T cells were isolated using negative selection (Stemcell Technologies #17951) from healthy donor leukopaks (AllCells) and were cultured in RPMI-1640 supplemented with IL-2 (PROLEUKIN, Prometheus, NDC #65483-116-07), IL-7 (BioLegend #581906), FBS, and penicillin / streptomycin (T cell media).

[0107] Construct cloning

[0108] DNA sequences encoding immunotoxin and control constructs w ere synthesized by GeneArt (ThermoFisher). Sequences were cloned into pcDNA3.1 (ThermoFisher #V79020) using suitable restriction enzymes. The pmaxGFP (Lonza #V4XP-3032) vector was used as a control for experiments reported in FIGs. 4A-4E. Construct sequences are described in Table 3.

[0109] [Table 3] - Construct Sequences

[0110] Testinz 293FT sensitivity to DT and PE

[0111] 293FT cells were plated in wells in 293FT media in the presence of increasing concentrations of the purified unnicked, native bacterial DT (List Biological Labs #150 or MilliporeSigma #322326) or PE (MilliporeSigma #P0184). Cells were quantified by CellTiter-Glo Luminescent Cell Viability Assay (Promega #G7570) at defined time points. Luminescence was quantified as relative light units (RLU) using a GloMAX luminescence microplate reader (Promega). Creating engineered 293FT cell lines

[0112] 293FT cells were edited with CRISPR / Cas9 to generate knockouts of DPH1-7 or to introduce an EEF2 p.G717 mutation using homology-directed repair. CRISPR / Cas9 guide RNA sequences and homology-directed repair templates are listed in Tables 1-2. S.p. Cas9 crRNA for DPH1-DPH7 (IDT custom synthesis) was reconstituted in Nuclease-Free Duplex Buffer (IDT # 11-01-03-01) and complexed with equimolar ratio of Alt-R CRISPR-Cas9 tracrRNA (IDT #1072534). RNA complexes for each individual DPH1-7 or EEF2 gene locus was incubated with Alt-R S.p. Cas9 Nuclease V3 (IDT #1081058) at a 5: 1 molar ratio for 15 minutes at room temperature to allow for Cas9 ribonucleoprotein (RNP) complex assembly. For homology -directed repair of EEF2, repair template DNA was synthesized as Ultramer DNA Oligonucleotides (IDT custom synthesis). Repair template DNA was incubated with EEF2-directed Cas9 RNP for 10 minutes at room temperature. 293FT w ild-type cells grown in 293FT media were treated with trypsin-EDTA 0.05% (ThermoFisher #25300054) to generate single cell suspensions. 293FT cells were pelleted by centrifugation at 90 xg for 10 minutes. Pelleted cells resuspended in SF buffer (Lonza SF Cell Line 4D-Nucleofector X Kit S #V4XC-2032) at a concentration of 10*106cells / mL. 20 pL of 293FT cells (0.2*106cells) were combined with 5 pl. of suspension containing Cas9 RNP or Cas9 RNP with HDRT for EEF2. 20 ml of the mixture of cells and RNPs w as transferred to each well of a 16-well electroporation cuvette (Lonza). Cells were electroporated using a 4D-Nucleofector X Unit (Lonza, #AAF-1003X) with pulse code CM130. Cells were rested for 5 minutes at room temperature. 80 pL pre-warmed 293FT media was added and cells were incubated for 15 minutes at 37°C. Cells were transferred to 96-well tissue culture treated plates (Coming #3598) at 20.000 cells / well in pre-warmed 293FT media and allowed to recover for 3 days. Following recovery from electroporation, edited 293FT cell pools were grown in 293FT media containing 1 pg / ml DT and 1 pg / ml PE for selection of resistant cells, ensuring resistance w as not driven by loss or mutation of the specific cell surface receptor targeted by each ADPRT. Cell proliferation was quantified by cell confluence via live-cell imaging (Sartorius Incucyte S3). At the end of the selection penod, all 293FT wild-type cells had died, showing no recovery during subsequent incubation in media without toxin. Edited and selected 293FT cell lines w ere then maintained in 293FT media without ADPRT. Testing engineered 293FT resistance to DT and TNFa

[0113] To evaluate the resistance to DT of edited 293FT cell lines, cells were seeded in plates in the presence of DT (MilliporeSigma #322326). Cell growth was longitudinally quantified by live-cell imaging (Sartorius Incucyte S3). To test the susceptibility7of engineered 293FT cell lines to TNFa, cells were seeded in plates in 293FT media containing 1 pg / ml DT. Incucyte Cytotox Red Dye (Sartorius, #4632), and increasing concentrations of human recombinant TNFa (Cell Signaling Technology7, #16769). Cells were quantified over time using live-cell imaging.

[0114] Engineered T cell creation, expansion, and co-culture

[0115] Analogous to engineering resistance in 293FT cells, an adapted CRISPR / Cas9 editing approach was employed to engineer resistance in primary human T cells. For this, primary human T cells were isolated using negative magnetic bead selection (Stemcell Technologies #17951) from healthy donor peripheral blood mononuclear cells (AllCells). Isolated T cells were activated using Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher, #1113 ID) at a ratio of 1: 1 beads:cells in T cell media. At 48 hours of stimulation, Dy nabeads were removed by magnet and activated T cells edited using CRISPR / Cas9 (Tables 1-2). S.p. Cas9 crRNA (IDT custom synthesis) was complexed with equimolar ratio of Alt-R CR1SPR-Cas9 tracrRNA (IDT #1072534). Complexed RNA (100 pmol / 106cells) was incubated with Alt-R S.p. Cas9 Nuclease V3 (50pmol / 106cells, IDT #1081058) at a 2: 1 molar ratio for 15 minutes at room temperature. For HDR of EEF2, 50 pmol of homology -directed repair template DNA (IDT custom synthesis) was incubated with EEF2-directed Cas9 RNP for 10 minutes at room temperature. Immediately prior to electroporation, T cells were pelleted by centrifugation at 90 xg for 10 minutes and resuspended in P3 buffer (Lonza P3 Primary Cell 4D-Nucleofector X Kit S #V4XP-3032) at a concentration of 50*106cells / mL. 20 mL of T cell suspension (l*106cells) was combined with Cas9 RNP (for DPH knockout) or Cas9 RNP with repair template (for EEF2 mutation). 20 ml of the mixture of cells and RNPs was transferred to each well of a 16-well electroporation cuvette (Lonza). Cells were electroporated using a 4D- Nucleofector X Unit (Lonza, #AAF-1003X) with pulse code EH115. Cells were rested for 5 min at room temperature, 80 uL of pre-warmed T cell media without IL-2 or IL-7 was added, and cells incubated for 15 minutes at 37°C. Electroporated T cells were then resuspended in 2 mL of T cell media and allowed to recover in 12-well tissue culture treated plates at 37 °C. After 3 days of recovery, edited and mock-edited (guide RNA control) T cells were resuspended in new T cell media containing no DT or 1 mg / ml DT. Cells were seeded in replicate plates or propagated in tissue culture flasks for ongoing selection. Cell proliferation in the presence of DT was quantified using CellTiter-Glo Luminescent Cell Viability’ Assay (Promega #G7570) at each day after the start of selection using one replicate plate for each time point.

[0116] For T cell antigen-specific functional assays, edited T cells were selected in T cell media containing DT for a total of 10 days. T cells were labeled with CellTrace Violet (ThermoFisher #C34557) and then seeded at 50,000 cells / well in T cell media in 96-well tissue culture plates. GFP+ NALM6 target cells expressing CD19 were added at 25,000 cells / well. 0-50 ng / mL of a CD19xCD3 T cell-engaging bispecific antibody (InvivoGen. #bimab-hcdl9cd3-01) were added to the wells. Cells were incubated for 6 days. At the end of the experiment, cells were pelleted by centrifugation at 350 xg for 5 minutes. Co-culture supernatants were harvested, centrifuged at 4000 xg to remove residual cell debris, and frozen at -80 °C for later cytokine assays. Pelleted cells were subjected to flow cytometry’ to determine T cell proliferation and cytotoxicity. To do so, cells were stained with LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher L34976), Human TruStain FcX (BioLegend #422301), and APC anti-CD3 (BioLegend). Data was acquired using an Intellicyt iQue Screener PLUS (Sartorius) instrument and analyzed using FlowJo v9 (BD Life Sciences). All experimental data was analyzed by gating on single, live cells.

[0117] NALM6 cells were identified as GFP+ and CD3-. T cells were identified as CD3+ and GFP-. The gating strategy is illustrated in FIG. 18. Expansion indices were calculated using fit curves for CellTrace Violet dilution. For cytokine analyses, supernatants were analyzed using the iQue Qpanel T Helper 6-plex kit for Intellicyt (Sartorius #90502) as per manufacturer’s instructions with the modification of diluting supernatants 1 :4 in assay buffer. Constructs used for transfection are described in Table 3, with pCI_GFP used as a transfection control and cll3-hUCHTlv9LHLH used as a control for FLAG-tag staining. 293FT wild-type and engineered cell lines were seeded in 96-well tissue culture plates at 20,000 cells / well. Reaching 40-50% confluence the next day, 293FT cell lines were transfected using Lipofectamine 3000 (ThermoFisher #L3000001). For this, immunotoxin or control plasmid DNA (100 ng / well) was diluted in P3000 reagent and Opti-MEM (ThermoFisher #31985062), Lipofectamine reagent w as diluted in Opti-MEM, and 10 mL of transfection mixture (1: 1 diluted DNA / P3000 and diluted Lipofectamine) was added to cells growing in 200 mL 293FT media.

[0118] At 48 hours, transfected 293FT cell lines were analyzed by flow cytometry to evaluate intracellular expression of immunotoxin or control proteins. For evaluation, transfected and control 293FT cells were treated with trypsin-EDTA 0.05% (ThermoFisher #25300054) to generate single-cell suspensions. Cells were stained with LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher #L34976). Cells were then fixed and permeabilized using the Cyto-Fast Fix / Perm Buffer Set (BioLegend #426803) and stained with Brilliant Violet 421 anti-DYKDDDDK tag (L5, BioLegend #637322). Data was acquired using an Intellicyt iQue Screener PLUS (Sartorius) instrument and analyzed using FlowJo v9 (BD Life Sciences). All experimental data was analyzed by gating on single, live cells. BV421 or GFP signal was quantified. The gating strategy is illustrated in FIG. 19.

[0119] Treatment ofNCI-H358 cells with conditioned media supernatant

[0120] 293FT cell lines were transfected with DNA constructs using Lipofectamine 3000 (ThermoFisher #L3000001) and were grown to near confluence in 293FT media. Culture supernatants were harvested, centrifuged to remove cell debris, and added to NCLH358 target cells. Grow th of NCI-H358 cells quantified by live-cell imaging (Sartorius Incucyte S3). At day 4, remaining NCI-H358 cells were quantified using the Steady -Gio Luciferase Assay System (Promega #E2510).

[0121] Transwell culture of 293FT with NCI-H358

[0122] 293FT cell lines were transfected with DNA constructs using Lipofectamine 3000 (ThermoFisher #L3000001). washed, and then seeded at 40,000 cells / well in the insert chamber of a 24-well transwell culture plate (Coming #3450) in 293FT media without Geneticin. NCLH358 target cells were seeded at 10,000 cells / well in the low er chamber. NCI-H358 cells were imaged (Sartorius Incucyte S3) at designated time points by temporarily removing the transwell insert, imaging the lower cell chamber, and then replacing the transwell insert for continued co-culture. In addition, NCI-H358 target cells were quantified at day 9 using the Steady -Gio Luciferase Assay System (Promega #E2510).

[0123] Co-culture of 293FT with NCI-H358

[0124] 293FT cell lines engineered for resistance were transfected with DNA constructs using Lipofectamine 3000 (ThermoFisher #L3000001), w ashed, and then added together with NCI- H358 target cells into wells of tissue culture plates. GFP-expressing NCI-H358 cells were quantified longitudinally with live-cell imaging (Sartorius Incucyte S3).

[0125] Creation of Jurkat DP Hl. KO cell line

[0126] 100 pmol sgRNA-1 targeting DPH1 (IDT, Table 1) was combined with Alt-R S.p. Cas9 Nuclease V3 (IDT #1081059) and 64 pmol Alt-R Cas9 Electroporation Enhancer (IDT #1075916) in a total volume of 2.5 ml to create an RNP. l*106Jurkat cells were centrifuged at 90 xg for 10 minutes and resuspended in 20 ml SE Buffer (Lonza SE Cell Line 4D- Nucleofector X Kit S #V4XC-1032). 2.5 ml of the RNP mix were combined with 20 ml of cells and 2.5 ml of Opti-MEM (ThermoFisher #31985070). 20 ml of the cell mixture w as transferred to the electroporation cuvette. Cells were electroporated using a 4D-Nucleofector X Unit (Lonza # AAF-1003X) with pulse code CL-120. 80 ml pre-warmed media was added to the cells and the cells were incubated for 30 minutes at 37°C. Cells were diluted to l*106cells / ml. After 8 days of culture, Jurkat cells were selected for resistance using 2 mg / ml DT (MilliporeSigma #322326).

[0127] Testing DT resistance in DP Hl. KO primary T cells

[0128] Primary human T cells w ere isolated from leukopaks and rested overnight. 100 pmol sgRNA- 1 targeting DPH1 (IDT, Table 1) was combined with Alt-R S.p. Cas9 Nuclease V3 (IDT #1081059) and 64 pmol Alt-R Cas9 Electroporation Enhancer (IDT #1075916) in a total volume of 2.5 ml to create an RNP mix. 1 * 106T cells were centrifuged at 90 xg for 10 minutes and resuspended in 20 ml P2 Buffer or P3 buffer (Lonza P2 Primary7Cell 4D- Nucleofector X Kit S #V4XP-2032, Lonza P3 Primary7Cell 4D-Nucleofector X Kit S #V4XP-3032). 2.5 ml of the RNP mix were combined with 20 ml of cells and 2.5 ml of Opti- MEM (ThermoFisher # 31985070). 20 ml of the cell mixture was transferred to the electroporation cuvette. Cells were electroporated using a 4D-Nucleofector X Unit (Lonza # AAF-1003X) w ith pulse code EH100 or EH111. 80 ml warm media was added to the cells and the cells were incubated for 30 minutes at 37°C. Cells were diluted to l*106cells / ml. After 5 days of culture, T cells were activated using Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher #11131D) at a ratio of 1 : 1 beads:cells. At 3 day7s post-activation, beads w ere removed from cells by passing the cells over a magnet. Cells were treated with 2 mg / ml DT. Cells were imaged using an Incucyte SX5 (Sartorius). Cells w ere quantified with total phase area per well. Co-culture of target cells with Jurkat effector cells

[0129] 1* 104target cells were plated in HTS Transwell-96 Permeable Support with 0.4 mm pore polycarbonate membrane plates (Coming #3391). 0.5*106Jurkat cells were centrifuged at 90 xg for 10 minutes and resuspended in 20 ml SE Buffer (Lonza SE Cell Line 4D-Nucleofector X Kit S #V4XC-1032). 1 mg of plasmid in a volume of 1 ml was added to the cells. 20 ml of the cell mixture was transferred to the electroporation cuvette. Cells were electroporated using a 4D-Nucleofector X Unit (Lonza # AAF-1003X) with pulse code CL-120. 80 ml warm media was added to the cells and the cells were incubated for 30 minutes at 37°C. Cells were diluted to 0.5*106cells / ml. The next day, 5*104electroporated Jurkat cells were added into the transwell insert. An equivalent volume of media or of DT in media for a final concentration of 2 mg / ml was added into the transwell insert of control wells. Cells were imaged using an Incucyte SX5 (Sartorius). Cells were quantified with total phase area per well.

[0130] Example 1 - Engineering Resistance to Adenosine Diphosphate-Ribosylating Immunotoxins (ADPRTs)

[0131] The first step in creating human effector cells that can efficiently express and secrete immunotoxins was to engineer resistance to ADPRT. 293FT model cells were treated with exogenous DT or PE and measured cell survival over time to determine sensitivity. Mammalian 293FT cells were sensitive to both DT and PE. with intoxication leading to arrest in cell proliferation and death (FIG. 1 A). Next, CRISPR / Cas9 was used to disrupt genes in the diphthamide biosynthesis pathway in 293FT cells, using guide RNAs to selectively target DPH1, DPH2, DPH3, DPH4, DPH5, DPH6. or DPH7, thereby preventing diphthamide synthesis at EEF2 H715. the shared catalytic target of ADPRTs. In an orthogonal approach, CRISPR / Cas9 homology-directed repair was used to introduce a p.G717R mutation in the endogenous EEF2 gene locus in 293FT cells (Table 2 and FIG. 6). The modified 293FT cells were treated with exogenous DT and measured cell growth over time. Disruption of DPH1, DPH2, DPH3, and DPH4 as well as introduction of mutant EEF2 p.G717R conferred resistance to DT (FIG. IB and FIG. 7). Knockout of DPH1 in the transformed T cell line Jurkat (FIG. 1C) and in primary human T cells (FIG. ID) similarly rendered cells resistant to exposure to exogenous DT, which invariably resulted in arrest of cell division and death in unedited T cells. This established that knockout of DPH genes as well as EEF2 p.G717R mutation could be used to confer resistance to immune effector cells expressing immunotoxins. Previous reports have described that disruption of diphthamide biosynthesis genes can impair cell growth in yeast. Impaired replication could be detrimental to human immune effector cells which generally rely on in-vivo expansion. To evaluate this, properties of modified effector cells were measured. CRISPR / Cas9 was used to disrupt DPH1, DPH2, DPH3, or DPH4, or to introduce an EEF2 p.G717R mutation in primary human T cells. Edited T cells were then expanded in the presence of exogenous DT for 9 days to select for resistant T cells. Engineered T cells proliferated under DT exposure whereas unedited cells did not (FIGs. 8A- 8B). Then, resting, edited T cells (after removal of DT for several days) and unedited T cells (not exposed to DT) were co-cultured with CD 19+ NALM6 B cells in the presence or absence of a CD19xCD3 bispecific antibody to activate the T cells and induce T cell- mediated killing of NALM6 target cells. Disruption of DPH genes or EEF2 mutation did not prevent T cell proliferation (FIG. 2A and FIG. 9), cytokine production (FIG. 2B), or T cell- mediated cytotoxicity (FIG. 2C) and allowed complete killing of NALM6 cells. Prior studies have also reported that diphthamide biosynthesis pathway disruption can predispose cells to TNFa-mediated apoptosis. If this effect were pronounced in human effector cells, this would be problematic for cellular therapies where TNF signaling is a key cytotoxicity mechanism. To assess sensitivity to TNFa, DPH1 or DPH2 were disrupted or mutant EEF2 p.G717R was introduced in 293FT cells. After selection for resistant edited cells with exogenous DT, resistant 293FT were cultured with exogenous human TNFa and cell growth was measured over time (FIG. 2D). It was found that TNFa exposure did not meaningfully decrease growth rates in cells with DPH gene disruption or EEF2 mutation compared to their wild-type counterparts. In addition, engineered resistance did not lead to increased 293FT cell death compared to wild-type cells under exposure to high TNFa concentrations (FIG. 10). These results collectively suggested that using DPH gene knockout or EEF2 mutation as a resistance mechanism may not overtly impair effector cell activity and growth.

[0132] With a resistance mechanism established, candidate immunotoxin constructs were then evaluated to determine which ones could be effectively expressed by mammalian cells (Table 3 and FIG. 11). Candidate immunotoxins were targeted against epidermal growth factor receptor (EGFR) using either an anti-EGFR antibody single chain variable fragment (scFv) derived from cetuximab or truncated epidermal growth factor (tEGF) as a ligand for EGFR. The targeting domains were fused to several toxin variants derived from the catalytic and translocation domains of DT or PE. After transfecting immunotoxin constructs into various resistance-modified 293FT cells, immunotoxin protein expression was measured using intracellular flow cytometry, with GFP constructs (FIG. 12) and a bispecific antibody with a FLAG-tag used as controls for transfection and staining. It was observed that all immunotoxin constructs could be expressed in ADPRT-resistant 293FT cells at levels similar to that of a cetuximab scFv control not fused to a toxin domain (FIG. 3A). Notably, immunotoxin expressing cells were almost completely depleted when active immunotoxin constructs were transfected into wild-ty pe, non-resistant 293FT cells, indicating that all fusion toxins were indeed actively cytotoxic.

[0133] To evaluate secretion and function of the immunotoxins, the same constructs were transfected into resistance-modified 293FT cells and the biological activity of the EGFR-targeted immunotoxins were tested on EGFR+ NCI-H358 target cells. First, conditioned media supernatants of resistant 293FT cells transfected with targeted immunotoxins were collected and the supernatant was applied to NCI-H358 targets. Supernatant of resistant 293FT cells transfected wi th various immunotoxins, but not supernatant of wild-type 293FT cells transfected with the same DNA, induced cell death in NCI-H358 cells (FIG. 3B and FIG. 13). NCI-H358 target cells treated with active immunotoxins exhibited cellular morphologies characteristic of ADPRT intoxication (FIGs. 14A-14B). Second, transfected 293FT cells w ere co-cultured across the barrier insert of a transwell plate from NCI-H358 cells (FIG. 3C and FIG. 15). Third, 293FT cells transfected with various immunotoxins were co-incubated in mixed co-culture with NCI-H358 cells (FIG. 3D). Overall, it w as consistently observed that the IEGF-PE38 immunotoxin, of all tested immunotoxin designs, had the greatest cytotoxicity in these assays. Fourth, a second set of constructs were tested with new designs additionally incorporating the EGFR-targeting 806 and D2C7ds scFv domains and the Vibrio cholerae exotoxin (CET) catalytic domain (Table 3, FIG. 16, FIGs. 17A-17B). Results revealed that the tEGF-PE38 immunotoxin in particular could be effectively expressed and secreted by resistant mammalian cells in an active conformation that effectively kills target cells. Other PE and CET immunotoxins showed comparatively lower activity7although they may have additional advantages for use in humans such as reduced immunogenicity.

[0134] Although DT-based immunotoxins show ed strong activity' when transfected into non-resistant 293FT cells, killing of NCI-H358 was not detected, indicating that the fusion protein is either not secreted or does not effectively reach the cytoplasm of these target cells.

[0135] To move towards a more clinically relevant format for cancer immunotherapy, CRISPR / Cas9 was then used to disrupt the DPH1 gene in Jurkat cells and thereby confer toxin resistance. Jurkat cells w ere transfected with a DNA construct to express the tEGF-PE38 immunotoxin. To test the specificity of the secreted immunotoxin, the immunotoxin-expressing Jurkat cells w ere co-cultured w ith target cells and the growth of target cells w as quantified using live-cell microscopy. The immunotoxin-expressing Jurkat cells did not exhibit cytotoxicity against target cells engineered for resistance with DPH1 gene disruption (FIG. 4A) or cells that do not express EGFR (FIG. 4B). They also did not exhibit cytotoxicity against cells that express EGFRvIII, which lacks the binding site for the tEGF targeting domain (FIG. 4C). However, the Jurkat effector cells did exhibit cytotoxicity against a panel of cells that express wild-type EGFR (FIG. 4D). These data indicate that the immunotoxin secreted by the Jurkat cells killed target cells by the expected mechanism of ADP ribosylation of diphthamide, dependent on DPH1 function, and was targeted selectively based on cognate surface receptor expression of EGFR. Next, the efficacy of immunotoxin expression was compared by wild-type Jurkat cells versus resistant Jurkat cells by co-culturing the Jurkat cells with target cells. Jurkat cells engineered for resistance and expressing the immunotoxin construct exhibited cytotoxicity against EGFR+ NCI-H358 and CFPAC-1 target cells. In contrast, wild-ty pe Jurkat cells expressing the immunotoxin did not exhibit cytotoxicity against any tested cell line (FIG. 4E). This demonstrated that the engineering of toxin resistance into the effector cells is a critical innovation to enable the efficacy of immunotoxin expression.

[0136] These experiments relied on transient expression of the immunotoxin construct via DNA transfection. In cell therapy, transient expression can have advantages over stable expression by limiting toxicities and adverse events. However, increased efficacy may be possible with stable expression considering that CAR-T cell persistence is associated with therapy success in some settings. As such, this work reveals the importance of engineering resistance into effector cells for immunotoxin delivery and introduces their potential as a powerful, specific anti-cancer therapy (FIG. 5).

Claims

WHAT IS CLAIMED IS:

1. An engineered effector cell comprising: a toxin expressing module, wherein the toxin expressing module expresses a toxin, and wherein the engineered effector cell is resistant to the toxin.

2. The engineered effector cell of claim 1, further comprising a chimeric antigen receptor (CAR), wherein the CAR binds specifically to a tumor antigen.

3. The engineered effector cell of claim 1 or 2, wherein the toxin comprises an ADP- ribosylating toxin (ADPRT).

4. The engineered effector cell of claim 3, wherein the toxin comprises a Pseudomonas aeruginosa exotoxin A (PE), Corynebacterium diphtheria toxin (DT), or Vibrio cholerae cholix toxin (ChxA) and parts thereof.

5. The engineered effector cell of any one of claims 1-4, further comprising a mutation in a gene in the diphthamide biosynthesis pathway.

6. The engineered effector cell of claim 5, wherein the gene in the diphthamide biosynthesis pathway comprises a DPH1, DPH2, DPH3, DPH4. DPH5, DPH6, or DPH7 gene.

7. The engineered effector cell of any one of claims 1-6, further comprising a mutation in an EEF2 gene.

8. The engineered effector cell of claim 7, wherein the mutation comprises a p.G717R mutation in the EEF2 gene.

9. A method of generating an engineered effector cell of any one of claims 1-8, the method comprising delivering a gene-editing agent into an effector cell, wherein the gene-editing agent alters a target gene of the effector cell, thereby generating the engineered effector cell that is resistant to the toxin.

10. The method of claim 9, wherein the altering comprises inactivating the target gene.

11. The method of claim 9 or 10, wherein the gene-editing agent comprises CRISPR / Cas9 components.

12. The method of claim 11. wherein the gene-editing agent comprises a guide RNA (gRNA), wherein the gRNA is targeted to the target gene of the effector cell.

13. The method of any one of claims 9-12, wherein the target gene comprises a gene in the diphthamide biosynthesis pathway.

14. The method of claim 13, wherein the target gene comprises a DPH1, DPH2, DPH3, DPH4, DPH5, DPH6, or DPH7 gene.

15. The method of claim 12. wherein the gRNA comprises a SEQ ID NO: 1-28.

16. The method of any one of claims 9-15, wherein the altering comprises introducing a mutation into the target gene.

17. The method of claim 1 , wherein the target gene comprises a EEF2 gene.

18. The method of claim 17, wherein the mutation comprises a p.G717R mutation in the EEF2 gene.

19. The method of claim 18, wherein the gRNA comprises a SEQ ID NO: 29-34.

20. A pharmaceutical composition comprising an engineered effector cell of any one of claims 1-8 and a pharmaceutically acceptable carrier.

21. A method of treating a subject having a disease, the method comprising administering to the subject an engineered effector cell of any one of claims 1-8, or a pharmaceutical composition of claim 20.

22. The method of claim 21, wherein the disease is a cancer.

23. The method of claim 22, wherein the cancer is selected from a bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, Hodgkin lymphoma, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.

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