Improved methods of making genome-edited immune effector cells

Abstract

The disclosure relates to chimeric antigen receptors and immune effector cells bearing chimeric antigen receptors (CARs). Disclosed herein are improved methods of making immune effector cells that use stimulation with or exposure to one or more antibodies specific for the TCR-CD3 complex, such as, for example, an anti-CD3 antibody. Stimulation / exposure is used either in place of or in combination with a magnetic depletion step. The immune effector cells produced by such methods are functionally comparable to and / or have fewer TCR+ cells and show efficacy and stability relative to immune effector cells sorted via magnetic depletion.

Description

Attorney Docket No. WGN0029-401-PCIMPROVED METHODS OF MAKING GENOME-EDITED IMMUNE EFFECTOR CELLS

[0001] This application claims the benefit of priority of U. S. Provisional Application No. 63 / 721,220, filed November 15, 2024, the disclosure of which is hereby incorporated by reference as if written herein in its entirety.

[0002] This application generally relates to methods of making genome-edited immune effector cells. In particular, the disclosure relates to treating the edited immune effector cells with one or more monoclonal antibodies in order to achieve a population of expanded T-cells with reduced numbers of unedited T-cells. The disclosed compositions and methods are particularly useful for the generation of allogeneic cell therapies.

[0003] Genome-edited immune effector cell therapies have shown dramatic outcomes for the treatment of hematologic malignancies. However, these therapies are not effective for all patients and not without associated toxicides. The challenge now is to optimize these products and their manufacture. The manufacturing process is complex and subject to numerous variabilities at each step. These variabilities can affect the critical quality attributes of the final product, and this can ultimately impact clinical outcomes.

[0004] Manufacturing genome-edited immune effector cells commonly begins with cell collection, followed by target cell selection and enrichment, optionally. CAR gene transfer, and cell expansion. At each step in manufacturing, several seemingly trivial details can impact the critical quality attributes of the final product, including safety, purity, potency, consistency, and durability. Safety includes not only negative sterility testing, but also producing a product lacking oncogenic or viral replicative potential; purity describes a product with a high concentration of CAR-T-cells and minimal contaminating cells; consistency includes producing products that meet lot release criteria time after time; potency is characterized by a product capable of eliminating tumor cells; and durability is indicated by a product that persists in circulation and maintains its anti-tumor effects. At most steps during manufacture, one or more of these parameters may be compromised, but careful selection of methods and procedures may minimize these vulnerabilities.Attorney Docket No. WGN0029-401-PC

[0005] Therefore, there remains a need for improved methods of making genome-edited immune effector cell-based immunotherapies, for more effective, safe, and efficient targeting of cancers, including T-cell associated malignancies.

[0006] Current methods of manufacturing genome-edited allogeneic immune effector cells require magnetic depletion of TRAC+ unedited cells during manufacturing to prevent graft versus host disease. It is estimated that 5-10% of the immune effector cells remain “unedited”. Additionally, magnetic depletion results in a loss of cells due to sort inefficiencies, and certain methods of depletion also have a current ceiling of approximately 80 billion cells. Magnetic depletion represents a potential failure point in manufacturing. The genome-edited immune effector cells also may be stressed after sorting and require overnight rest before cryopreservation. To address the issues of purity and stability in the population of genome-edited immune effector cells, it would be beneficial to develop improved methods of depleting the cells without magnetic separation.

[0007] Disclosed herein are methods of making genome-edited immune effector cells with improved purification methods. In some embodiments, certain depletion steps comprise stimulation with or exposure to one or more antibodies, or bivalent binding fragments thereof, which may result in activation induced cell death (AICD) of unedited cells. In some embodiments, such antibody-mediated depletion results in a purified cell population that is stable compared to magnetic depletion. In some embodiments, such antibody-depleted cells made by the methods disclosed herein are functionally comparable to magnetically depleted cells. In some embodiments, cells are stimulated with / exposed to one or more antibodies, or antigen binding fragments thereof, that are specific for the TCR-CD3 complex, such as. for example, an anti-CD3 antibody. In some embodiments, stimulation / exposure is used either in place of or in combination with a magnetic depletion step. In some embodiments, the immune effector cells produced by such methods are functionally comparable to and / or have fewer TCR+ cells and show efficacy and stability relative to immune effector cells sorted via magnetic depletion.

[0008] Disclosed herein is a method of making a population of genome-edited immune effector cells which binds to one or more proteins, comprising:a. optionally, activating a population of immune effector cells comprising a T-cell receptor (TCR); thenAttorney Docket No. WGN0029-401-PCb. editing the genome of the immune effector cells to reduce expression of TCR to a negligible level; thenc. treating the genome-edited immune effector cells with one or more antibodies, or bivalent binding fragments thereof, which bind to a polypeptide that forms part of the TCR-CD3 complex; and thend. expanding the edited immune effector cells.

[0009] In some embodiments, editing the genome comprises reducing expression of one or more of T-cell constant β chain (TRBC), CD3ε, CD3δ, CD3ζ, and / or CD3γ to a negligible level.

[0010] In some embodiments, editing the genome comprises reducing expression of the TCR a chain (TRAC) to a negligible level.

[0011] In some embodiments, the immune effector cells are chosen from iNKT-cells or T-cells, such as, for example, cytotoxic T-cells.

[0012] In some embodiments, the immune effector cells are primary cells derived from healthy donors.

[0013] In some embodiments, the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

[0014] Also disclosed herein are methods treating the edited immune effector cells with one or more antibodies for the purpose of depletion, purification and / or enrichment.

[0015] In some embodiments, the antibodies, or bivalent binding fragments thereof, bind CD3 and / or epitopes on the TCR-CD3 complex, such as, for example, CD3ε, CD3δ, CD3γ, and / or TCRβ.

[0016] In some embodiments, the antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 polypeptides and the LCVR comprises LCDR1, LCDR2, and LCDR3 polypeptides, wherein:a. HCDR1 is GYTFTRYT (SEQ ID NO:21),b. HCDR2 is INPSRGYT (SEQ ID NO:22),c. HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23),d. LCDR1 is SSVSY (SEQ ID NO:24).e. LCDR2 is DTS (SEQ ID NO:25), andAttorney Docket No. WGN0029-401-PCf. LCDR3 is QQWSSNPFT (SEQ ID NO:26).

[0017] In some embodiments, the antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:a. HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10;b. HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12;c. HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;d. HCVR of SEQ ID NO: 15 and LCVR of SEQ ID NO: 16;e. HCVR of SEQ ID NO: 17 and LCVR of SEQ ID NO: 18; orf. HCVR of SEQ ID NO: 19 and LCVR of SEQ ID NO:20.

[0018] In some embodiments, the antibodies, or bivalent binding fragments thereof, comprise an anti-CD3 antibody chosen from teplizumab, humanized OKT3, muromonab, foralumab, otelixizumab, visilizumab, TR66, SK7, gOKT3-5, UCHT1, 145-2C11 and SP34-2.

[0019] In some embodiments, said genome-edited immune effector cells express a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to CD2 or CD7.

[0020] In some embodiments, after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than <1%, <0.5%, <0.4%, <0.3%, or <0.25% per 900 x 108cells. In some embodiments the percentage of TCRa / p positive T cells is no more than <0.4% per 900 x 108cells.

[0021] Further provided herein is a method of treating cancer in a patient comprising administering a population of immune effector cells made according to the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] Figure 1 A shows a scatter plot of the number of TR AC+ T-cells (x-axis) versus the number of CD3+ T-cells (y-axis) in a typical, population of T-cell with no genomic modifications (unedited). 98.4% of T-cells are TRAC+ and CD3+.Attorney Docket No. WGN0029-401-PC

[0024] Figure IB shows a scatter plot of the number of TRAC+ T-cells (x-axis) versus the number of CD3+ T-cells (y-axis) on T-cells wherein the TRAC gene has been knocked out (TRAC KO).

[0025] Figure 1C shows a scatter plot of the number of TRAC+ T-cells (x-axis) versus the number of CD3+ T-cells (y-axis) after the TRAC gene is knocked out and after T-cells are depleted with teplizumab. wherein there are zero (0) cells (upper right-hand quadrant) that continue to express both TRAC and CD3 after treatment with an anti-CD3 antibody, suggesting strong efficacy.

[0026] Figure 2A is a graphed plot showing that dosing with 2 micrograms / mL teplizumab (diamond) demonstrates stable depletion at day 6 and day 14 in culture, versus 0.1 micrograms / mL (triangle), 0.2 micrograms / mL (square), and 0 micrograms / mL (circle). Data was averaged across 2-3 donors as represented by shapes. T-cells were split from the standard process (prodigy isolation and activation) on day 4 of manufacturing. Samples were treated with the indicated concentrations of teplizumab and CD3+ / TCR+ cells were tracked over the course of manufacturing.

[0027] Figure 2B shows that dosing with 2 micrograms / mL teplizumab (diamond) leads to fewer TCR+ cells when compared to 0.1 micrograms / mL (triangle) or 0.2 micrograms / mL (square), relative to magnetic depletion (dotted line).

[0028] Figure 3A shows the results of 4-Hour Killing of CAR-T cells vs CCRF-CEM (CD7+) and indicates that both methods of TCRa0 depletion yielded similar cytotoxicity profiles, where “E: T” stands for effector to target ratio.. Cells from each depletion method were thawed and challenged against the standard target cell line (CCRF-CEM). The graph indicates comparable cytotoxicity between depletion methods and that the addition of teplizumab does not negatively affect cytotoxicity.

[0029] Figure 3B shows the results of a Rechallenge Assay of CAR-T vs CCRF-CEM (CD7+). T-cells were challenged against CCRF-CEM cells at 1:4 E: T every 48 hours until they exhausted. The graph indicates comparable cytotoxicity between depletion methods and that the addition of teplizumab does not negatively affect cytotoxicity.

[0030] Figure 4A shows that expansion of TRAC+ cells is similar during rechallenge and comparable between both methods of depletion, with no aberrant TCR outgrowth. ShapesAttorney Docket No. WGN0029-401-PCrepresent matched donors where the depletion method used a dose of 2 micrograms / mL teplizumab.

[0031] Figure 4B shows CAR-T cell expansion through a rechallenge assay. The graph indicates that depletion with 2 micrograms / mL teplizumab (open circles) gave comparable expansion kinetics and no negative effect on CAR-T cell persistence. Dotted vertical lines indicate feed with cytokine or targets.

[0032] Figures 5A shows no aberrant growth of TCR+ cells when cultured with cytokines. Cells were thawed and cultured in media containing cytokines. Figure 5B shows that CAR-T cells do not expand in the absence of cytokines (no treatment), regardless of depletion method, and also show similar kinetics in expansion in the presence of cytokines (IL7 / 15).

[0033] Figure 6A versus Figure 6B shows a scatter plot of edited cells on days 6 and 14 (post magnetic depletion), respectively.

[0034] Figure 7A versus Figure 7B shows a scatter plot of the results of depleting with Foralumab on days 6 and 14, respectively. With foralumab, a large population of CD3+ / TCRaP+ T cells returns by Day 14 (FIG. 7B, upper right-hand quadrant).

[0035] Figure 8A versus Figure 8B shows a scatter plot of the results of depleting with Visilizumab on days 6 and 14, respectively. With visilizumab, a 0.79% TCR population returns by day 14, which is above FDA specifications for the maximum number of TCRa[3+ cells in an allogeneic cell product (FDA specifies <0.4% TCRaP+ cells per 9 x 108of cells dosed).

[0036] Figure 9A versus Figure 9B shows a scatter plot of the results of depleting with muromonab (OKT3) on days 6 and 14, respectively, muromonab (OKT3) appeared promising by day 14.

[0037] Figure 10 shows that all 3 tested antibodies (Foralumab, Visilizumab, Muromab) failed to completely deplete TCRab-i- cells by Day 14 despite initial success depleting on Day 6.

[0038] Figure 11 shows that muromonab (OKT3) treated CAR T cells (squares) have a higher recurrence of TCRa|3+ cells upon activation with target cells as compared to teplizumab, treated CAR T cells (open circles) and against cells depleted magnetically (closed circles). In a rechallenge assay, T-cells depleted with muromonab (OKT3) have more TCR outgrowth than magnetically-depleted or teplizumab-depleted cells. Thus, muromonab (OKT3) depletion of TRAC+ cells resulted in cells that are less stable after challenge.Attorney Docket No. WGN0029-401-PC

[0039] Figure 12A shows untreated edited cells in a CAR2 model. T-cells were transduced with CAR targeting CD2.

[0040] Figure 12B shows that addition of teplizumab 24 hours after TRAC / CD2 knock-out and viral transduction satisfactorily depleted TCRocP+ cells. The addition of teplizumab markedly decreased the amount of CD3+ cells at end of manufacturing. Thus, the addition of teplizumab to CAR2 samples depleted CD3 better than knockout (KO) alone across 4 donors, as assessed by FACs on day 14 at cell harvest.

[0041] Figure 12C shows the TCR+ / CD3+ percentage at the end of manufacturing in a CAR2 model. Data is averaged across 4 donors.

[0042] Figure 13 shows a schematic timeline of the bioprocess described herein.

[0043] Figure 14 shows a schematic timeline of the bioprocess using humanized OKT3 antibodies described herein.

[0044] Figure 15 shows treatment with humanized OKT3 antibody and CD3 column depletion brings the percentage of TCR or CD3 positive cells below the 0.4% threshold.

[0045] Figure 16 shows that there are no significant changes in expansion seen with the use of humanized OKT3 antibody depletion.

[0046] Figure 17 shows humanized OKT3 antibody treated CAR-T cells have similar cytotoxicity to untreated cells against CD7+ targets. Figure 17A shows data for biological sample D329992. Figure 17B shows data for biological sample 110046481. D329992 and 110046481 refer to different biological samples (different donors) to demonstrate robustness of the approach.

[0047] Figure 18 shows humanized OKT3 antibody treated CAR-T cells have similar cytotoxicity to untreated cells against CD7- targets. Figure 18A shows data for biological sample D329992. Figure 18B shows data for biological sample 110046481. As indicated for Figure 16, D329992 and 110046481 refer to different biological samples (different donors) to demonstrate repeatability of the approach.DETAILED DESCRIPTIONDefinitions

[0048] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinaryAttorney Docket No. WGN0029-401-PCskill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.

[0049] As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0050] The phrase “and / or,” as used herein, means “either or both” of the elements so conjoined, / .<?., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0051] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. The term “about,” as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a range. When no particular range, such as a margin of error or a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean the greater of the range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures, and the range which would encompass the recited value plus or minus a degree of error. Exemplary degrees of error are within about 20 percent (%), typically, within about 10%, and more typically, within about 5% of a given value or range of values.

[0052] The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically

[0053] The term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0054] As used herein the term “antibody” or “immunoglobulin” have the same meaning and will be used equally in the present disclosure. The term “antibody” as used herein refers to a protein, or polypeptide sequence derived from immunoglobulin molecules and immunologicallyAttorney Docket No. WGN0029-401-PCactive portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immuno- specifically binds an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.

[0055] As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (A) and kappa (K).

[0056] There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four to five domains, a variable domain (VH) and three to four constant domains (CHI, CH2, CH3 and CH4 collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The antibodies of the present disclosure can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).

[0057] The constant domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fc fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs).

[0058] The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to. Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid domains,Attorney Docket No. WGN0029-401-PCmulti-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies. v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U. S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0059] The term “antibody heavy chain,” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0060] The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (K) and lambda (X) light chains refer to the two major antibody light chain isotypes.

[0061] The term “antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen, and may be soluble or cell membrane bound in particular but not restricted to molecular entities that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, T- and NK-cell engagers, or any other molecule that can execute binding to a structure with high affinity. It is readily apparent that an antigen can be generated by synthesis or can be derived from a biological sample or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0062] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T-cells mayAttorney Docket No. WGN0029-401-PCrecognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0063] The term “autologous” refers to any material derived from the same individual into whom it is later to be re-introduced.

[0064] The term “binding” as used herein refers to a direct association between two molecules, due to. for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD). which is used to evaluate and rank the strength of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the ligand for its target. As used herein, the term “binding” in the context of the binding of an antibody to a predetermined target molecule (e.g., an antigen or epitope) typically is a binding with an affinity corresponding to a KD of about 10'7M or less, such as about IO"8M or less, such as about 10"' M or less, about 10"" M or less, or about 10"1M or even less. The concepts of binding affinity, association constant, and dissociation constant are well known.

[0065] As used herein, the term “binding domain” or “antibody molecule” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain” or “antibody molecule” encompasses antibodies and antibody fragments. In an embodiment, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.

[0066] The term “bioequivalent” refers to an amount of an agent other than the reference compound, required to produce an effect equivalent to the effect produced by the reference dose or reference amount of the reference compound.

[0067] The term “cancer” refers to a disease characterized by the uncontrolled, sometimes rapid, growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer,Attorney Docket No. WGN0029-401-PClymphoma, leukemia, lung cancer and the like. The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant. as well as malignant cancers and tumors. Examples of various cancers are described herein and include sub-types of lymphoma and leukemia.

[0068] The terms “cancer associated antigen” or “tumor antigen” interchangeably refers to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC / peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC / peptide), and not synthesized or expressed on the surface of a normal cell.

[0069] The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation.

[0070] The term “chemotherapy” refers to the treatment of cancer (cancerous cells) with one or more cytotoxic anti-neoplastic drugs (“chemotherapeutic agents” or “chemotherapeutic drugs”) as part of a standardized regimen. Chemotherapy may be given with a curative intent, or it may aim to prolong life or palliate symptoms. It is often used in conjunction with other cancer treatments, such as radiation therapy, surgery, and / or hyperthermia therapy. Traditional chemotherapeutic agents act by killing cells that divide rapidly, one of the main properties of most cancer cells. This means that chemotherapy also harms cells that divide rapidly under normal circumstances, such as cells in the bone marrow, digestive tract, and hair follicles. This results in the most common side-effects of chemotherapy, such as myelosuppression (decreasedAttorney Docket No. WGN0029-401-PCproduction of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

[0071] The term “anti-cancer effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.

[0072] The term “complementary” as used in connection with nucleic acid, refers to the pairing of bases, A with Tor U, and G with C. The term complementary refers to nucleic acid molecules that are completely complementary, that is, from A to T or U pairs and G to C pairs across the entire reference sequence, as well as molecules that are at least about 80%, 85%, 90%.95%, 99% complementary.

[0073] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein.

[0074] It is understood that the molecules of the present disclosure may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions. Table 1 shows exemplary conservative amino acid substitutions. In someAttorney Docket No. WGN0029-401-PCembodiments, the antibody constant domain can comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgGl antibody and differ by one or more substitution(s) where all the amino acid positions in an Fc domain or hinge region disclosed herein are numbered according to EU numbering.Table 1Original amino acid residue One and three-letter codes Conservative substitutionAlanine A or Ala Gly; Ser Arginine R or Arg Lys; His Asparagine N or Asn Gin; His Aspartic acid D or Asp Gin; Asn Cysteine C or Cys Ser; Ala Glutamine Q or Gin AsnGlutamic acid E or Glu Asp: Gin Glycine G or Gly AlaHistidine H or His Asn; Gin Isoleucine I or He Leu; Vai Leucine L or Leu He; val Lysine K or Lys Arg; His Methionine M or Met Leu; He; Tyr Phenylalanine F or Phe Tyr; Met; Leu Proline P or Pro AlaSerine S or Ser Thr Threonine T or Thr SerTry ptophan W or Trp Tyr; Phe Tyrosine Y or Tyr Trp; PheAttorney Docket No. WGN0029-401-PCValine V or Vai He; Leu

[0075] The terms “CRISPR system,” “Cas system” or “CRISPR / Cas system” refer to a set of molecules comprising an RNA-guided nuclease or other effector molecule and a gRNA molecule that together are necessary and sufficient to direct and effect modification of nucleic acid at a target sequence by the RNA-guided nuclease or other effector molecule. In one embodiment, a CRISPR system comprises a gRNA and a Cas protein, e.g., a Cas9 protein. Such systems comprising a Cas9 or modified Cas9 molecule are referred to herein as “Cas9 systems” or “CRISPR / Cas9 systems.” In one example, the gRNA molecule and Cas molecule may be complexed, to form a ribonuclear protein (RNP) complex.

[0076] The term “effective amount”, as used herein, means an amount that leads to measurable effect, e.g., antigen-dependent cell proliferation, cytokine secretion, cytotoxic killing, etc. The effective amount may be determined by using the methods known in the art and / or described in further detail in the examples.

[0077] An “effective dose” or “therapeutically effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0078] The term “effector function” refers to a specialized function of a differentiated cell. An effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. An effector function in a naive, memory, or memory-type T-cell may also include antigen-dependent proliferation.

[0079] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.Attorney Docket No. WGN0029-401-PC

[0080] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0081] The term “enrich” as used herein in relation to cells means to concentrate, purify, or isolate for further analysis or use. Enriched and purified cell populations comprise a majority of the desired cell, and a negligible fraction of other cells.

[0082] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0083] The term “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0084] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including plasmids and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0085] “Fc region” refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C -terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge, a CH2 domain, and a CH3 domain. Two Fc regions that are dimerized are referred to as “Fc” or “Fc dimer.” An Fc region may be a naturally occurring Fc region, or a naturally occurring Fc region in which one or more amino acids have been substituted, added or deleted, provided that the Fc region has the desired biological properties. A desired biological activity may be a natural biological activity, an enhanced biological activity or a reduced biological activity relative to that of the naturally occurring domain.

[0086] The term “flexible polypeptide linker” or “linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1.Attorney Docket No. WGN0029-401-PC

[0087] In some embodiments, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. The prefix “hum,” “hu,” “Hu,” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions can be included to increase affinity, increase stability of the humanized antibody, remove a post-translational modification or for other reasons.

[0088] “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0089] A “genome-edited immune effector cell” refers to an immune cell, such as a T-cell or Natural Killer cell, that has been genetically modified using genome editing technology (e.g., CRISPR-Cas9) to alter its DNA sequence, thereby enhancing its ability to target and destroy specific cells, such as cancer cells. CAR-T-cells encompassed by the present disclosure are genome-edited to be deficient in an antigen to which the chimeric antigen receptor specifically binds and are therefore fratricide-resistant.

[0090] Similarly, the term “genetic modification” or “genetically modified” refers to the alteration of the nucleic acid content including but not restricted to the genomic DNA of a cell. This includes but is not restricted to the alteration of a cell’s genomic DNA sequence by introduction exchange or deletion of single nucleotides or fragments of nucleic acid sequence. The term also refers to any introduction of nucleic acid into a cell independent of whether that leads to a direct or indirect alteration of the cells genomic DNA sequence or not.

[0091] The terms “guide RNA,” “guide RNA molecule,” “gRNA molecule” or “gRNA” are used interchangeably, and refer to a set of nucleic acid molecules that promote the specific directing of an RNA-guided nuclease or other effector molecule (typically in complex with the gRNA molecule) to a target sequence. In some embodiments, said directing is accomplished through hybridization of a portion of the gRNA to DNA (e.g., through the gRNA targeting domain), and by binding of a portion of the gRNA molecule to the RNA-guided nuclease or other effector molecule (e.g., through at least the gRNA tracr).

[0092] The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNAAttorney Docket No. WGN0029-401-PCmolecules or two RNA molecules, or between two polypeptide molecules. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g.. if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[0093] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0094] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g.. alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0095] The term “immune cell,” or “immune effector cell,” or “effector cell” as that term is used herein, refers to a cell that may be part of the immune system and executes a particular effector function such as alpha-beta T cells, NK cells (including memory NKs. ML-NKs, and CIML-NKs), NKT cells (including iNKT cells), B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes and macrophages. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells (including memory NKs, ML-NKs, and CIML-NKs), NKT cells (including iNKT cells), ILC, CIK cells, LAK cells or gamma-delta T cells.

[0096] An “indel,” as the term is used herein, refers to a nucleic acid comprising one or more insertions of nucleotides, one or more deletions of nucleotides, or a combination of insertions and defections of nucleotides, relative to a reference nucleic acid, that results after being exposedAttorney Docket No. WGN0029-401-PCto a composition comprising a gRNA molecule, for example a CRISPR system. Indels can be determined by sequencing nucleic acid after being exposed to a composition comprising a gRNA molecule, for example, by NGS. With respect to the site of an indel. an indel is said to be “at or near” a reference site (e.g., a site complementary to a targeting domain of a gRNA molecule) if it comprises at least one insertion or deletion within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s) of the reference site, or is overlapping with part or all of said reference site (e.g., comprises at least one insertion or deletion overlapping with, or within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of a site complementary to the targeting domain of a gRNA molecule, e.g., a gRNA molecule described herein).

[0097] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule, such as a chimeric antigen receptor (CAR). The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CAR T-cell. Examples of immune effector function, e.g., in a CAR T-cell, include cytolytic activity and helper activity, including the secretion of cytokines. Non-limiting examples of suitable intracellular domains include the zeta chain of the T-cell receptor or any of its homologs (e.g., zeta, delta, gamma, or epsilon), MB 1 chain, B29, Fc RIM, Fc Rl, and combinations of signaling molecules, such as CD3 and CD28, CD27, 4-1BB, DAP-10, 0X40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins may be used, such as Fey RIM and Fes RI.

[0098] Invariant natural killer T (iNKT) cells are also referred to as type I or classical NKT- cells. iNKT-cells represent a distinct population of T-cells that express an invariant a T-cell receptor (TCR) and various cell surface molecules in common with natural killer (NK) cells.

[0099] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0100] A “malignant T-cell” is a T-cell derived from a T-cell malignancy. The term “T-cell malignancy” refers to a broad, highly heterogeneous grouping of malignancies derived from T-cell precursors, mature T-cells, or natural killer cells. Non-limiting examples of T-cellAttorney Docket No. WGN0029-401-PCmalignancies include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), T-cell lymphoblastic lymphoma (T-LBL), T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1-positive (HTLV-1 +) adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and various peripheral T-cell lymphomas (PTCLs), including but not limited to angioimmunoblastic T-cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma, and ALK-negative anaplastic large cell lymphoma. For instance, by way of nonlimiting example, CD2, CD3, CD4, CD5, CD7, CD19, CD20 and CD30 and may be suitable antigens expressed on the surface of a malignant T-cell.

[0101] As used herein, the term “negligible” refers to an insignificant or unimportant amount that is so small as to be not worth considering. For example, a negligible level of expression of the T-cell receptor (TCR) may not be detectable or no different than the level of expression of a control without TCR outgrowth. In the context of the present disclosure, Cas9 induces doublestranded breaks (DSBs) at locations determined by guide RNA (gRNA). Repair of the DSBs by non-homologous end joining (NHEJ) is the basis for CRISPR genome editing. Repair causes small insertions and deletions (indels) near the DNA DSBs. This can result in a “negligible level” of a protein product being expressed. Thus, Cas9 gene editing may result in the deletion of a gene, or a portion thereof, which in turn results in a “negligible level” of the gene product being expressed. A “negligible level” may be within the error of experimental determination, may not have any medical or experimental consequences, may be insufficient to noticeably affect activity, sorting and / or purification, or it may be less than about 5% as measured using FACs or genome sequencing techniques known in the art.

[0102] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form.Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions).

[0103] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein orAttorney Docket No. WGN0029-401-PCan RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0104] The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

[0105] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.

[0106] The term “persistence” as sued herein refers to the ability of cells, especially adoptively transferred into a subject, to continue to live.

[0107] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides.

[0108] As used herein, the term “prevent” refers to obtaining beneficial or desired results including, but not limited to, prophylactic benefit.

[0109] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0110] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3. and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.Attorney Docket No. WGN0029-401-PC

[0111] In general, the term “receptor” refers to a biomolecule that may be soluble or attached to the cell surface membrane and specifically binds a defined structure that may be attached to a cell surface membrane or soluble. Receptors include but are not restricted to antibodies and antibody-like structures, adhesion molecules, transgenic or naturally occurring TCRs or CARs. The term “antigen-recognizing receptor” or “antigen-binding receptor” as used herein may be a membrane bound or soluble receptor such as a natural TCR, a transgenic TCR. a CAR, a scFv, a Fab-fragment or multimers thereof, an antibody or multimers thereof, a bi-specific T cell enhancer (BiTE), a bi-specific killer cell enhancer (BiKE), a diabody, or any other molecule that can execute specific binding with high affinity.

[0112] “Refractory” as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer.

[0113] “Relapsed” as used herein refers to the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, e.g., after prior treatment of a therapy, e.g., cancer therapy.

[0114] The term “recombinant antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, technology which is available and well known in the art.

[0115] The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.Attorney Docket No. WGN0029-401-PC

[0116] The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0117] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0118] The term “soluble” as used herein, when referring to an antibody, or functional fragment thereof, refers to a protein that is not surface-bound, membrane-bound, or cell-associated. A soluble antibody, or functional fragment thereof, is a free-floating protein dissolved in fluid, where the fluid may be any fluidic sample, or any mammalian bodily fluid, including intracellular fluid (ICF), which may comprise cytosolic fluid, and extracellular fluid (ECF), which may include interstitial fluid, intravascular fluid (blood and blood plasma), lymphatic fluid, or transcellular fluid (e.g., cerebrospinal fluid, synovial fluid, aqueous humor, and serous fluid).

[0119] The term “specifically binds,” refers to a molecule recognizing and binding with a binding partner (e.g., a protein or nucleic acid) present in a sample, but which molecule does not substantially recognize or bind other molecules in the sample.

[0120] Antibody “specificity” is defined according to the paratope-epitope interaction. When specificity is low, an antibody may bind several different epitopes. Specificity indicates that an antibody binds almost exclusively to one particular antigen or epitope thereon, with minimal binding to anything else, even closely related antigens. Thus, specific binding relates to an antibody’s ability to target a particular epitope on an antigen, and implies a very precise interaction, whereas selective binding means favoring one antigen over others, even if there may be minor cross-reactivity with similar antigens present in a mixture. Essentially, “specific” is a stricter definition than “selective” in the context of antibody binding.

[0121] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) with its cognate ligand (or tumorAttorney Docket No. WGN0029-401-PCantigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3.

[0122] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[0123] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0124] The term “surface-bound,” as used herein, when referring to an antibody, or functional fragment thereof, means immobilized on laboratory tools, where their fixed position is intended to capture target molecules from a sample for detection and / or analysis. The surface may be any support or substrate, such as beads, columns, plates, bags, resins, magnetic particles, microspheres, and / or nanoparticles.

[0125] The T-cell receptor (TCR) is a key molecule on the surface of T lymphocytes that recognizes antigens presented by other cells. It is comprised of a and p chains that closely associate with the s- 6- y- and ^-chains of CD3. Antigen-mediated activation of the a and P chains induces downstream signaling. Accordingly, the T-cell receptor (TCR)-CD3 complex is composed of a diverse ap TCR heterodimer noncovalently associated with invariant CD3 dimers CD3ey, CD3e5. and CD3c

[0126] The “T-cell constant P chain (TRBC)” refers to a specific protein region on the P chain of the TCR. The TRBC is the constant part of the chain that is expressed by all T-cells. There are two main isoforms of TRBC, called TRBC1 and TRBC2. which are mutually exclusive, meaning a single T-cell will only express one of them. Thus, “T-cell constant £ chain (TRBC)” refers specifically to the constant region of the T-cell receptor beta chain (TCR£), meaning it is a part of the TCR protein that is not involved in antigen recognition, whereas “TCRP” encompasses the entire beta chain of the T-cell receptor, including both the variable region (which binds to antigen) and the constant region (TRBC).Attorney Docket No. WGN0029-401-PC

[0127] The term “target sequence” refers to a sequence of nucleic acids complimentary, for example fully complementary to a gRNA targeting domain. In embodiments, the target sequence is disposed on genomic DNA. In an embodiment the target sequence is adjacent to (either on the same strand or on the complementary strand of DNA) a protospacer adjacent motif (PAM) sequence recognized by a protein having nuclease or other effector activity, e.g., a PAM sequence recognized by Cas9.

[0128] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state. This may be manifested by a decrease in the number of malignant cells, an increase in life expectancy, or amelioration of various physiological symptoms associated with the malignant condition.

[0129] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0130] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to. adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0131] In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”,Attorney Docket No. WGN0029-401-PC“treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0132] In the context of the present invention, “tumor antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.

[0133] In one embodiment “variants” or “functional variants” of a reference antibody show sequence variation at one or more CDRs when compared to corresponding reference CDR sequences. Thus, a functional antibody variant may comprise a functional variant of a CDR. Where the term “functional variant” is used in the context of a CDR sequence, this means that the CDR has at most 2, preferably at most 1 amino acid difference when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) enables the variant antibody to bind to the same target antigen as the reference antibody.

[0134] For example, a variant antibody may comprise: a light chain CDR 1 having at most 1-2 amino acid differences when compared to SEQ ID NO:24; a light chain CDR2 having at most 1-2 amino acid differences when compared to SEQ ID NO:25; a light chain CDR3 having at most 1-2 amino acid differences when compared to SEQ ID NO:26; wherein the variant antibody binds to CD3.

[0135] For example, a variant antibody may comprise: a heavy chain CDR 1 having at most 1-2 amino acid differences when compared to SEQ ID NO:21; a heavy chain CDR2 having at most 1-2 amino acid differences when compared to SEQ ID NO:22; a heavy chain CDR3 having at most 1-2 amino acid differences when compared to SEQ ID NO:23; wherein the variant antibody binds to CD3.

[0136] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0137] The term “4- IBB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Ace. No. AAA62478.2, or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In one aspect, the “4- IBB costimulatory domain”.Attorney Docket No. WGN0029-401-PCTreatment Applications

[0138] Compositions of matter disclosed herein can be used in the treatment or prevention of progression of proliferative diseases such as cancers and myelodysplastic syndromes. The cancer may be a hematologic malignancy or solid tumor.

[0139] Hematologic malignancies include leukemias, lymphomas, multiple myeloma, and subtypes thereof. Lymphomas can be classified various ways, often based on the underlying type of malignant cell, including Hodgkin’s lymphoma (often cancers of Reed- Sternberg cells, but also sometimes originating in B cells; all other lymphomas are non-Hodgkin’s lymphomas), nonHodgkin’s lymphomas, B-cell lymphomas, T-cell lymphomas, mantle cell lymphomas, Burkitt’s lymphoma, follicular lymphoma, and others as defined herein and known in the art.Myelodysplastic syndromes comprise a group of diseases affecting immature leukocytes and / or hematopoietic stem cells (HSCs); MDS may progress to AML.

[0140] B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and others as defined herein and known in the art.

[0141] T-cell lymphomas include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and others as defined herein and known in the art.

[0142] Leukemias include acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL) hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.

[0143] Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0144] Solid tumors include melanomas, neuroblastomas, gliomas or carcinomas such as tumors of the brain, head and neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive tract (e.g., ovary), upper digestive tract, gastroesophageal adenocarcinoma (GEA, also known as bowel cancer, colon cancer, or rectal cancer), pancreas, liver, renal system (e.g., kidneys), bladder, prostate and colorectum.

[0145] Head and neck cancers include squamous cell carcinoma of head and neck (SCCHN, a heterogeneous group of epithelial neoplasms that arise from upper aerodigestive tract), softAttorney Docket No. WGN0029-401-PCtissue sarcomas, nasopharyngeal cancer, laryngeal cancer, paranasal sinus cancer, salivary gland cancer, and nasal cavity cancer.

[0146] In embodiments, the CAR recognizes an antigen chosen from: CD2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70, CD123, CD171, CD174, CD274, CD276; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule- 1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3): Tumor- associated glycoprotein 72 (TAG72): CD38: CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (GDI 17); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-llRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit 1; Latent Membrane Protein 2 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5): high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH);Attorney Docket No. WGN0029-401-PCmammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-la);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostate carcinoma tumor antigen- 1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA 17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein (ACRBP); sp32 (OY-TES1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation End products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a: CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like modulecontaining mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75);Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).Attorney Docket No. WGN0029-401-PC

[0147] Treatments and therapeutic (including immunotherapeutic) compositions and kits comprising compositions of matter disclosed herein may be administered in any order or contemporaneously.

[0148] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, who has been diagnosed with, suspected of having, or at risk for developing, or at rick of progressing to a later stage of, cancer. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue.Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans, or other animals such as chickens. For example, the subject can be a human subject. Generally, a safe and effective amount of a therapy is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.

[0149] According to the methods described herein, administration can be intradermal, intramuscular, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, intranasal, epidural, or ophthalmic. Where the therapy is a biologic or cell therapy, the mode of administration will likely be via intravenous injection or infusion.

[0150] Additional features of the CAR molecules, and methods of the invention are described in detail below.I. CAR-T CELLS

[0151] One aspect of the present disclosure encompasses T-cells comprising a chimeric antigen receptor, wherein the T-cells are deficient in an antigen to which the chimeric antigen receptor specifically binds, i.e., fratricide-resistant CAR-T-cells. In some embodiments, editing the genome comprises reducing expression of CD2, CD7, TRBC, CD3E, CD35, CD3^, and / or CD3y to a negligible level. In some embodiments, editing the genome comprises reducing expression of the TCRoc chain (TRAC) to a negligible level.

[0152] A CAR-T-cell is a T-cell that expresses a chimeric antigen receptor. The phrase “chimeric antigen receptor (CAR),” as used herein and generally used in the art, refers to a recombinant fusion protein that has an antigen- specific extracellular domain coupled to anAttorney Docket No. WGN0029-401-PCintracellular domain that directs the cell to perform a specialized function upon binding of an antigen to the extracellular domain. The terms “artificial T-cell receptor,” “chimeric T-cell receptor,” and “chimeric immunoreceptor” may each be used interchangeably herein with the term “chimeric antigen receptor.” Chimeric antigen receptors are distinguished from other antigen binding agents by their ability to both bind MHC-independent antigen and transduce activation signals via their intracellular domain. The extracellular and intracellular portions of a CAR are discussed in more detail below.

[0153] The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. An antigen-specific extracellular domain specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 pM, preferably about 0.1 pM to about 1 pM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An antigenspecific extracellular domain suitable for use in a CAR of the present disclosure may be any antigen-binding polypeptide, a wide variety of which are known in the art, and which may be referred to a tumor-associated antigen (TAA) expressed on cancer cells. In some instances, the antigen-binding domain is a single chain Fv (scFv).

[0154] In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4 IBB (i.e., CD137), CD27 and / or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signalingAttorney Docket No. WGN0029-401-PCdomain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0155] The portion of the CAR of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody or bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989; Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird etal., 1988, Science 242:423-426).

[0156] Other antibody-based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing VaVP) are also suitable for use.Attorney Docket No. WGN0029-401-PC

[0157] In some embodiments, the CARs of the present invention include CARs comprising an antigen binding domain (e.g., antibody or antibody fragment) that binds to an MHC presented peptide. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. A CAR that comprises an antigen binding domain (e.g., a scFv) that targets a specific tumor marker, such as CD7, is also referred to as CD7CAR.

[0158] Other suitable antigens may include T-cell-specific antigens and / or antigens that are not specific to T-cells. In a preferred embodiment, an antigen specifically bound by the chimeric antigen receptor of a CAR-T-cell, and the antigen for which the CAR-T-cell is deficient, is an antigen expressed on a malignant T-cell, more preferably an antigen that is overexpressed on a malignant T-cell in comparison to a non-malignant T-cell.

[0159] For instance, by way of non-limiting example, CD2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70. CD123, CD171, CD174, CD274, CD276. CD319, EGFR. EGFR vIII, GD2. Mesothelin, PSA. FLT3, CEA, HER2, TNFRSF17, IL3RA, SDC1, MS4A1, TNFRSF8, NCAM1, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EPCAM, EPHA2, ERBB2, GPC3, MUC1, PDCD1, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, SLAMF7, PSCA, GPNMB, CSPG4, CD133, TEM1, and DLL3 may be suitable antigens expressed on the surface of a malignant T-cell. The tumor antigen may be a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell.

[0160] In some embodiments, a CAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD2. In some embodiments, a CAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD3. In some embodiments, a CAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD4. In some embodiments, a CAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD5. In some embodiments, a CAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD7. In still yet another embodiment, aAttorney Docket No. WGN0029-401-PCCAR-T-cell of the present disclosure comprises an extracellular domain of a chimeric antigen receptor that specifically binds to CD30.

[0161] As described above, in one embodiment the tumor associated antigen is CD7. CD7 is a T-cell surface membrane-associated glycoprotein. CD7 may be overexpressed in T-cell malignancies including T-cell lymphoblastic lymphoma (T-LBL), T-cell acute lymphoblastic leukemia (T-ALL) and non-Hodgkin’s T-cell lymphoma (NHL). CAR-T-cells of the present disclosure may be used to target malignant T-cells that overexpress CD7.

[0162] A chimeric antigen receptor of the present disclosure also comprises an intracellular domain that provides an intracellular signal to the T-cell upon antigen binding to the antigenspecific extracellular domain. The intracellular signaling domain of a chimeric antigen receptor of the present disclosure is responsible for activation of at least one of the effector functions of the T-cell in which the chimeric receptor is expressed.

[0163] While usually the entire intracellular domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0164] Typically, the antigen- specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a transmembrane domain. A transmembrane domain traverses the cell membrane, anchors the CAR to the T-cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the T-cell surface. Chimeric antigen receptors may also further comprise one or more costimulatory domain and / or one or more spacer. A costimulatory domain is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and / or persistence in vivo. A spacer connects (i) the antigen- specific extracellular domain to the transmembrane domain, (ii) the transmembrane domain to a costimulatory domain, (iii) a costimulatory domain to the intracellular domain, and / or (iv) the transmembrane domain to the intracellular domain. For example, inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain may affectAttorney Docket No. WGN0029-401-PCflexibility of the antigen-binding domain and thereby CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art.

[0165] In some embodiments, the antigen of the T-cell is modified such that the chimeric antigen receptor no longer specifically binds the modified antigen. For example, the epitope of the antigen recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen. In other embodiments, expression of the antigen is reduced in the T-cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. Methods for decreasing the expression of a protein are known in the art and include, but are not limited to, modifying or replacing the promoter operably linked to the nucleic acid sequence encoding the protein.

[0166] In still other embodiments, the T-cell is modified such that the antigen is not expressed, e.g., by deletion or disruption of the gene encoding the antigen. In each of the above embodiments, the CAR-T-cell may be deficient in one or preferably all the antigens to which the chimeric antigen receptor specifically binds. Methods for genetically modifying a T-cell to be deficient in an antigen are well known in art, and non-limiting examples are provided above. In an exemplary embodiment, CRISPR / Cas9 gene editing can be used to modify a T-cell to be deficient in an antigen, for example as described herein in the Methods for Examples 2 and 5, and in PCT / US2017 / 045304, which is incorporated by reference herein in its entirety.

[0167] CAR-T-cells encompassed by the present disclosure may further be deficient in endogenous T-cell receptor (TCR) signaling. In various embodiments it may be desirable to decrease or eliminate endogenous TCR signaling in CAR-T-cells disclosed herein. For example, decreasing or eliminating endogenous TCR signaling in CAR-T-cells may prevent or reduce graft versus host disease (GvHD) when allogenic T-cells are used to produce the CAR-T-cells. Methods for decreasing or eliminating endogenous TCR signaling are known in the art and include, but are not limited to, modifying a part of the TCR receptor (e.g., the TCR receptor a chain (TRAC), etc.). TRAC modification may block TCR mediated signaling. TRAC modification may thus permit the safe use of allogeneic T-cells as the source of CAR-T-cells without inducing life-threatening GvHD.

[0168] Alternatively, or in addition, CAR-T-cells encompassed by the present disclosure may further comprise one or more suicide genes. As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a CAR-T-cell by standard methods known in the art that,Attorney Docket No. WGN0029-401-PCwhen activated, results in the death of the CAR-T-cell. Suicide genes may facilitate effective tracking and elimination of the CAR-T-cells in vivo if required. Facilitated killing by activating the suicide gene may occur by methods known in the art. Suitable suicide gene therapy systems known in the art include, but are not limited to, various the herpes simplex virus thymidine kinase (HSV-tk) / ganciclovir (GCV) suicide gene therapy systems or inducible caspase 9 protein. In an exemplary embodiment, a suicide gene is a CD34 / thymidine kinase chimeric suicide gene.

[0169] In an exemplary embodiment, the disclosure provides a T-cell comprising a chimeric antigen receptor that specifically binds CD7, wherein the T-cell is deficient in CD7 (e.g., CD7ACART7 cell). In non-limiting examples the deficiency in CD7 resulted from (a) modification of CD7 expressed by the T-cell such that the chimeric antigen receptor no longer specifically binds the modified CD7. (b) modification of the T-cell such that expression of the antigen is reduced in the T-cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the T-cell such that CD7 is not expressed (e.g., by deletion or disruption of the gene encoding CD7). In further embodiments, the T-cell comprises a suicide gene and / or a modification such that endogenous T-cell receptor (TCR) mediated signaling is blocked in the T-cell. In non-limiting examples the suicide gene expressed in the CD7ACART7 cells encodes a modified Human-Herpes Simplex Virus- 1 -thymidine kinase (TK) gene fused inframe to the extracellular and transmembrane domains of the human CD34 cDNA and the modification resulting in blocked TCR is a modification to endogenous T-cell Receptor Alpha Chain (TRAC).

[0170] In another exemplary embodiment, the disclosure provides a T-cell comprising a chimeric antigen receptor that specifically binds CD2, wherein the T-cell is deficient in CD2 (e.g., CD2ACART2 cell). In non-limiting examples the deficiency in CD2 resulted from (a) modification of CD2 expressed by the T-cell such that the chimeric antigen receptor no longer specifically binds the modified CD2, (b) modification of the T-cell such that expression of the antigen is reduced in the T-cell by at least 50%, at least 60%, at least 70%, at least 80%. at least 90% or more, or (c) modification of the T-cell such that CD2 is not expressed (e.g., by deletion or disruption of the gene encoding CD2). In further embodiments, the T-cell comprises a suicide gene and / or a modification such that endogenous T-cell receptor (TCR) mediated signaling is blocked in the T-cell. In non-limiting examples the suicide gene expressed in the CD2ACART2 cells encodes a modified Human-Herpes Simplex Virus- 1 -thymidine kinase (TK) gene fused inAttorney Docket No. WGN0029-401-PCframe to the extracellular and transmembrane domains of the human CD34 cDNA and the modification resulting in blocked TCR is a modification to endogenous T-cell Receptor Alpha Chain (TRAC).

[0171] In an exemplary embodiment, the tumor associated antigen (TAA)-binding domain comprises an scFv which binds a TAA chosen from CD2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70, CD123. CD171, CD174, CD274. CD276, CD319, EGFR, EGFR vIII, GD2, Mesothelin, PSA, FLT3, CEA, HER2, TNFRSF17, IL3RA, SDC1, MS4A1, TNFRSF8, NCAM1, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EPCAM, EPHA2, ERBB2, GPC3, MUC1, PDCD1, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, SLAMF7, PSCA, GPNMB, CSPG4, CD133, TEM1, and DLL3.

[0172] Methods for CAR design, delivery and expression in T-cells, and the manufacturing of clinical-grade CAR-T-cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res., 2012, 18(10): 2780-90, hereby incorporated by reference in its entirety. For example, the engineered CARs may be introduced into T-cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. An exemplary method for the viral vector production is described herein in the Methods to Example 4. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., type I. type II, or type III systems using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, etc.).

[0173] CAR-T-cells may be generated from any suitable source of T-cells known in the art including, but not limited to, T-cells collected from a subject. The subject may be a patient with a T-cell malignancy in need of CAR-T-cell therapy or a subject of the same species as the subject with the T-cell malignancy in need of CAR-T-cell therapy. The subject may also be a healthy donor. The collected T-cells may be expanded ex vivo using methods commonly known in the art before transduction with a CAR to generate a CAR-T-cell.Attorney Docket No. WGN0029-401-PC

[0174] The use of autologous T-cells for the generation of CAR-T-cells, while possible, may present unique challenges. The subjects in need of CAR-T-cell therapy may be undergoing treatment for malignancies and this treatment may have affected the number and function of T-cells of the patient, thereby reducing the number of T-cells that may be efficiently engineered into CAR-T-cells. Additionally, T-cell hematologic malignancies and normal T effectors may co-express many of the same surface antigens making it very difficult to purify normal T effectors away from the malignant T-cells for genetic editing and lentiviral transduction. Also, if the process of purification is not absolute, there may be a risk of deleting the target antigen such as CD7 in the malignant T-cells resulting in the generation of a population of contaminating T-cell cancers that are potentially resistant to the fratricide CAR-T-cell. Thus, to avoid contamination risk of normal effector T-cells with malignant T-cell, the use of patient-derived T-cells to generate CAR-T-cells for T-cell malignancies may not be desirable.

[0175] To overcome the contamination risk, T-cells from another subject (a healthy donor subject), without T-cells malignancies may be used to generate CAR-T-cells for allogeneic therapy. The T-cells for allogeneic therapy may be collected from a single subject or multiple subjects. Methods of collecting blood cells, isolating and enriching T-cells, and expanding them ex vivo may be by methods known in the art.

[0176] In an exemplary embodiment, the CAR for a CD7 specific CAR T-cell may be generated by cloning a commercially synthesized anti-CD7 single chain variable fragment (scFv) into a 3rd generation CAR backbone with CD28 and 4-1 BB internal signaling domains. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. An exemplary method of generating a CAR specific for CD7 is described herein in the methods for Example 5. A similar method may be followed for making CARs specific for other malignant T-cell antigens, such as CD2, CD3, CD4. CD5, CD7, CD19, CD20 and / or CD30.

[0177] In a further aspect, a CAR-T-cell control may be created. The control CAR-T-cell may include an extracellular domain that binds to an antigen not expressed on a malignant T-cell. The antigen the control CAR-T-cell control binds to may be CD19. CD19 is an antigen expressed on B cells but not on T-cells, so a CAR-T-cell with an extracellular domain adapted to bind to CD 19 will not bind to T-cells. These CAR-T-cells may be called CART 19 cells and may be used as controls to analyze the binding efficiencies and non-specific binding of CART7 cells.Attorney Docket No. WGN0029-401-PCII. METHOD OF USING CAR-T CELLS

[0178] In another aspect, the present disclosure provides a method of killing a malignant T-cell, the method comprising contacting the malignant T-cell with an effective amount of a T-cell comprising a chimeric antigen receptor (CAR-T-cell), wherein the CAR-T-cell is deficient in an antigen to which the chimeric antigen receptor specifically binds, and wherein the chimeric antigen receptor specifically binds an antigen expressed on a malignant cell. In various embodiments, the malignant cell is a malignant T-cell. In further embodiments, the antigen is CD2, CD3, CD4, CD5, CD7, CD 19, CD20 and / or CD30, or any combination thereof. Suitable CAR-T-cells are described in detail herein. In exemplary embodiments, the CAR-T-cells may be CD2ACART2 cells, CD4ACART4 cells. CD5ACART5 cells, CD7ACART7 cells CD30ACART30 cells, or any combination thereof.

[0179] Contacting a malignant cell with an effective amount of a CAR-T-cell generally involves admixing the CAR-T-cell and the malignant cell for a period of time sufficient to allow the chimeric antigen receptor of the CAR-T-cell to bind its cognate antigen on the surface of the malignant cell. This may occur in vitro or ex vivo.

[0180] In another aspect, the present disclosure provides a method for treating a subject having a T-cell malignancy. In some embodiments, the T-cell malignancy is a hematological malignancy. In some embodiments, the T-cell malignancy is T-cell lymphoblastic lymphoma (T-LBL), T-cell acute lymphoblastic leukemia (T-ALL) or a T-cell non-Hodgkin’s lymphoma (T-NHL). The method comprises administering to the subject a therapeutically effective amount of plurality of chimeric antigen receptor T (CAR-T) cells, each CAR-T-cell comprising the same chimeric antigen receptor, wherein the CAR-T-cells are deficient in an antigen specifically recognized by the chimeric antigen receptor, and wherein the chimeric antigen receptor specifically binds an antigen expressed on a malignant T-cell. In various embodiments, the antigen may be CD2, CD3, CD4, CD5, CD7, CD19, CD20 and / or CD30, or any combination thereof. Suitable subjects include any mammal, preferably a human. Suitable CAR-T-cells are described in detail herein. In exemplary embodiments, the CAR-T-cells may be CD2ACART2 cells, CD7ACART7 cells, CD5ACART5 cells, CD30ACART30 cells, CD4ACART4 cells, or any combination thereof. The method may comprise allogenic CAR-T-cell therapy or autologous CAR-T-cell therapy, though allogenic CAR-T-cell therapy may be preferred for the reasonsAttorney Docket No. WGN0029-401-PCdiscussed herein. The CAR-T-cell therapy may be accompanied by other therapies, including but not limited to immunotherapy, chemotherapy or radiation therapy.

[0181] In another aspect, the present disclosure provides a method for treating a subject having a non-T-cell myeloid or lymphoid malignancy. The method comprises administering to the subject a therapeutically effective amount of a plurality of chimeric antigen receptor T (CAR-T) cells, each CAR-T-cell comprising the same chimeric antigen receptor, wherein the CAR-T-cells are deficient in an antigen specifically recognized by the chimeric antigen receptor, and wherein the chimeric antigen receptor specifically binds an antigen expressed on the non-T-cell myeloid or lymphoid malignancy. Suitable subjects include any mammal, preferably a human. Suitable CAR-T-cells are described in detail herein. In an exemplary embodiment the CAR-T-cells are CD7ACART7 cells. The method may comprise allogenic CAR-T-cell therapy or autologous CAR-T-cell therapy, though allogenic CAR-T-cell therapy may be preferred for the reasons discussed herein. The CAR-T-cell therapy may be accompanied by other therapies, including but not limited to immunotherapy, chemotherapy or radiation therapy.

[0182] In another aspect, the present disclosure provides a method for preventing or reducing graft versus host disease in subject in need of CAR-T-cell therapy. In some embodiment, the subject in need of CAR-T-cell therapy is a subject with a T-cell malignancy, a non-T-cell myeloid malignancy, or lymphoid malignancy. The method comprises administering to the subject a therapeutically effective amount of a plurality of chimeric antigen receptor T (CAR-T) cells, each CAR-T-cell comprising (a) the same chimeric antigen receptor and (b) a suicide gene and / or a modification such that endogenous T-cell receptor (TCR) mediated signaling is blocked in the CAR-T-cell; wherein the CAR-T-cells are deficient in an antigen specifically recognized by the chimeric antigen receptor, and wherein the chimeric antigen receptor specifically binds an antigen expressed on the malignancy. In various embodiments, the malignant cell is a malignant T-cell. In further embodiments, the antigen is CD2, CD3, CD4, CD5, CD7, CD19, CD20 and / or CD30, or any combination thereof. Suitable subjects include any mammal, preferably a human. The method may comprise allogenic CAR-T-cell therapy or autologous CAR-T-cell therapy, though allogenic CAR-T-cell therapy may be preferred for the reasons discussed herein. The CAR-T-cell therapy may be accompanied by other therapies, including but not limited to immunotherapy, chemotherapy or radiation therapy.Attorney Docket No. WGN0029-401-PC

[0183] In another aspect, the present disclosure provides a method for preventing or reducing alloreactivity in a subject in need of allogenic CAR-T-cell therapy. In some embodiment, the subject in need of allogenic CAR-T-cell therapy is a subject with a T-cell malignancy, a non-T-cell myeloid malignancy, or lymphoid malignancy. The method comprises administering to the subject a therapeutically effective amount of a plurality of chimeric antigen receptor T (CAR-T) cells, each CAR-T-cell comprising (a) the same chimeric antigen receptor and (b) a suicide gene and / or a modification such that endogenous T-cell receptor (TCR) mediated signaling is blocked in the CAR-T-cell; wherein the CAR-T-cells are deficient in an antigen specifically recognized by the chimeric antigen receptor, and wherein the chimeric antigen receptor specifically binds an antigen expressed on the malignancy. In various embodiments, the malignant cell is a malignant T-cell. In further embodiments, the antigen is CD2, CD3, CD4, CD5, CD7. CD 19. CD20 and / or CD30, or any combination thereof. Suitable subjects include any mammal, preferably a human. The CAR-T-cell therapy may be accompanied by other therapies, including but not limited to immunotherapy, chemotherapy or radiation therapy.

[0184] In various embodiments of the above aspects, the plurality of CAR-T-cells may be a plurality of CD2ACART2 cells, a plurality of CD4ACART4 cells, a plurality of CD5ACART5 cells, a plurality of CD7ACART7 cells, a plurality of CD30ACART30 cells, or any combination thereof. In further embodiments, the CAR-T-cells may comprise a suicide gene and / or a modification such that endogenous T-cell receptor (TCR) mediated signaling is blocked in the CAR-T-cell.

[0185] CAR-T-cells may be administered to a subject by an intravenous route, for instance, by an intravenous infusion. The CAR-T-cells may be administered in a single dose or in multiple doses. The CAR-T-cells may be injected in a pharmaceutical composition suitable for intravenous administration. Suitable pharmaceutical compositions for IV administration are known in the art. A pharmaceutical composition of the present disclosure may further comprise additional components. For instance, such components may be used to sustain the viability and / or activity of injected CAR-T-cells. In one embodiment, the CAR-T-cell composition may include IL-2 to sustain the CAR-T-cells.

[0186] The CAR-T-cells may be administered in effective doses. The effective dose may be either one or multiple doses and are sufficient to produce the desired therapeutic effect. A typical dose of CAR-T-cells may range from about 1.0 x 105- 1.0 x 109cells / Kg body weight of theAttorney Docket No. WGN0029-401-PCsubject receiving therapy, and all integer values within this range, such as, for example 900 million cells provided as a flat dose intravenous infusion. The effective dose may be calculated based on the stage of the malignancy, the health of the subject, and the type of malignancy. In the situation where multiple doses are administered, that dose and the interval between the doses may be determined based on the subject’s response to therapy.

[0187] As used herein, the term “CDS” has its general meaning in the art and refers to the protein complex associated with the T-cell receptor. It is composed of four distinct chains. In mammals, the complex contains two CD3e chains, a CD3y chain, a CD38 chain, and two CD3g chains.

[0188] As used herein, the term “anti-CD3 antibody” include antibodies and antigen-binding fragments thereof, that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies, and antigen-binding fragments thereof, that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). An anti-CD3 antibody of the present disclosure may bind to the epsilon (e) chain of the TCR-CD3 complex present at the surface of all peripheral T-cells.

[0189] A number of anti-CD3 antibodies are known, including but not limited to:- hOKT3Yl (teplizumab) (MGA031) (Herold et al., NEJM 346 (22): 1692- 1698 (2002)) OKT3 (muromonab) Orthoclone OKT3TM, Ortho Biotech, Raritan, NJ; U. S. Patent No.4,361,549)- gOKT3-5 (Alegre et al., J. Immunol. 148 ( 11):3461-8 (1992) U. S. Patent No. 5,885,573) - 28F11 (foralumab)- TRX4 (otelixizumab)- HuM291 (visilizumab) (Nuvion™, Protein Design Labs, Fremont, CA)- 1F4 (Tanaka et al., J. Immunol. 142: 2791-2795 (1989)- G4. I8 (Nicolls etal., Transplantation 55:459-468 (1993)145-2C11 (Davignon et aL, J. Immunol. 141 (6): 1848-54 (1988)); and as describedin Frenken et al., Transplantation 51 (4):881-7 (1991); U. S. Patent Nos. 6,491,9116, 6,406,696, and 6,143,297).

[0190] In some embodiments, the anti-CD3 antibody of the present disclosure comprises a variable heavy (VH) and a variable light (VL) domain selected from Table 2.Table 2Attorney Docket No. WGN0029-401-PCName VH domain (HCVR) VL domain (LCVR)teplizumab QVQLVQ SGGGWQP GRS LRL SCKASGYTFTRYT DIQMTQSPSSLSASVGDRVTITCSASSSVSY MHWVRQAP GKGLEWI G YINPSRGYTN YNQKVKD MNWYQQTPGKAPKRWIYDTSKLASGVPSRFS RFTTSRDNSKNTAFLQMDSLRPEDTGVYFC GSGSGTDYTFTI SSLQPEDIATYYC ARYYDDHYCLDYWGQGTPVTVS S QQWSSNPFTFGQGTKLQIT(SEQ ID NO: 9) (SEQ ID NO: 10)muromonab QVQLQQSGAELARPGASVKMSCKASGYTFTRYT QIVLTQSPAIMSASPGEKVTMTCSASSSVSY (OKT3) MHWVKQRP GQGLEWI GYINPSRGYTN YNQKFKD MNWYQQKSGTSPKRWIYDTSKLASGVPAHFR KATLTTDKSSSTAYMQLSSLTSEDSAVYYC GSGSGTSYSLTISGMEAEDAATYYC ARYYDDHYCLDYWGQGT T LTVS S QQWSSNPFTFGSGTKLEIK(SEQ ID NO: 11) (SEQ ID NO: 12)gOKT3-5 QVQLVQSGGGWQPGRSLRLSCKASGYTFTRYT QIVMTQSPSSLSASVGDRVTITCSASSSVSY MHWVRQAP GKGLEWI GYINPSRGYTN YNQKFKD MNWYQQTPGKAPKRWIYDTSKLASGVPSRFS RFTISTDKSKSTAFLQMDSLRPEDTAVYYC GSGSGTDYTFTI SSLQPEDIATYYC ARYYDDHYCLDYWGQGTPVTVS S QQWSSNPFTFGQGTKLQITR(SEQ ID NO: 13) (SEQ ID NO: 14 )visilizumab QVQLVQSGAEVKKPGASVKVSCKASGYT DIQMTQSPSSLSASVGDRVTITCSASSS FISYTMHWVRQAPGQGLEWMGYINPRSG VSYMNWYQQKPGKAPKRLI YDTSKLASG YTHYNQKLKDKATLTADKSASTAYMELS VPSRFSGSGSGTDFTLTISSLQPEDFAT S LRS E D T AVY YCARSAYYDYDGFAYWGQ YYCQQWSSNPPTFGGGTKVEIK GTLVTVSS (SEQ ID NO: 15) (SEQ ID NO: 16)foralumab QVQLVESGGGWQPGRSLRLSCAASGFK EIVLTQSPATLSLSPGERATLSCRASQS FSGYGMHWVRQAPGKGLEWVAVIWYDGS VSSYLAWYQQKPGQAPRLLIYDASNRAT KKYYVDSVKGRFTI SRDNSKNTLYLQMN GIPARFSGSGSGTDFTLTI SSLEPEDFA S LRAE D T AVY YCARQMGYWHFDLWGRGT VYYCQQRSNWPPLTFGGGTKVE I K LVTVSS (SEQ ID NO: 17 ) (SEQ ID NO: 18 )otelixizumab EVQLLE S GGGLVQP GGS LRL S CAASGFT DIQLTQPNSVSTSLGSTVKLSCTLSSGN FSSFPMAWVRQAPGKGLEWVS TISTSGG IENNYVHWYQLYEGRSPTTMI YDDDKRP RTYYRDSVKGRFTI SRDNSKNTLYLQMN DGVPDRFSGSIDRSSNSAFLTIHNVAIE S LRAE D T AVY YCAKFRQYSGGFDYWGQG DEAI YFCHSYVSSFNVFGGGTKLTVL TLVTVSS (SEQ ID NO: 19) (SEQ ID NO: 20 )In some embodiments, the anti-CD3 antibody of the present disclosure is teplizumab having a heavy chain as set forth in SEQ ID NO:9 and a light chain as set forth in SEQ ID NO: 10.Attorney Docket No. WGN0029-401-PCIn some embodiments, the anti-CD3 antibody of the present disclosure is muromonab having a heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO:12.The amino acid sequences of exemplary heavy and light chain complementarity determining regions (CDRs) are shown in Table 3.Table 3Sequence CDR Residues Ab NameSEQ ID NO: 21 HCDR1 ( IMGT ) GYTFTRYT tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 27 HCDR1 ( IMGT ) GYTFI SYT visilizumabSEQ ID NO: 33 HCDR1 ( IMGT ) GFKFSGYG for alum abSEQ ID NO: 39 HCDR1 ( IMGT ) GFTFSGYG otelixi zumabSEQ ID NO: 22 HCDR2 ( IMGT ) INPSRGYT tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 28 HCDR2 ( IMGT ) INPRSGYT visilizumabSEQ ID NO: 34 HCDR2 ( IMGT ) IWYDGSKK f oralumabSEQ ID NO: 40 HCDR2 ( IMGT ) ISTSGGRT otelixi zumabSEQ ID NO: 23 HCDR3 ( IMGT ) ARYYDDHYCLDY tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 29 HCDR3 ( IMGT ) ARSAYYDYDGFAY visilizumabSEQ ID NO: 35 HCDR3 ( IMGT ) ARQMGYWHFDL f oralumabSEQ ID NO: 41 HCDR3 ( IMGT ) AKFRQYSGGFDY otelixizumabSEQ ID NO: 24 LCDR1 ( IMGT ) SSVSY tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 30 LCDR1 ( IMGT ) SSVSY visilizumabSEQ ID NO: 36 LCDR1 ( IMGT ) QSVSSY f oralumabSEQ ID NO: 42 LCDR1 ( IMGT ) SGNIENNY otelixizumabSEQ ID NO: 25 LCDR2 ( IMGT ) DTS tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 31 LCDR2 ( IMGT ) DTS visilizumabSEQ ID NO: 37 LCDR2 ( IMGT ) DAS f oralumabSEQ ID NO: 43 LCDR2 ( IMGT ) DDD otelixi zumabSEQ ID NO: 26 LCDR3 ( IMGT ) QQWSSNPFT tepli zumab / OKT3 / gOKT3-5 SEQ ID NO: 32 LCDR3 ( IMGT ) QQWSSNPPT visilizumabSEQ ID NO: 38 LCDR3 ( IMGT ) QQRSNWPPLT f oralumabSEQ ID NO: 44 LCDR3 ( IMGT ) HSYVSSFNV otelixizumab

[0191] Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fc region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3, respectively. An antigen-binding site, therefore, typicallyAttorney Docket No. WGN0029-401-PCincludes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat etal., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al ').

[0192] In certain embodiments, the CDRs are identified using the IMGT numbering system (the international ImMunoGeneTics information system®) as described on the website “www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.htm]”, also accessible at “www.imgt.org” (accessed on November 3, 2024). The IMGT numbering system is used in the present specification.

[0193] The Kabat and IMGT residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the Kabat and IMGT numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.

[0194] The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (HCDR1), residues 50-65 (HCDR2) and residues 95-102 (HCDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (LCDRl ), residues 50-56 (LCDR2) and residues 89-97 (LCDR3) according to the Kabat numbering system.

[0195] The CDRs of the heavy chain variable domain are located at residues 27-38 (IICDR1), residues 56-65 (HCDR2) and residues 105-117 (HCDR3) according to the IMGT numbering system. The CDRs of the light chain variable domain are located at residues 27-38 (LCDRl ), residues 56-65 (LCDR2) and residues 105-117 (LCDR3) according to the IMGT numbering system.

[0196] Exemplary polypeptide chains of heavy and light chain constant regions of the IgGl, IgG2, and IgG4 subclasses are shown below in SEQ ID NO:45 through SEQ ID NO:50 in TableAttorney Docket No. WGN0029-401-PC4. An example of an IgGl mutated sequence has the amino acid sequence set forth in SEQ ID NO:46.Table 4Exemplary Constant DomainsSEQ ID NO: 45 IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS constant region GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN CH1-CH2-CH3 HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTFc underlined KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 46 IgGl heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS constant region GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN (mutated) CH1- HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK CH2-CH3 PKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIFc underlined EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 47 IgG2 heavy chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS constant region GALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVD CH1-CH2-CH3 HKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREFc underlined EQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISV EWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLW TT SEQ ID NO: 48 IgG4 heavy chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS constant region GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVD CH1-CH2-CH3 HKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKD TLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRFc underlined EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLELQLEESCAEAQDGELDGL WTTITIFITLFLLSVCYSATVTFFKVKWIFSSWDLKQTIVPD YRNMIRQGA SEQ ID NO: 49 IgG light chain RTVAAP S VF I FP P S DEQLKSGTASWC LLNNF YP REAKVQWKV constant region DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA kappa CEVTHQGLSSPVTKSFNRGEC(K) CLSEQ ID NO: 50 IgG light chain GQP KANP T VT LF P P S S EE LQANKAT LVC L I S D F YP GAVT VAWK constant region ADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSlambda (X) CL

[0197] In some embodiments, the anti-CD3 antibody of the present disclosure comprises two heavy chains, each heavy chain comprising a variable domain and a constant domain, the heavy chain variable domain comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and two light chains, each light chain comprising a variable domain and a constantAttorney Docket No. WGN0029-401-PCdomain, the light chain variable domain comprising complementarity determining regions LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-CD3 anti body of the present disclosure comprises a heavy chain constant region that is an Ig gamma- 1 chain C region (e.g., NCBI ACCESSION: P01857 ), and a light chain constant region that is an Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).Enumerated Embodiments

[0198] Accordingly, although other embodiments may be found throughout the disclosure, provided herein are the following embodiments.

[0199] Embodiment 1. A method of making a population of genome-edited immune effector cells comprising:a) editing the genome of immune effector cells to reduce expression of T-cell receptor (TCR) to a negligible level: thenb) treating the genome-edited immune effector cells with one or more antibodies, or bivalent binding fragments thereof, which bind to a polypeptide that forms part of the TCR-CD3 complex; and thenc) expanding the edited immune effector cells.

[0200] Embodiment 2. The method of embodiment 1, wherein editing the genome comprises reducing expression of one or more of the TCR ex chain (TRAC), T-cell constant 0 chain (TRBC), CD3e, CD35, CD3^, and / or CD3yto a negligible level.

[0201] Embodiment 3. The method of embodiment 1. wherein editing the genome comprises reducing expression of TCR a chain (TRAC) to a negligible level.

[0202] Embodiment 4. The method of any one of embodiments 1-3, wherein the immune effector cells are chosen from T-cells and iNKT-cells.

[0203] Embodiment 5. The method of any one of embodiments 1-4, wherein the immune effector cells are primary cells derived from healthy donors.

[0204] Embodiment 6. The method of any one of embodiments 1-5, wherein the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

[0205] Embodiment 7. The method of any one of embodiments 1-6, wherein the immune effector cells are T-cells.Attorney Docket No. WGN0029-401-PC

[0206] Embodiment 8. The method of embodiment 7, wherein the T-cells are cytotoxic T-cells.

[0207] Embodiment 9. The method of any one of embodiments 1-8, wherein the one or more antibodies, or bivalent binding fragments thereof, bind CD3E, CD35, CD3y, and / or TCR[3; or binds CD3e.

[0208] Embodiment 10. The method of any one of embodiments 1-10, wherein the one or more antibodies, or bivalent binding fragments thereof, are soluble.

[0209] Embodiment 11. The method of any one of embodiments 1-11, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises human or humanized intervening framework regions.

[0210] Embodiment 12. The method of any one of embodiments 9-11, wherein the polypeptide is CD3E.

[0211] Embodiment 13. The method of any one of embodiments 1-12, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 polypeptides and the LCVR comprises LCDR1, LCDR2, and LCDR3 polypeptides wherein:a. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 wherei. Xi is Y or F;ii. X? is T or K;iii. Xs is T, l or S;iv. X4 is R, S or G: andv. X5 is T or G:b. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 wherei. Xiis N, W or S;ii. Xz is P, Y orT;iii. X3 is S, R or D;iv. X4 is R, S or G;v. X5 is S or G;vi. Xe is Y, K or R; andvii. X7 is T or K:Attorney Docket No. WGN0029-401-PCc. HCDR3 is A-R-XI-X2-X?-X4-X5-Y-X6-X7X8-X9wherei. Xi is Y or S:ii. X2 is Y or A:iii. X3 is D or Y;iv. X4 is D or Y;v. X5is H or D;vi. X<5 is C or D;vii. X7 is L or G:viii. X8is D or F; andix. X9 is Y or A;d. LCDR1 is X1-S-V-S-X2 wherei. Xi is S or Q andii. X? is Y or S;e. LCDR2 is D-X1-X2 where Xi is T, A or D: and X2 is S or D; and f. LCDR3 is Q-Q-X1-S-X2-X3-P-X4-X5 wherei. Xi is W or R;ii. X2 is S or N:iii. Xa is N or W;iv. X4 is F or P; andv. X5 is T or L.

[0212] Embodiment 14. The method of embodiment 13, wherein:a. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 wherei. Xi is Y;ii. X2 is T or K;iii. X3 is T;iv. X4 is R;v. X5 is T or G.

[0213] Embodiment 15. The method of any one of embodiments 13-14, wherein:b. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 wherei. Xi is N, W or S;ii. X2is P, Y or T;Attorney Docket No. WGN0029-401-PCiii. X3 is S, R orD;iv. X4 is R;v. X5 is S or G:vi. Xg is Y, K or R; andvii. X? is T or K.

[0214] Embodiment 16. The method of any one of embodiments 13-15. wherein:c. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9wherei. Xi is Y;ii. X2 is Y;iii. X3 is D;iv. X4 is D;v. X5 is H;vi. Xg is C or D;vii. X7 is L;viii. Xg is D or F; andix. X9 is Y or A.

[0215] Embodiment 17. The method of any one of embodiments 13-16, wherein:d. LCDR3 is Q-Q-X1-S-X2-X3-P-X4-X5 wherei. Xi is W or R;ii. X2 is S or N;iii. Xj is N orW;iv. X4 is F; andv. X5 is T or L.

[0216] Embodiment 18. The method of any one of embodiments 1-13, wherein:a) HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26); b) HCDR1 is GYTFISYT (SEQ ID NO:27), HCDR2 is INPRSGYT (SEQ ID NO:28), HCDR3 is ARSAYYDYDGFAY (SEQ ID NO:29), LCDR1 is SSVSY (SEQ ID NO:30), LCDR2 is DTS (SEQ ID NO:31), and LCDR3 is QQWSSNPPT (SEQ ID NO:32);Attorney Docket No. WGN0029-401-PCc) HCDR1 is GFKFSGYG (SEQ ID NO:33), HCDR2 is IWYDGSKK (SEQ ID NO:34), HCDR3 is ARQMGYWHFDL (SEQ ID NO:35), LCDR1 is QSVSSY (SEQ ID NO:36), LCDR2 is DAS (SEQ ID NO:37), and LCDR3 is QQRSNWPPLT (SEQ ID NO:38); or d) HCDR1 is GFTFSGYG (SEQ ID NO:39), HCDR2 is ISTSGGRT (SEQ ID NO:40), HCDR3 is AKFRQYSGGFDY (SEQ ID NO:41), LCDR1 is SGNIENNY (SEQ ID NO:42), LCDR2 is DDD (SEQ ID NO:43), and LCDR3 is HSYVSSFNV (SEQ ID NO:44).

[0217] Embodiment 19. The method of embodiment 18, wherein HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26).

[0218] Embodiment 20. The method of embodiment 19, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10;b) HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12;c) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;d) HCVR of SEQ ID NO: 15 and LCVR of SEQ ID NO: 16;e) HCVR of SEQ ID NO: 17 and LCVR of SEQ ID NO: 18; orf) HCVR of SEQ ID NO: 19 and LCVR of SEQ ID NO:20;or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%. or at least 99% identity to any of the foregoing.

[0219] Embodiment 21. The method of embodiment 20, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10; orb) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;Attorney Docket No. WGN0029-401-PCor are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

[0220] Embodiment 22. The method of any one of embodiments 1-21, wherein the one or more antibodies, or bivalent binding fragments thereof, further comprises an Fc domain and the Fc domain is of an IgGl, IgG2 or an IgG4 subclass.

[0221] Embodiment 23. The method of embodiment 22, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 234 (L234A).

[0222] Embodiment 24. The method of embodiment 22, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 235 (L235A).

[0223] Embodiment 25. The method of any one of embodiments 1-24, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises:a) two heavy chains, each heavy chain comprising a heavy chain variable region (HCVR) and a constant region; andb) two light chains, each light chain comprising a light chain variable region (LCVR) and a constant region.

[0224] Embodiment 26. The method of embodiment 25, wherein the heavy chain constant region has an amino acid sequence as set forth as SEQ ID NO:45 and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID NO:49.

[0225] Embodiment 27. The method of embodiment 9, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise an anti-CD3e antibody chosen from teplizumab, humanized OKT3, muromonab, foralumab, otelixizumab, visilizumab, TR66, SK7, gOKT3-5, UCHT1, 145-2C11, and SP34-2.

[0226] Embodiment 28. The method of any one of embodiments 1-27, wherein the genome-edited immune effector cells express at least one chimeric antigen receptor (CAR) which binds to one or more proteins.

[0227] Embodiment 29. The method of any one of embodiments 1-27, wherein editing the genome comprises editing the genome of the immune effector cells to reduce the expression of the one or more proteins to which the CAR binds.

[0228] Embodiment 30. The method of any one of embodiments 1-29, wherein the population of immune effector cells is activated prior to step a.Attorney Docket No. WGN0029-401-PC

[0229] Embodiment 31. The method of any one of embodiments 1 -30, wherein after 3- 14 days of culture, the percentage of TCRa / p positive T cells is no more than about <1%, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

[0230] Embodiment 32. The method of any one of embodiments 1-30, for use in the manufacture of an allogeneic cell product, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5. 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the percentage of TCRa / p positive T cells is no more than about <1%, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

[0231] Embodiment 33. The method of any one of embodiments 1-31, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5. 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the percentage of TCRa / p positive T cells is no more than about <0.4% per 9.0 x 108cells.

[0232] Embodiment 34. The method of any one of embodiments 1-31, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5. 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the number of TCRa / p positive T cells is no more than 3.6 x 106cells.

[0233] Embodiment 35. The method of any one of embodiments 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL, about 1.0 pg / mL, about 1.5 pg / mL, about 1.6 pg / mL, about 1.7 pg / mL, about 1.8 pg / mL, about 1.9 pg / mL, about 2.0 pg / mL, about 2.2 pg / mL, and about 2.5 pg / mL human or humanized a-CD3 antibody.

[0234] Embodiment 36. The method of any one of embodiments 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from at least 0.5 pg / mL, at least 0.6 pg / mL, at least 0.7 pg / mL, at least 0.8 pg / mL, at least 0.9 pg / mL, at least 1.0 pg / mL, at least 1.1 pg / mL, at least 1.2 pg / mL, at least 1.3 pg / mL, at least 1.4 pg / mL, at least 1.5 pg / mL, at least 1.6 pg / mL, at least 1.7 pg / mL, at least 1.8 pg / mL, at least 1.9 pg / mL, or at least 2.0 pg / mL human or humanized a-CD3 antibody.

[0235] Embodiment 37. The method of any one of embodiments 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL to about 5.0 pg / mL, about 1.0 pg / mL to about 4.0 pg / mL, about 1.0 pg / mL to about 3.0 pg / mL, and about 1.5 to about 2.5 pg / mL.Attorney Docket No. WGN0029-401-PC

[0236] Embodiment 38. The method of any of embodiments 1-37, wherein the human or humanized a-CD3 antibody is chosen from humanized OKT3 and teplizumab.

[0237] Embodiment 39. The method of any one of embodiments 1-38, wherein the time of treating with the antibody is chosen from at least 18 hours after editing and / or transduction, at least 24 hours after editing and / or transduction, at least 36 hours after editing and / or transduction, and at least 48 hours after editing and / or transduction.

[0238] Embodiment 40. The method of embodiment 39, wherein the time of treating with the antibody is chosen from 18-48 hours after editing and / or transduction, 24-48 hours after editing and / or transduction, 36-48 hours after editing and / or transduction, and 24-36 hours after editing and / or transduction.

[0239] Embodiment 41. The method of embodiment 39, wherein the time of treating with the antibody is 24-48 hours after editing and / or transduction.

[0240] Embodiment 42. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from at least 3 days, at least 4 days, at least 5 days, at least 6 days, and at least 7 days.

[0241] Embodiment 43. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 3-11 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, and 3-4 days.

[0242] Embodiment 44. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 4-11 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, and 4-5 days.

[0243] Embodiment 45. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, and 5-6 days.

[0244] Embodiment 46. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 6-11 days, 6-10 days, 6-9 days, 6-8 days, and 6-7 days.

[0245] Embodiment 47. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 7-11 days, 7-10 days, 7-9 days, and 7-8 days.

[0246] Embodiment 48. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is chosen from 8-11 days, 8-10 days, and 8-9 days.Attorney Docket No. WGN0029-401-PC

[0247] Embodiment 49. The method of any one of embodiments 1 -41, wherein incubation time with the a-CD3 antibody is chosen from 9-11 days, and 9-10 days.

[0248] Embodiment 50. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is 10-11 days.

[0249] Embodiment 51. The method of any one of embodiments 1-41, wherein incubation time with the a-CD3 antibody is 3-11 days.

[0250] Embodiment 52. The method of any one of embodiments 1-51, wherein there are no more than 100,000 TCRa / p positive T cells per kilogram patient weight.

[0251] Embodiment 53. The method of any one of embodiments 1-52, wherein there are no more than 70,000 TCRa / p positive T cells per kilogram patient weight.

[0252] Embodiment 54. The method of any one of embodiments 1-53, wherein the method does not comprise a magnetic separation (MACS) step.

[0253] Embodiment 55. The method of any one of embodiments 1-53, wherein the method additionally comprises a magnetic separation (MACS) step.

[0254] Embodiment 56. The method of any one of embodiments 1-55, wherein the T cells are in the resting state.

[0255] Embodiment 57. The method of any one of embodiments 1-55, wherein the T cells are in the activated state.

[0256] Embodiment 58. The method of any one of embodiments 1-55, wherein the T cells are in the partially activated state.

[0257] Embodiment 59. A method of:making a population of genome-edited immune effector cells expressing a chimeric antigen receptor (CAR) which binds to one or more proteins; orselecting TCR-negative immune effector cells from a population of immune effector cells expressing a chimeric antigen receptor (CAR), ormaking a population of at least 80 billion genome-edited immune effector cells expressing a chimeric antigen receptor (CAR) which binds to one or more proteins from one batch of donor cells;the method comprising:a) optionally, activating a population of immune effector cells comprising a T-cell receptor (TCR); thenAttorney Docket No. WGN0029-401-PCb) editing the genome of the immune effector cells to reduce expression of the TCR a chain (TRAC) to a negligible level, and, optionally, editing the genome of the immune effector cells to reduce expression of one or more proteins to which the CAR binds to a negligible level; thenc) transducing the cells with nucleic acid encoding the CAR; thend) treating the edited immune effector cells with one or more antibodies, or bivalent binding fragments thereof, which bind to the extracellular domain of a polypeptide that forms part of the TCR-CD3 complex; and thene) expanding the edited immune effector cells.

[0258] Embodiment 60. The method of embodiment 59, wherein editing the genome comprises reducing expression of the TCR a chain (TRAC), T-cell constant P chain (TRBC), CD3E, CD38, CD3i^, and / or CD3yto a negligible level.

[0259] Embodiment 61. The method of embodiment 59, wherein editing the genome comprises reducing expression of the TCR a chain (TRAC) to a negligible level.

[0260] Embodiment 62. The method of any one of embodiments 59-61, wherein the immune effector cells are chosen from T-cells and iNKT-cells.

[0261] Embodiment 63. The method of any one of embodiments 59-62, wherein the immune effector cells are primary cells derived from healthy donors.

[0262] Embodiment 64. The method of any one of embodiments 59-62, wherein the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

[0263] Embodiment 65. The method of any one of embodiments 59-64, wherein the immune effector cells are T-cells.

[0264] Embodiment 66. The method of embodiment 65, wherein the T-cells are cytotoxic T-cells.

[0265] Embodiment 67. The method of any one of embodiments 59-66, wherein the one or more antibodies, or bivalent binding fragments thereof, bind CD3E, CD38, CD3y, and / or TCR£.

[0266] Embodiment 68. The method of embodiment 67, wherein the one or more antibodies, or bivalent binding fragments thereof, binds CD3e.Attorney Docket No. WGN0029-401-PC

[0267] Embodiment 69. The method of any one of embodiments 59-68, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises human or humanized intervening framework regions.

[0268] Embodiment 70. The method of any one of embodiments 59-69, wherein the one or more antibodies, or bivalent binding fragments thereof, are soluble.

[0269] Embodiment 71. The method of embodiment 70, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein:a. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 wherei. Xj is Y or F;ii. X2 is T or K;iii. X3 is T, l or S;iv. X4 is R, S or G: andv. X5 is T or G:b. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 wherei. Xris N, W or S:ii. X2 is P, Y or T;iii. X3is S, R or D;iv. X4 is R. S or G;v. X5 is S or G;vi. X& is Y, K or R: andvii. X? is T or K;c. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9wherei. Xi is Y or S;ii. X? is Y or A;iii. Xj is D or Y;iv. X4 is D or Y;v. X5is H or D;vi. Xe is C or D;vii. X? is L orG;viii. Xs is D or F; andAttorney Docket No. WGN0029-401-PCix. Xo is Y or A;d. LCDR1 is X1-S-V-S-X2 wherei. Xi is S or Q andii. Xiis Y orS;e. LCDR2 is D-X1-X2 where Xi is T, A or D: and X2is S or D; and f. LCDR3 is Q-Q-X j -S-X2-X3-P-X4-X5 wherei. Xi is W or R;ii. X? is S orN;iii. X3 is N or W;iv. X4 is F or P; andv. X5 is T or L.

[0270] Embodiment 72. The method of embodiment 71, wherein:a. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 wherei. Xi is Y;ii. X? is T or K;iii. Xs is T;iv. X4 is R;v. X5 is T or G.

[0271] Embodiment 73. The method of any one of embodiments 71-72, wherein:b. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 wherei. Xiis N, W or S;ii. X2is P, Y or T;iii. X3is S, R or D;iv. X4 is R;v. X5 is S or G:vs. Xe is Y, K or R; andvii. X? is T or K.

[0272] Embodiment 74. The method of any one of embodiments 71-73, wherein:c. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9where i. Xi is Y;ii. X2is Y;Attorney Docket No. WGN0029-401-PCiii. X3 is D;iv. X4 is D;v. X5 is H;vi. Xg is C or D;vii. X? is L;viii. Xg is D or F; andix. X9 is Y or A.

[0273] Embodiment 75. The method of any one of embodiments 71-74, wherein:d. LCDR3 is Q-Q-X1-S-X2-X3-P-X4-X5 wherei. Xj is W or R;ii. X2 is S or N:iii. X3 is N or W;iv. X4 is F; andv. X5 is T or L.

[0274] Embodiment 76. The method of embodiment 71, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 polypeptides and the LCVR comprises LCDR1, LCDR2, and LCDR3 polypeptides, wherein:a) HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24). LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26);b) HCDR1 is GYTFISYT (SEQ ID NO:27), HCDR2 is INPRSGYT (SEQ ID NO:28), HCDR3 is ARSAYYDYDGFAY (SEQ ID NO:29), LCDR1 is SSVSY (SEQ ID NO:30), LCDR2 is DTS (SEQ ID NO:31), and LCDR3 is QQWSSNPPT (SEQ ID NO:32);c) HCDR1 is GFKFSGYG (SEQ ID NO:33), HCDR2 is IWYDGSKK (SEQ ID NO:34), HCDR3 is ARQMGYWHFDL (SEQ ID NO:35), LCDR1 is QSVSSY (SEQ ID NO:36), LCDR2 is DAS (SEQ ID NO:37), and LCDR3 is QQRSNWPPLT (SEQ ID NO:38); or d) HCDR1 is GFTFSGYG (SEQ ID NO:39), HCDR2 is ISTSGGRT (SEQ ID NO:40), HCDR3 is AKFRQYSGGFDY (SEQ ID NO:41), LCDR1 is SGNIENNY (SEQ ID NO:42), LCDR2 is DDD (SEQ ID NO:43). and LCDR3 is HSYVSSFNV (SEQ ID NO:44).

[0275] Embodiment 77. The method of embodiment 77, wherein HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDYAttorney Docket No. WGN0029-401-PC(SEQ TD NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26).

[0276] Embodiment 78. The method of any one of embodiments 59-77, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10;b) HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12;c) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;d) HCVR of SEQ ID NO: 15 and LCVR of SEQ ID NO: 16;e) HCVR of SEQ ID NO; 17 and LCVR of SEQ ID NO: 18; orf) HCVR of SEQ ID NO: 19 and LCVR of SEQ ID NO:20;or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

[0277] Embodiment 79. The method of embodiment 78, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10; orb) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

[0278] Embodiment 80. The method of any one of embodiments 78-79, wherein the one or more antibodies, or bivalent binding fragments thereof, further comprises an Fc domain and the Fc domain is of an IgGl or an IgG4 subclass.

[0279] Embodiment 81. The method of embodiment 80, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 234 (L234A).

[0280] Embodiment 82. The method of embodiment 80, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 235 (L235A).

[0281] Embodiment 83. The method of any one of embodiments 59-82, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises:Attorney Docket No. WGN0029-401-PCa) two heavy chains, each heavy chain comprising a heavy chain variable region (HCVR) and a constant region; andb) two light chains, each light chain comprising a light chain variable region (LCVR) and a constant region.

[0282] Embodiment 84. The method of embodiment 83, wherein the heavy chain constant region has an amino acid sequence as set forth as SEQ ID NO:47 and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID NO:49.

[0283] Embodiment 85. The method of any one of embodiments 59-84, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise an anti-CD3e antibody chosen from teplizumab, humanized OKT3, muromonab, foralumab, otelixizumab, visilizumab, TR66, SK7, gOKT3-5, UCHT1, 145-2C11, and SP34-2.

[0284] Embodiment 86. The method of any one of embodiments 59-85, wherein the genome-edited immune effector cells express at least one chimeric antigen receptor (CAR) which binds to one or more proteins.

[0285] Embodiment 87. The method of any one of embodiments 59-86, wherein editing the genome of the immune effector cells to delete the one or more proteins to which the CAR binds.

[0286] Embodiment 88. The method of any one of embodiments 59-87, wherein the population of immune effector cells is activated prior to step a.

[0287] Embodiment 89. The method of any one of embodiments 59-88, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5, 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the percentage of TCR / p positive T cells is no more than about <1%, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

[0288] Embodiment 90. The method of any one of embodiments 59-88, for use in the manufacture of an allogeneic cell product, wherein after 3-14 (e.g., 7-14 days, or 3, 4, 5, 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) days of culture, the percentage of TCRa / p positive T cells is no more than about <1%, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

[0289] Embodiment 91. The method of any one of embodiments 59-88, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5, 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the percentage of TCRa / p positive T cells is no more than about <0.4% per 9.0 x 108cells.Attorney Docket No. WGN0029-401-PC

[0290] Embodiment 92. The method of any one of embodiments 59-91, wherein after 3-14 days (e.g., 7-14 days, or 3, 4, 5, 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days) of culture, the number of TCRot / p positive T cells is no more than 3.6 x 106cells.

[0291] Embodiment 93. The method of any one of embodiments 59-92, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL, about 1.0 pg / mL, about 1.5 pg / mL, about 1.6 pg / mL, about 1.7 pg / mL, about 1.8 pg / mL, about 1.9 pg / mL, about 2.0 pg / mL, about 2.2 pg / mL, and about 2.5 pg / mL human or humanized a-CD3 antibody.

[0292] Embodiment 94. The method of any one of embodiments 59-93, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from at least 0.5 pg / mL, at least 0.6 pg / mL, at least 0.7 pg / mL, at least 0.8 pg / mL, at least 0.9 pg / mL, at least 1.0 pg / mL, at least 1.1 pg / mL, at least 1.2 pg / mL, at least 1.3 pg / mL, at least 1.4 pg / mL, at least 1.5 pg / mL, at least 1.6 pg / mL, at least 1.7 pg / mL, at least 1.8 pg / mL, at least 1.9 pg / mL, or at least 2.0 pg / mL human or humanized a-CD3 antibody.

[0293] Embodiment 95. The method of any one of embodiments 59-94, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL to about 5.0 pg / mL, about 1.0 pg / mL to about 4.0 pg / mL, about 1.0 pg / mL to about 3.0 pg / mL, and about 1.5 to about 2.5 pg / mL.

[0294] Embodiment 96. The method of either of embodiments 94 or 95, wherein the human or humanized a-CD3 antibody is chosen from humanized OKT3 and teplizumab.

[0295] Embodiment 97. The method of any one of embodiments 59-96, wherein the time of treating with the antibody is chosen from at least 18 hours after editing and / or transduction, at least 24 hours after editing and / or transduction, at least 36 hours after editing and / or transduction, and at least 48 hours after editing and / or transduction.

[0296] Embodiment 98. The method of embodiment 97, wherein the time of treating with the antibody is chosen from 18-48 hours after editing and / or transduction, 24-48 hours after editing and / or transduction, 36-48 hours after editing and / or transduction, and 24-36 hours after editing and / or transduction.

[0297] Embodiment 99. The method of embodiment 98, wherein the time of treating with the antibody is 24-48 hours after editing and / or transduction.Attorney Docket No. WGN0029-401-PC

[0298] Embodiment 100. The method of any one of embodiments 59-99, wherein incubation time with the a-CD3 antibody is chosen from at least 3 days, at least 4 days, at least 5 days, at least 6 days, and at least 7 days.

[0299] Embodiment 101. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 3-11 days, 3-10 days, 3-9 days. 3-8 days, 3-7 days. 3-6 days, 3-5 days, and 3-4 days.

[0300] Embodiment 102. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 4-11 days, 4-10 days, 4-9 days. 4-8 days, 4-7 days, 4-6 days, and 4-5 days.

[0301] Embodiment 103. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, and 5-6 days.

[0302] Embodiment 104. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 6-11 days, 6-10 days, 6-9 days, 6-8 days, and 6-7 days.

[0303] Embodiment 105. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 7-11 days, 7-10 days, 7-9 days, and 7-8 days.

[0304] Embodiment 106. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 8-11 days, 8-10 days, and 8-9 days.

[0305] Embodiment 107. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is chosen from 9-11 days, and 9-10 days.

[0306] Embodiment 108. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is 10-11 days.

[0307] Embodiment 109. The method of any one of embodiments 59-100, wherein incubation time with the a-CD3 antibody is 3-11 days.

[0308] Embodiment 110. The method of any one of embodiments 59-109, wherein there are no more than 100,000 TCRa / p positive T cells per kilogram patient weight.

[0309] Embodiment 111. The method of any one of embodiments 59-110, wherein there are no more than 70.000 TCRa / p positive T cells per kilogram patient weight.Attorney Docket No. WGN0029-401-PC

[0310] Embodiment 112. The method of any one of embodiments 59-111, wherein the method does not comprise a magnetic separation (MACS) step.

[0311] Embodiment 113. The method of any one of embodiments 59-111, wherein the method additionally comprises a magnetic separation (MACS) step.

[0312] Embodiment 114. The method of any one of embodiments 59-111, wherein the T cells are in the resting state.

[0313] Embodiment 115. The method of any one of embodiments 59-111, wherein the T cells are in the activated state.

[0314] Embodiment 116. The method of any one of embodiments 59-111, wherein the T cells are in the partially activated state.

[0315] Embodiment 117. The method of any one of embodiments 59-116, wherein the one or more proteins bound by the CAR is / are antigen(s) overexpressed on the surface a malignant T-cell.

[0316] Embodiment 118. The method of embodiment 117 wherein the proteins are tumor-associated antigens and chosen from CD2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70, CD123, CD171, CD174, CD274, CD276, CD319, EGFR, EGFR vIII, GD2, Mesothelin, PSA, FLT3, CEA, HER2, TNFRSF17, IL3RA, SDC1, MS4A1, TNFRSF8, NCAM1, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EPCAM, EPHA2, ERBB2, GPC3, MUC1, PDCD1, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, SLAMF7, PSCA, GPNMB, CSPG4, CD133, TEM1, and DLL3.

[0317] Embodiment 119. The method of any one of embodiments 59-118, wherein the CAR is designed to bind to one protein.

[0318] Embodiment 120. The method of embodiment 119, wherein the CAR selectively binds one protein.

[0319] Embodiment 121. The method of embodiment 119, wherein the CAR specifically binds one protein.

[0320] Embodiment 122. The method of embodiment 121, wherein the CAR binds to CD7.

[0321] Embodiment 123. The method of any one of embodiments 59-122, wherein the genome of the immune effector cell is edited to delete the protein to which the CAR binds.

[0322] Embodiment 124. The method of embodiment 123, wherein the genome of the immune effector cell is edited to delete CD7.Attorney Docket No. WGN0029-401-PC

[0323] Embodiment 125. The method of any one of embodiments 59-124, wherein the genome is edited using a CRISPR associated protein (CRISPR / Cas), a transcription activator-like effector nuclease (TALEN), or a zinc-finger nuclease (ZFN).

[0324] Embodiment 126. The method of embodiment 125, wherein the genome is edited using a CRISPR / Cas9 associated protein.

[0325] Embodiment 127. The method of embodiment 125, wherein the genome is edited using a base editor.

[0326] Embodiment 128. The method of embodiment 126, wherein the Cas9 is delivered into the cell as mRNA or protein.

[0327] Embodiment 129. The method of embodiment 128, wherein the Cas9 is delivered into the cell as protein.

[0328] Embodiment 130. The method of embodiment 126, wherein the Cas9 is delivered contemporaneously with at least one guide RNA (gRNA) targeting the one or more gene(s) to be edited.

[0329] Embodiment 131. The method of any one of embodiments 59-130, wherein the population of expanded T-cells contains less than about 40%, less than about 30%. less than about 20%, less than about 10%, or less than about 5% unedited T-cells.

[0330] Embodiment 132. The method of any one of embodiments 59-131, wherein the population of genome-edited chimeric antigen receptor (CAR) T-cells undergoes a sorting step.

[0331] Embodiment 133. The method of any one of embodiments 59-131, wherein the population of genome-edited chimeric antigen receptor (CAR) T-cells does not undergo a sorting step.

[0332] Embodiment 134. The method of any one of embodiments 59-133, wherein the population of genome-edited chimeric antigen receptor (CAR) T-cells is activated for up to five days.

[0333] Embodiment 135. The method of any one of embodiments 59-134, wherein the population of genome-edited chimeric antigen receptor (CAR) T-cells is expanded for greater than or equal to about 3-14 days (e.g., 7-14 days, or 3, 4, 5, 6, 7, 8, 9,k 10, 11, 12, 13 or 14 days).

[0334] Embodiment 136. The method of any one of embodiments 59-135, wherein the method is performed at a temperature of between about 25°C and about 40°C.Attorney Docket No. WGN0029-401-PC

[0335] Embodiment 137. A population of genome-edited CAR-T-cells made by the method of any one of embodiments 59-136.

[0336] Embodiment 138. A method of treating a solid organ tumor or hematologic malignancy in a patient comprising administering the population of genome-edited CAR-T-cells of embodiment 137.

[0337] Embodiment 139. The method of embodiment 138, wherein the hematologic malignancy is a T-cell malignancy.

[0338] Embodiment 140. The method of embodiment 139, wherein the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

[0339] Embodiment 141. The method of embodiment 139, wherein the T-cell malignancy is T-cell lymphoblastic lymphoma (T-LBL).

[0340] Embodiment 142. The method of embodiment 139, wherein the T-cell malignancy is any one of: T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1-positive (HTLV-1 +) adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and various peripheral T-cell lymphomas (PTCLs), angioimmunoblastic T-cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma, or ALK-negative anaplastic large cell lymphoma.

[0341] Embodiment 143. The method of embodiment 139, wherein the T-cell malignancy is non-Hodgkin’s lymphoma.

[0342] Embodiment 144. The method of embodiment 138, wherein the hematologic malignancy is a B-cell malignancy.

[0343] Embodiment 145. The method of embodiment 144, wherein the B-cell malignancy is a B cell lymphoma.

[0344] Embodiment 146. The method of embodiment 144, wherein the B-cell malignancy is a B cell leukemia.

[0345] Embodiment 147. The method of embodiment 144, wherein the hematologic malignancy is a myeloid malignancy.

[0346] Embodiment 148. The method of embodiment 138, wherein the hematologic malignancy is acute myeloid leukemia.

[0347] Embodiment 149. The method of any one of embodiments 1-148, wherein the cells express SEQ ID NO:51.Attorney Docket No. WGN0029-401-PC

[0348] Embodiment 150. The method of any one of embodiments 1-149, wherein the cells comprise DNA encoding SEQ ID NO:51.

[0349] Embodiment 151. The method of any one of embodiments 1-150, wherein the DNA comprises SEQ ID NO:52.

[0350] Embodiment 152. The method of any one of embodiments 1-151, wherein the CAR comprises SEQ ID NO:53.

[0351] Embodiment 153. The method of any one of embodiments 1-152, wherein the population of genome-edited immune effector cells comprise DNA encoding SEQ ID NO:53.

[0352] Embodiment 154. The method of any one of embodiments 1-153, wherein the DNA comprises SEQ ID NO:54.EXAMPLES

[0353] The following examples are intended to further illustrate certain non-limiting embodiments of the disclosure and are not intended to limit in any way the scope of the disclosure.Example 1 - Gene editing of CAR-T-cells

[0354] T-cells can be genetically modified to express chimeric antigen receptors (CARs). which are fusion proteins comprised of an antigen recognition moiety and T-cell activation domains. CAR-T-cells demonstrate exceptional clinical efficacy against B cell malignancies. However, the development of CAR-T therapy against T-cell malignancies has proven problematic, due in part to the expression of target antigens shared between T-cell malignancies and effector T-cells. Expression of target antigens on CAR-T-cells may induce fratricide of CAR-T-cells, leading to loss of efficacy and reduced clinical benefit. Through gene editing of CAR-T-cells, it was demonstrated that efficient deletion of T-cell-specific target antigens that are normally expressed on CAR-T-cells (and on the cell surface of T-cell malignancies) can result in the effective expansion of CAR-T without significant “fratricide” and effective killing of tumor targets using gene edited CAR-T-cells. The development of a T-cell product that has CD7 bi-allelically deleted and which overexpresses a CD7-CAR in T-cells that have been gene-edited to delete CD7 is described in PCT / US2017 / 045304, which is incorporated by reference herein in its entirety.

[0355] This approach can be extended to encompass other T-cell antigens such as CD5, CD4, and CD2 that are expressed on various T-cell cancers and on normal T-cells. In addition,Attorney Docket No. WGN0029-401-PCthe incorporation of a suicide gene in lentiviral and retroviral constructs expressing CARs will not only be used to protect against both insertional mutagenesis and leukemogenesis but also against long term T-cell and NK cell cytopenias. In an embodiment, purified T-cells from peripheral blood are sorted via antibody selection (IE: CD4 / 8 positive selection). Sorted T-cells are activated with anti-CD3 and anti-CD28 reagents (IE: Transact™). T-cells are then cultured in appropriate culture media (IE: Texmacs™ +3% HAB, lOng / mL IL7 / 15).Example 2 - Gene editing of the CD7 locus resulted in loss of CD7 expression

[0356] The adhesion molecule CD7 is highly expressed on T-ALL (T-cell acute lymphoblastic leukemia; 98%) and other T-cell malignancies and proves to be an attractive target for immunotherapy of T-cell cancers. CD7, however, is highly expressed on activated T-cells (>86%). CRISPR / Cas9 was used to delete CD7 expression on CAR-T-cells. Guide RNAs (gRNA) targeting hCD7 were designed and validated for activity by the Washington University genome engineering core. The gRNA with the greatest activity was commercially synthesized, incorporating modified bases (2'0me and phosphorothiate) to increase gRNA stability. To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into a backbone of a 3rd generation CAR with CD28 and 4-1 BB internal signaling domains (provided by Dr. C. June, University of Pennsylvania). The construct was modified to express a cytoplasmic truncation mutant of human CD34 (or the CD34-TK75 chimeric suicide gene; Eissenberg et al. Molecular Therapy, 2015) via a P2A peptide to enable detection of CAR following viral transduction. In an embodiment, human primary T-cells are activated using anti-CD3 / CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA (20pg) and Cas9 mRNA ( 15(tg). T-cells were then rested for 24hrs. On day three, T-cells were transduced with lentivirus particles encoding either CD7-CAR, CD7-CAR-P2A-CD34, CD7-CAR-P2A-CD34-TK75, or control CD19-CAR and allowed to expand for a further 6 days. Transduction efficiency and CD7 ablation was confirmed by flow cytometry. Gene editing of the CD7 locus resulted in loss of CD7 expression in >90% of T-cells compared to control T-cells see, e.g., PCT / US2017 / 045304, which is incorporated by reference herein in its entirety).

[0357] The foregoing methods can be used to edit the loci of other genomic targets, using gRNA appropriate to the targets, to reduce / delete target expression.Attorney Docket No. WGN0029-401-PCExample 3 - CAR Design

[0358] CD7-CAR was generated by using commercial gene synthesis of an anti-CD7 single chain variable fragment (scFv) and cloned into a backbone of a 3rd generation CAR with CD28 and 4-1 BB internal signaling domains. The Efl a pELNS lentiviral plasmid was a kind gift from Dr. Carl June (University of Pennsylvania). The construct was modified to express the extracellular domain of hCD34 via a P2A peptide to enable both detection of CAR following viral transduction and, if required, purification of CAR-T using anti-hCD34 magnetic beads. CART19 were used as a non-targeting control, as described in detail in PCT / US2019 / 035010, which is incorporated by reference herein in its entirety).

[0359] Alternately or in addition, disclosed below are exemplary embodiments of CAR amino acid sequences, additional engineered components, and their encoding polynucleotides.

[0360] Table 6. Sequences of Chimeric Antigen Receptors (CARs) and Additional Engineered ComponentsDescription Sequence SequenceIdentifierAmino Acid SEQ ID NO:51 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDR VTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSG VPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPY TFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVKPGGSLKLSCAASGLTFSS YAMSWVRQTPEKRLE WVAS ISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSL RSEDTAMYYCARDEVRGYLDVWGAGTTVTVSPRASTTTP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDFWVLWVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPRRTDGSGATNFSL LKQAGDVEENPGPVSEAMPRGWTALCLLSLLPSGFMSLD NNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLH PVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQS QTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTST SLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGIC LEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVC SLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLK KLGILDFTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPIAttorney Docket No. WGN0029-401-PCNucleic Acid SEQ ID NO:52 atggccttaccagtgaccgccttgctcctgccgctggcc ttgctgctccacgccgccaggccggatatccagatgaca cagactacatcctccctgtctgcctctctgggagacaga gtcaccatcagttgcagtgcaagtcagggcattagcaat tatttaaactggtatcagcagaaaccagatggaactgtt aaactcctgatctattacacatcaagtttacactcagga gtcccatcaaggttcagtggcagtgggtctgggacagat tattctctcaccatcagcaacctggaacctgaagatatt gccacttattattgtcagcagtatagcaagcttccgtac acgttcggaggggggaccaagctggaaataaaacgtggt ggtggtggttctggtggtggtggttctggcggcggcggc tccggtggtggtggatccgaggtgcaactggtggagtct gggggaggcttagtgaagcctggggggtccctgaaactc tcctgtgcagcctctggactcactttcagtagctatgcc atgtcttgggttcgccagactccagagaagaggctggag tgggtcgcatccattagtagtggtggtttcacctactat ccagacagtgtgaagggccgattcaccatctccagagat aatgccaggaacatcctgtatctgcaaatgagcagtctg aggtctgaggacacggccatgtattactgtgcaagagac gaggtacgggggtacctcgatgtctggggcgcagggacc acggtcaccgtttcccctaggGCTAGCACCACGACGCCA GCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCG CAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCG GCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCC TGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATA T T C AAACAAC CAT T T AT GAGAC CAGT AC AAAC T ACT CAA GAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAGCTAAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTC TATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCCGGACCGATGGAAGCGGAGCTACTAACTTCAGCCTG CTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT GTTTCTGAAGCCATGCCGCGGGGCTGGACCGCGCTTTGC TTGCTGAGTTTGCTGCCTTCTGGGTTCATGAGTCTTGAC AACAACGGTACTGCTACCCCAGAGTTACCTACCCAGGGA ACATTTTCAAATGTTTCTACAAATGTATCCTACCAAGAA ACTACAACACCTAGTACCCTTGGAAGTACCAGCCTGCAC CCTGTGTCTCAACATGGCAATGAGGCCACAACAAACATC AC AGAAAC GACAGT CAAAT T CACAT C T ACC T C T GT GAT A ACCTCAGTTTATGGAAACACAAACTCTTCTGTCCAGTCA CAGACCTCTGTAATCAGCACAGTGTTCACCACCCCAGCC AACGTTTCAACTCCAGAGACAACCTTGAAGCCTAGCCTGTCACCTGGAAATGTTTCAGACCTTTCAACCACTAGCACTAttorney Docket No. WGN0029-401-PCAGCCTTGCAACATCTCCCACTAAACCCTATACATCATCT T C T C CT AT CC T AAGT GAC AT CAAGGC AGAAAT CAAAT GT TCAGGCATCAGAGAAGTGAAATTGACTCAGGGCATCTGC CTGGAGCAAAATAAGACCTCCAGCTGTGCGGAGTTTAAG AAGGACAGGGGAGAGGGCCTGGCCCGAGTGCTGTGTGGG GAGGAGCAGGCTGATGCTGATGCTGGGGCCCAGGTATGC TCCCTGCTCCTTGCCCAGTCTGAGGTGAGGCCTCAGTGT CTACTGCTGGTCTTGGCCAACAGAACAGAAATTTCCAGC AAACTCCAACTTATGAAAAAGCACCAATCTGACCTGAAA AAGCTGGGGATCCTAGATTTCACTGAGCAAGATGTTGCA AGCCACCAGAGCTATTCCCAAAAGACCCTGATTGCACTG GTCACCTCGGGAGCCCTGCTGGCTGTCTTGGGCATCACT GGCTATTTCCTGATGAATCGCCGCAGCTGGAGCCCCAtt taaAmino Acid SEQ ID NO:53 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDR VTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSG VPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPY TFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVKPGGSLKLSCAASGLTFSS YAMSWVRQTPEKRLE WVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSL RSEDTAMYYCARDEVRGYLDVWGAGTTVTVSPRASTTTP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDFWVLWVGGVLACYSLLVTVAFIIFWVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPRNucleic Acid SEQ ID NO:54 atggccttaccagtgaccgccttgctcctgccgctggcc ttgctgctccacgccgccaggccggatatccagatgaca cagactacatcctccctgtctgcctctctgggagacaga gtcaccatcagttgcagtgcaagtcagggcattagcaat tatttaaactggtatcagcagaaaccagatggaactgtt aaactcctgatctattacacatcaagtttacactcagga gtcccatcaaggttcagtggcagtgggtctgggacagat tattctctcaccatcagcaacctggaacctgaagatatt gccacttattattgtcagcagtatagcaagcttccgtac acgttcggaggggggaccaagctggaaataaaacgtggt ggtggtggttctggtggtggtggttctggcggcggcggc tccggtggtggtggatccgaggtgcaactggtggagtct gggggaggcttagtgaagcctggggggtccctgaaactc tcctgtgcagcctctggactcactttcagtagctatgcc atgtcttgggttcgccagactccagagaagaggctggag tgggtcgcatccattagtagtggtggtttcacctactat ccagacagtgtgaagggccgattcaccatctccagagat aatgccaggaacatcctgtatctgcaaatgagcagtctg aggtctgaggacacggccatgtattactgtgcaagagac gaggtacgggggtacctcgatgtctggggcgcagggacc acggtcaccgtttcccctaggGCTAGCACCACGACGCCA GCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCG CAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAttorney Docket No. WGN0029-401-PCTGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT TTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATA T T C AAAC AAC C AT T T AT GAGAC C AGT AC AAAC T AC T C AA GAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAGCTAAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTC T AT AACGAGC T C AAT C T AGGAC GAAGAGAGGAGT AC GAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTAC AATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCExample 4 - Viral vector production

[0361] To produce lentivirus, the Lenti-X 293T Cell Line (Takara Bio, Mountain View. CA) was transfected with CAR lentiviral vector and the packaging plasmids, pMD. Lg / pRRE, pMD. G, pRSV-Rev using the CalPhos™ Mammalian Transfection Kit (Takara) per the manufacturer’s instructions. Virus was harvested 36 hrs post transfection, filtered to remove cell debris, and concentrated by ultracentrifugation for 90 mins at 25000 rpm, 4°C (Optima LE-80K Ultracentrifuge, Beckman Coulter, Indianapolis IN). Virus was re-suspended in phosphate buffered saline, snap frozen in liquid nitrogen and stored at -80°C in single use aliquots.Example 5 - CRISPR / Cas9 gene editing

[0362] Guide RNAs (gRNA) targeting antigens of interest, such as, for example, CD2, CD7 and TRAC, were designed and validated for activity by the Washington University genome engineering core. The gRNA with the greatest activity was commercially synthesized, incorporating modified bases (2'0me and phosphorothiate) to increase gRNA stability. T-cells were cultured in Xcyte media supplemented with 50 micrograms / mL IL-2, 10ng / ml IL-2, 10ng / ml IL-15, and 10ng / ml IL7 in the presence of anti-CD3 / CD28 beads (Bead to cell ratio 3:1). On day +2 post activation, stimulation was removed and 1x107T-cells were electroporated in 100 pl MaxCyte™ buffer containing 15 pg spCas9 (Trilink CA) and 20 pg of each gRNA (IDT) using a MaxCyte™ GT, program ‘expanded T-cell 2’. The cells were transduced with CD2-28(^ or CD 19-28^ lentiviral particles in the presence of polybrene (Sigma Aldrich, St Louis MO) (final concentration 6 pg / ml) on day +3. The cells were then expanded for 6 days followingAttorney Docket No. WGN0029-401-PCtransduction before being used in later experiments, as described in detail in PCT / US2019 / 035010, which is incorporated by reference herein in its entirety).

[0363] Exemplary guide RNAs (gRNA) targeting antigens of interest for use in the above CRISPR / Cas9 gene editing protocol are listed below:CD7 gRNA4:5' 2'0Me(A(ps)U(ps)C(ps))ACGGAGGUCAAUGUCUAGUUUUAGAGCUAGAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGC2'OMe(U(ps)U(ps)U(ps)U_3' (SEQ ID NO:1)CD7 gRNAlO:5' 2'0Me(G(ps)U(ps)A(ps))ACAUUGACCUCCGUGAGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGC2'OMe(U(ps)U(ps)U(ps)U_3' (SEQ ID NO:2)CD2 gRNA:5' 2'0Me(A(ps)C(ps)A(ps))GCUGACAGGCUCGACACGUUUUAGAGCUAGAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGC2'OMe(U(ps)U(ps)U(ps)U_3' (SEQ ID NO:3)TRAC gRNA:5' 2'0Me(G(ps)A(ps)G(ps))AAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAU AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGC2'OMe(U(ps)U(ps)U(ps)U_3' (SEQ ID NO:4)(ps) indicates phosphorothioate. Underlined bases denote targeting domains.

[0364] Other guide RNAs are known in the art and may be used in the methods disclosed herein, for example in WO2018027036.Example 6 - Targeted deep sequencing to confirm editing

[0365] The CD7 locus was amplified with primers forward primer GCCTGCGTGGGATCTACCTGAGGCA (SEQ ID NO:5), and reverse primer AGCTATCTAGGAGGCTGCTGGGGGC (SEQ ID NO:6). The TRAC Locus was amplified with forward primer TGGGGCAAAGAGGGAAATGA (SEQ ID NO:7), and reverse primer GTCAGATTTGTTGCTCCAGGC (SEQ ID NO:8). PCR products were sequenced using theAttorney Docket No. WGN0029-401-PCIllumina MiSeq™ platform (San Diego, CA). Editing efficiencies were determined as a percentage of sequencing reads with indels aligned to reads obtained from WT-cells. Removal of both CD7 and TRAC was successful, described in detail in PCT / US2019 / 035010, which is incorporated by reference herein in its entirety).Example 7 - CAR-Bearing Immune Effector Cells

[0366] CAR-bearing immune effector cells were constructed, optionally with a genome editing step, as described herein, to effect deletion or suppression of one or more surface proteins. Such surface proteins included, for example, CD7 and those that form part of the TCR complex (TRAC) as described in PCT / US2017 / 045304, which is incorporated by reference herein in its entirety. Such surface proteins may induce GvHD if the cells are administered to patients in the allogeneic setting. Additionally, surface proteins that are the target antigen of the CAR, may induce fratricide if expression of the antigen on CAR-T-cells is not suppressed.

[0367] For example, in one protocol, on Day 0, CD4+ CD8+ T-cells were thawed in a cell culture media. The required number of cells were centrifuged at 200xg for 10 minutes at room temperature. Supernatant was removed completely, and cells were resuspended in cell culture media (TexMacs) supplemented with IL-7 (10 ng / ml) and IL- 15 (10 ng / ml) at concentration of 1x106 / ml. T-cells were then stimulated with Miltenyi™ research grade TransAct™ (10µl / ml).

[0368] On day 1, the required amount of viral vector comprising a nucleic acid sequence encoding the CAR was added to the activated cells at the required M. O. I (Multiplicity of Infection). Cells and virus were mixed and placed back in the incubator at 37°C. On day 3, activated cells were washed to remove stimulation.

[0369] Where genome editing was desired, cells were harvested and counted. The required number of cells were centrifuged at 100xg for 10 minutes at room temperature.Supernatant was removed completely, cells resuspended in electroporation buffer (1ml) (e.g., Maxcyte™ EP buffer) and transferred to a microcentrifuge tube and centrifuged at 100xg for 10 minutes at room temperature. Supernatant was removed completely, and cells then resuspended in electroporation buffer (e.g., MaxCyte™ EP buffer), at the desired concentration (e.g., 5 x 107 / ml).

[0370] Commercially-available Cas9 Protein (10 pg) and commercially synthesized gRNA (20 pg) were complexed at room temperature for 10 minutes. Cells (100 pl) wereAttorney Docket No. WGN0029-401-PCtransferred to the tube containing complexed Cas9 / gRNA, gently mixed, and everything transferred into a MaxCyte™ OCIOO cuvette. Electroporation was thereafter commenced using Maxcyte™ program Expanded T-cell 2. After this procedure, the activated cells were transferred to 10 ml of pre-warmed media and returned to the incubator to expand for an additional 7-12 days. FACS analysis was used to show the purity of CAR-transduced cells (CAR expression and target gene deletion).Example 8 - Depletion via anti-CD3 antibody

[0371] A schematic timeline of the bioprocess described herein is illustrated in FIG. 13.24-48 hours following gene-editing and transduction, cells were further stimulated with an anti-CD3 antibody, such as, for example, teplizumab, foralumab, visilizumab or muromonab at for example, 2.0 micrograms / mL final concentration, and expanded to establish pure cell lines. Roughly 3 days after addition of an anti-CD3 antibody, CD3+ T-cells were assayed for depletion by flow cytometry or genomic analysis. Cells were primarily assayed by flow cytometry. 1E6 cells were stained in a volume of 100 uL FACS buffer. Anti-CD3 (clone OKT3) conjugated to a BV785 fluorophore and anti-TCRocP conjugated to a BV511 fluorophore (clone IP26) were tested. Both antibodies were used in a 1:100 dilution. The instrument used was a ThermoFisher Attune NxT.

[0372] Dosing with 2 micrograms / mL teplizumab demonstrated stable depletion at day 6 and day 14 in culture, versus 0.1 micrograms / mL, 0.2 micrograms / mL and 0 micrograms / mL (FIG. 2A). T-cells were split from the standard process (prodigy isolation and activation) on day 4 of manufacturing and data was averaged across 2-3 donors. Samples were treated with the indicated concentrations of teplizumab and CD3+ / TCR+ cells and tracked over the course of manufacturing. Dosing with 2 micrograms / mL teplizumab led to fewer TCR+ cells when compared to 0.1 micrograms / mL, 0.2 micrograms / mL, or magnetic depletion (FIG. 2B).

[0373] The addition of teplizumab did not negatively affect cytotoxicity and there was a comparable level of cytotoxicity observed between depletion methods (FIG. 3A).

[0374] By day 10. depletion with 2 micrograms / mL teplizumab resulted in comparable or greater fold expansion. The similar expansion kinetics observed indicate that regardless of the depletion strategy use (magnetic or anti-CD3) treatment did not negatively affect T-cell persistence (FIG. 4B).Attorney Docket No. WGN0029-401-PC

[0375] With foralumab treatment, some CD3+ / TCRa|3+ T cells returned by Day 14 (FIG. 7B, upper right-hand quadrant).

[0376] With visilizumab treatment, a 0.79% TCR population returned by day 14, which is above FDA specifications for the maximum number of TCRa|3+ cells in an allogeneic cell product (FIG. 8B). Another humanized clone of OKT3, the parental clone of teplizumab, was also tested (data not shown). huOKT3 is structurally similar to teplizumab, having the same complementarity determining regions, and successfully depleted TCRoc[3-i- cells.

[0377] While muromonab (OKT3) treatment appeared promising by day 14 (FIG. 9B), as compared to teplizumab, muromonab (OKT3) depletion of TRAC+ cells resulted in cells that were less stable after challenge (FIG. 10).

[0378] In a CD2CAR model, T-cells transformed with CAR targeting CD2 were treated with teplizumab 24 hours after TRAC / CD2 knock-out and viral transduction. The addition of teplizumab to CAR2 samples depleted CD3 better than knockout (KO) alone across 4 donors, as assessed by FACs on day 14 at cell harvest (FIG. 1 IB).

[0379] Alternatively, a commercially-available, humanized OKT3 antibody was tested for depletion efficacy compared to teplizumab. A schematic timeline of the bioprocess described herein is illustrated in FIG. 13. Approximately 72 hours following gene-editing and transduction, cells were further stimulated cells with an anti-CD3 antibody, GMP humanized OKT3 antibody from BioTechne (Catalog MAB11411-GMP) (hu-OKT3) at for example, 1.0 or 2.0 micrograms / mL final concentration, and expanded to establish pure cell lines. Roughly 7 days after addition of the anti-CD3 antibody, CD3+ T-cells were assayed for depletion by flow cytometry or genomic analysis. Cells were primarily assayed by flow cytometry. 1E6 cells were stained in a volume of 100 pL FACS buffer. Anti-CD3 (clone OKT3) conjugated to a BV785 fluorophore and anti-TCRocP conjugated to a BV511 fluorophore (clone IP26) were tested. Both antibodies were used in a 1:100 dilution. The instrument used was a ThermoFisher Attune NxT.

[0380] Dosing with 1 microgram / mL teplizumab demonstrated stable depletion at day 14. T-cells were split from the standard process (prodigy isolation and activation) on day 3 of manufacturing and data was averaged across 2-3 donors. Samples were treated with the indicated concentrations of hu-OKT3 and CD3+ / TCR+ cells and tracked over the course of manufacturing. Analysis of cells post-sort showed that the percentage of TCR or CD3 positive cells is less than 0.4% (FIG. 14).Attorney Docket No. WGN0029-401-PCExample 9 - Expansion

[0381] After 72 hours cells were cultured in a 24 well GREX™ plate at 37°C for 7 days, with media replacements and additions as necessary until Day 14. On Day 14, CD3+ cells were harvested, counted, and phenotyped via flow cytometry or genomic analysis, then cryopreserved. No significant changes in expansion seen with the use of huOKT3 depletion were observed (FIG.15A and 15B). Samples of anti-CD3 depleted or magnetically depleted cells can be thawed for in vitro cytotoxicity experiments.

[0382] The addition of hu-OKT3 did not negatively affect cytotoxicity and there was a comparable level of cytotoxicity to untreated cells against CD7+ targets (FIG. 16A and B).Similarly, the addition of hu-OKT3 did not negatively affect cytotoxicity and there was a comparable level of cytotoxicity to untreated cells against CD7- targets (FIG. 17A and B).Example 10 - Genomic Analysis

[0383] Table 5 provides a summary of the genomic analysis results of cells that were treated with or without teplizumab. The results indicate comparable purity with magnetic-based depletion of TRAC-edited cells. Cell samples were collected at the end of manufacturing and assayed via targeted Amplicon (AMP) sequencing. Briefly, the genomic region surrounding the expected TRAC Crispr / CAS9 site was amplified by PCR; the resultant PCR product was then sequenced using the Illumina hi-seq platform. Referring to Table 5, ‘% mutant’ refers to the number of reads showing an indel within this amplicon compared to the wildtype sequence. Compared to un-edited WT cells, a high percentage of cells comprise an indel within the TRAC locus regardless of the purification method used, showing that anti-CD3-based depletion does not affect the physical characteristics of the cell product.Table 5Sample Target Reads Mutant Reads Mutant %Donor 1 + teplizumab 142123 137840 96.99%Donor 1 Magnetic 144151 133555 92.65%Donor 2 + teplizumab 77133 74051 96.0%Attorney Docket No. WGN0029-401-PCDonor 2 Magnetic 89292 80389 90.03%Donor 3 + teplizumab 115993 102973 88.78%Donor 3 Magnetic 117086 110111 94.04%WT T Cells 203867 3000 1.47%

[0384] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

Claims

1. Attorney Docket No. WGN0029-401-PC2.CLAIMS3.What is claimed is:

1. A method of making a population of genome-edited immune effector cells comprising:5.a) editing the genome of immune effector cells to reduce expression of T-cell receptor (TCR) to a negligible level; then6.b) treating the genome-edited immune effector cells with one or more antibodies, or bivalent binding fragments thereof, which bind to a polypeptide that forms part of the TCR-CD3 complex: and then7.c) expanding the edited immune effector cells.

2. The method of claim 1, wherein editing the genome comprises reducing expression of one or more of T-cell constant 0 chain (TRBC), CD3e, CD35, CD3i^, and / or CD3y to a negligible level.

3. The method of claim 1, wherein editing the genome comprises reducing expression of TCR a chain (TRAC) to a negligible level.

4. The method of claim 1, wherein the immune effector cells are chosen from T-cells and iNKT-cells.

5. The method of claim 1, wherein the immune effector cells are primary cells derived from healthy donors.

6. The method of claim 1, wherein the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

7. The method of claim 1, wherein the immune effector cells are T-cells.

8. The method of claim 7, wherein the T-cells are cytotoxic T-cells.

9. The method of claim 1, wherein the one or more antibodies, or bivalent binding fragments thereof, bind CD3E, CD35, CD3y, and / or TCR0.

10. The method of claim 9, wherein the one or more antibodies, or bivalent binding fragments thereof, are soluble.

11. The method of claim 9, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises human or humanized intervening framework regions.

12. The method of any of claims 9-11, wherein the polypeptide is CD3E.

13. The method of claim 12, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chainAttorney Docket No. WGN0029-401-PC19.variable region (LCVR), wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 polypeptides and the LCVR comprises LCDR1, LCDR2, and LCDR3 polypeptides wherein::20.a. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 where21.i. Xi is Y or F;22.ii. X-2 i s T or K;23.iii. X3 is T, l or S;24.iv. X4 is R, S or G; and25.v. X5 is T or G:26.b. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 where27.i. Xiis N, W or S;28.ii. X2is P, Y or T;29.iii. X3 is S, R or D;30.iv. X4 is R, S or G;31.v. Xs is S or G;32.vi. X6 is Y, K or R: and33.vii. X7 is T or K;34.c. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9 where35.i. Xi is Y or S;36.ii. X2is Y or A;37.iii. X3 is D or Y;38.iv. X4is D or Y;39.v. X5 is H or D;40.vi. X6 is C or D;41.vii. X7 is L or G;42.viii. Xg is D or F; and43.ix. Xo is Y or A;44.d. LCDR1 is X1-S-V-S-X2 where45.i. Xi is S or Q and46.ii. X2is Y or S; Attorney Docket No. WGN0029-401-PC47.e. LCDR2 is D-X1-X2 where Xi is T, A or D; and X2 is S or D; and f. LCDR3 is Q-Q-X1-S-X2-X3-P-X4-X5 where48.i. Xi is W or R;49.ii. X2 is S or N;50.iii. X3 is N or W;51.iv. X4 is F or P; and52.v. X5 is T or L.

14. The method of claim 13, wherein:54.e. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 where55.i. Xi is Y;56.ii. X2 is T or K;57.iii. X is T;58.iv. X4is R:59.v. X5 is T or G.

15. The method of claim 14, wherein:61.f. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 where62.i. Xi is N, W or S;63.ii. X2 is P, Y or T;64.iii. X3is S, R or D;65.iv. X4 is R;66.v. X5 is S or G:67.vi. Xe is Y, K or R; and68.vii. X? is T or K.

16. The method of claim 15, wherein:70.g. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9where i. Xi is Y:71.ii. X2 1S Y;72.iii. X3is D;73.iv. X4 is D:74.v. X5is H; Attorney Docket No. WGN0029-401-PC75.vi. Xgis C orD;76.vii. X7 is L;77.viii. Xg is D or F; and78.ix. Xg is Y orA.

17. The method of claim 16, wherein:80.h. LCDR3 is Q-Q-X1-S-X2-X3-P-X4-X5 where81.vi. Xi is W or R;82.vii. Xz is S orN:83.viii. X? is N or W;84.ix. X4 is F; and85.x. X5 is T or L.

18. The method of claim 13, wherein:87.a) HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26);88.b) HCDR1 is GYTFISYT (SEQ ID NO:27), HCDR2 is INPRSGYT (SEQ ID NO:28), HCDR3 is ARSAYYDYDGFAY (SEQ ID NO:29), LCDR1 is SSVSY (SEQ ID NO:30), LCDR2 is DTS (SEQ ID NO:31), and LCDR3 is QQWSSNPPT (SEQ ID NO:32);89.c) HCDR1 is GFKFSGYG (SEQ ID NO:33), HCDR2 is IWYDGSKK (SEQ ID NO:34), HCDR3 is ARQMGYWHFDL (SEQ ID NO:35). LCDR1 is QSVSSY (SEQ ID NO:36), LCDR2 is DAS (SEQ ID NO:37), and LCDR3 is QQRSNWPPLT (SEQ ID NO:38); or90.d) HCDR1 is GFTFSGYG (SEQ ID NO:39), HCDR2 is ISTSGGRT (SEQ ID NO:40), HCDR3 is AKFRQYSGGFDY (SEQ ID NO:41), LCDR1 is SGNIENNY (SEQ ID NO:42), LCDR2 is DDD (SEQ ID NO:43). and LCDR3 is HSYVSSFNV (SEQ ID NO:44).

19. The method of claim 13, wherein HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1Attorney Docket No. WGN0029-401-PC92.is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26).

20. The method of claim 19, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:94.a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10;95.b) HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12;96.c) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;97.d) HCVR of SEQ ID NO: 15 and LCVR of SEQ ID NO: 16;98.e) HCVR of SEQ ID NO: 17 and LCVR of SEQ ID NO: 18; or99.f) HCVR of SEQ ID NO: 19 and LCVR of SEQ ID NO:20;100.or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

21. The method of claim 20, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:102.a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10; or103.b) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;104.or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%. or at least 99% identity to any of the foregoing.

22. The method of claim 19 or claim 20, wherein the one or more antibodies, or bivalent binding fragments thereof, further comprises an Fc domain and the Fc domain is of an IgGl, IgG2 or an IgG4 subclass.

23. The method of claim 22, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 234 (L234A).

24. The method of claim 22, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 235 (L235A).

25. The method of any one of claims 1-24, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises:109.a) two heavy chains, each heavy chain comprising a heavy chain variable region (HCVR) and a constant region; and Attorney Docket No. WGN0029-401-PC110.b) two light chains, each light chain comprising a light chain variable region (LCVR) and a constant region.

26. The method of claim 25, wherein the heavy chain constant region has an amino acid sequence as set forth as SEQ ID NO:45 and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID NO:49.

27. The method of claim 9, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise an anti-CD3£ antibody chosen from teplizumab, humanized 0KT3, muromonab. foralumab, otelixizumab, visilizumab, TR66, SK7, gOKT3-5, UCHT1, 145-2C11, and SP34-2.

28. The method of any one of claims 1-27, wherein the genome-edited immune effector cells express at least one chimeric antigen receptor (CAR) which binds to one or more proteins.

29. The method of any one of claims 1-27, wherein editing the genome comprises editing the genome of the immune effector cells to reduce the expression of the one or more proteins to which the CAR binds.

30. The method of any one of claims 1-29, wherein the population of immune effector cells is activated prior to step a.

31. The method of any one of claims 1-30, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than about <1%. no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

32. The method of any one of claims 1-30, for use in the manufacture of an allogeneic cell product, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than about <1%, no more than about <0.5%. no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

33. The method of any one of claims 1-31, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than about <0.4% per 9.0 x 108cells.

34. The method of any one of claims 1-31, wherein after 3-14 days of culture, the number of TCRa / p positive T cells is no more than 3.6 x 106cells.

35. The method of any one of claims 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL, about 1.0 pg / mL, aboutAttorney Docket No. WGN0029-401-PC1.5 pg / mL, about 1.6 pg / mL, about 1.7 pg / mL, about 1.8 pg / mL, about 1.9 pg / mL, about 2.0 pg / mL, about 2.2 pg / mL, and about 2.5 pg / mL human or humanized a-CD3 antibody.

36. The method of any one of claims 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from at least 0.5 pg / mL, at least 0.6 pg / mL, at least 0.7 pg / mL, at least 0.8 pg / mL, at least 0.9 pg / mL, at least 1.0 pg / mL, at least 1.1 pg / mL, at least 1.2 pg / mL, at least 1.3 pg / mL, at least 1.4 pg / mL, at least 1.5 pg / mL, at least 1.6 pg / mL, at least 1.7 pg / mL, at least 1.8 pg / mL, at least 1.9 pg / mL, or at least 2.0 pg / mL human or humanized a-CD3 antibody.

37. The method of any one of claims 11-34, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL to about 5.0 pg / mL, about 1.0 pg / mL to about 4.0 pg / mL, about 1.0 pg / mL to about 3.0 pg / mL, and about 1.5 to about 2.5 pg / mL.

38. The method of any one of claims 32-34, wherein the human or humanized a-CD3 antibody is chosen from humanized OKT3 and teplizumab.

39. The method of any one of claims 1-38, wherein the time of treating with the antibody is chosen from at least 18 hours after editing and / or transduction, at least 24 hours after editing and / or transduction, at least 36 hours after editing and / or transduction, and at least 48 hours after editing and / or transduction.

40. The method of claim 39, wherein the time of treating with the antibody is chosen from 18-48 hours after editing and / or transduction, 24-48 hours after editing and / or transduction, 36-48 hours after editing and / or transduction, and 24-36 hours after editing and / or transduction.

41. The method of claim 40, wherein the time of treating with the antibody is 24-48 hours after editing and / or transduction.

42. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from at least 3 days, at least 4 days, at least 5 days, at least 6 days, and at least 7 days.

43. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 3-11 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, and 3-4 days.Attorney Docket No. WGN0029-401-PC44. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 4-11 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, and 4-5 days.

45. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, and 5-6 days.

46. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 6-11 days. 6-10 days. 6-9 days, 6-8 days, and 6-7 days.

47. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 7-11 days, 7-10 days. 7-9 days, and 7-8 days.

48. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 8-11 days, 8-10 days, and 8-9 days.

49. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is chosen from 9-11 days, and 9-10 days.

50. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is 10-11 days.

51. The method of any one of claims 1-41, wherein incubation time with the a-CD3 antibody is 3-11 days.

52. The method of any one of claims 1-51, wherein there are no more than 100,000 TCRo / p positive T cells per kilogram patient weight.

53. The method of any one of claims 1-52, wherein there are no more than 70,000 TCRa / p positive T cells per kilogram patient weight.

54. The method of any one of claims 1-53, wherein the method does not comprise a magnetic separation (MACS) step.

55. The method of any one of claims 1-53, wherein the method additionally comprises a magnetic separation (MACS) step.

56. The method of any one of claims 1-53, wherein the T cells are in the resting state.

57. The method of any one of claims 1-53, wherein the T cells are in the activated state.

58. The method of any one of claims 1-53, wherein the T cells are in the partially activated state.

59. A method of making a population of genome-edited immune effector cells expressing a chimeric antigen receptor (CAR) which binds to one or more proteins; orAttorney Docket No. WGN0029-401-PC145.selecting TCR-negative immune effector cells from a population of immune effector cells expressing a chimeric antigen receptor (CAR), or146.making a population of at least 80 billion genome-edited immune effector cells expressing a chimeric antigen receptor (CAR) which binds to one or more proteins from one batch of donor cells; the method comprising:147.a) optionally, activating a population of immune effector cells comprising a T-cell receptor (TCR); then148.b) editing the genome of the immune effector cells to reduce expression of the TCR a chain (TRAC) to a negligible level, and, optionally, editing the genome of the immune effector cells to reduce expression of one or more proteins to which the CAR binds to a negligible level; then149.c) transducing the cells with nucleic acid encoding the CAR; then150.d) treating the edited immune effector cells with one or more antibodies, or bivalent binding fragments thereof, which bind to the extracellular domain of a polypeptide that forms part of the TCR-CD3 complex; and then151.e) expanding the edited immune effector cells.

60. The method of claim 59, wherein editing the genome comprises reducing expression of T-cell constant 0 chain (TRBC), CD3e, CD35, CD3i^, CD3y to a negligible level.

61. The method of claim 59, wherein editing the genome comprises reducing expression of the TCR a chain (TRAC) to a negligible level.

62. The method of claim 59, wherein the immune effector cells are chosen from T-cells and iNKT-cells.

63. The method of claim 59, wherein the immune effector cells are primary cells derived from healthy donors.

64. The method of claim 59, wherein the immune effector cells are derived from induced pluripotent stem cells (iPSCs).

65. The method of claim 59, wherein the immune effector cells are T-cells.

66. The method of claim 65, wherein the T-cells are cytotoxic T-cells.

67. The method of claim 59, wherein the polypeptide is chosen from CD3e, CD35, CD3y. and TCR0.

68. The method of claim 68, wherein the polypeptide is CD3e.Attorney Docket No. WGN0029-401-PC69. The method of any one of claims 59-69, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises human or humanized intervening framework regions.

70. The method of any one of claims 59-69, wherein the one or more antibodies, or bivalent binding fragments thereof, are soluble.

71. The method of claim 70, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein:164.g. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 where165.vi. Xi is Y or F;166.vii. X? is T or K;167.viii. X3 is T, l or S;168.ix. X4 is R, S or G; and169.x. X5 is T or G:170.h. HCDR2 is I-X1-X2-X3-X4-X5-X6-X7 where171.viii. Xiis N, W or S:172.ix. X2 is P, Y orT;173.x. X3 is S, R or D;174.xi. X4 is R, S or G;175.xii. X5 is S or G;176.xiii. Xb is Y, K or R: and177.xiv. X7 is T or K;178.i. HCDR3 is A-R-Xj -X2-X3-X4-X5- Y-X6-X7X8-X9where179.x. Xi is Y or S:180.xi. X2 is Y or A;181.xii. X3 is D or Y;182.xiii. X4 is D or Y;183.xiv. X5 is H or D;184.xv. Xe is C or D;185.xvi. X-? is L or G:186.xvii. X8is D or F; and Attorney Docket No. WGN0029-401-PC187.xviii. Xo is Y or A;188.j. LCDR 1 is X1-S-V-S-X2 where189.iii. Xi is S or Q and190.iv. Xi is Y or S;191.k. LCDR2 is D-X1-X2 where Xi is T, A or D: and X> is S or D; and l. LCDR3 is Q-Q-X j -S-X2-X3-P-X4-X5 where192.vi. Xi is W or R;193.vii. X? is S or N;194.viii. X3 is N or W;195.ix. X4 is F or P; and196.x. X5 is T or L.

72. The method of claim 71, wherein:198.e. HCDR1 is G-X1-X2-F-X3-X4-Y-X5 where199.vi. Xi is Y;200.vii. X2 is T or K;201.viii. X3 is T;202.ix. X4 is R;203.x. X5 is T or G.

73. The method of claim 71, wherein:205.f. HCDR2 is I— Xi— X2— X3— X4— X5— X6— X7where206.viii. Xi is N, W or S;207.ix. X2 is P, Y or T;208.x. Xj is S. R or D:209.xi. X4 is R;210.xii. X5 is S or G;211.xiii. X<5 is Y, K or R: and212.xiv. Xy is T or K.

74. The method of claim 71, wherein:214.g. HCDR3 is A-R-X1-X2-X3-X4-X5-Y-X6-X7 X8-X9 where Xi is Y; Attorney Docket No. WGN0029-401-PC215.xi. X2 is Y;216.xii. X3 is D;217.xiii. X4 is D;218.xiv. X5 is H;219.xv. X& is C or D;220.xvi. X? is L;221.xvii. Xs is D or F; and222.xviii. X9 is Y or A.

75. The method of claim 71, wherein:224.h. LCDR3 is Q-Q-XJ-S-X2-X3-P-X4-X5 where225.vi. Xi is W or R;226.vii. X is S orN;227.viii. X3 is N or W;228.ix. X4 is F; and229.x. X5 is T or L.

76. The method of claim 71, wherein the HCVR comprises HCDR1, HCDR2, and HCDR3 polypeptides and the LCVR comprises LCDR1, LCDR2, and LCDR3 polypeptides, wherein:231.a) HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26);232.b) HCDR1 is GYTFISYT (SEQ ID NO:27), HCDR2 is INPRSGYT (SEQ ID NO:28), HCDR3 is ARSAYYDYDGFAY (SEQ ID NO:29), LCDR1 is SSVSY (SEQ ID NO:30), LCDR2 is DTS (SEQ ID NO:31), and LCDR3 is QQWSSNPPT (SEQ ID NO:32);233.c) HCDR1 is GFKFSGYG (SEQ ID NO:33). HCDR2 is IWYDGSKK (SEQ ID NO:34), HCDR3 is ARQMGYWHFDL (SEQ ID NO:35), LCDR1 is QSVSSY (SEQ ID NO:36), LCDR2 is DAS (SEQ ID NO:37), and LCDR3 is QQRSNWPPLT (SEQ ID NO:38); or Attorney Docket No. WGN0029-401-PC234.d) HCDR1 is GFTFSGYG (SEQ ID NO:39), HCDR2 is TSTSGGRT (SEQ ID NO:40), HCDR3 is AKFRQYSGGFDY (SEQ ID N0:41), LCDR1 is SGNIENNY (SEQ ID NO:42), LCDR2 is DDD (SEQ ID NO:43). and LCDR3 is HSYVSSFNV (SEQ ID NO:44).

77. The method of claim 71, wherein HCDR1 is GYTFTRYT (SEQ ID NO:21), HCDR2 is INPSRGYT (SEQ ID NO:22), HCDR3 is ARYYDDHYCLDY (SEQ ID NO:23), LCDR1 is SSVSY (SEQ ID NO:24), LCDR2 is DTS (SEQ ID NO:25), and LCDR3 is QQWSSNPFT (SEQ ID NO:26).

78. The method of any one of claims 59-77, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:237.a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10;238.b) HCVR of SEQ ID NO: 11 and LCVR of SEQ ID NO: 12;239.c) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;240.d) HCVR of SEQ ID NO: 15 and LCVR of SEQ ID NO: 16;241.e) HCVR of SEQ ID NO: 17 and LCVR of SEQ ID NO: 18; or242.f) HCVR of SEQ ID NO: 19 and LCVR of SEQ ID NO:20243.or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

79. The method of claim 78, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR and LCVR are polypeptides selected from:245.a) HCVR of SEQ ID NO:9 and LCVR of SEQ ID NO: 10; or246.b) HCVR of SEQ ID NO: 13 and LCVR of SEQ ID NO: 14;247.or are polypeptides having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of the foregoing.

80. The method of any one of claims 78-79, wherein the one or more antibodies, or bivalent binding fragments thereof, further comprises an Fc domain and the Fc domain is of an IgGl or an IgG4 subclass.Attorney Docket No. WGN0029-401-PC81. The method of claim 80, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 234 (L234A).

82. The method of claim 80, wherein the Fc domain is of an IgGl subclass and comprises a point mutation from leucine to alanine at residue 235 (L235A).

83. The method of any one of claims 59-82, wherein the one or more antibodies, or bivalent binding fragments thereof, comprises:252.a) two heavy chains, each heavy chain comprising a heavy chain variable region (HCVR) and a constant region: and253.b) two light chains, each light chain comprising a light chain variable region (LCVR) and a constant region.

84. The method of claim 83, wherein the heavy chain constant region has an amino acid sequence as set forth as SEQ ID NO:47 and / or the light chain constant region has an amino acid sequence as set forth in SEQ ID NO:49.

85. The method of any one of claims 59-84, wherein the one or more antibodies, or bivalent binding fragments thereof, comprise an anti-CD3e antibody chosen from teplizumab, humanized OKT3. muromonab, foralumab, otelixizumab, visilizumab, TR66, SK7, gOKT3-5, UCHT1, 145-2C11, and SP34-2.

86. The method of any one of claims 59-85, wherein the genome-edited immune effector cells express at least one chimeric antigen receptor (CAR) which binds to one or more proteins.

87. The method of any one of claims 59-86, wherein editing the genome of the immune effector cells to delete the one or more proteins to which the CAR binds.

88. The method of any one of claims 59-87, wherein the population of immune effector cells is activated prior to step a.

89. The method of any one of claims 59-88, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than about <1%, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

90. The method of any one of claims 59-88, for use in the manufacture of an allogeneic cell product, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells isAttorney Docket No. WGN0029-401-PC261.no more than about <1 %, no more than about <0.5%, no more than about <0.4%, no more than about <0.3%, or no more than about <0.25% per 9.0 x 108cells.

91. The method of any one of claims 59-88, wherein after 3-14 days of culture, the percentage of TCRa / p positive T cells is no more than about <0.4% per 9.0 x 108cells.

92. The method of any one of claims 59-91, wherein after 3-14 days of culture, the number of TCRa / p positive T cells is no more than 3.6 x 106cells.

93. The method of any one of claims 59-92, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL, about 1.0 pg / mL, about 1.5 pg / mL, about 1.6 pg / mL, about 1.7 pg / mL, about 1.8 pg / mL, about 1.9 pg / mL, about 2.0 pg / mL, about 2.2 pg / mL, and about 2.5 pg / mL human or humanized a-CD3 antibody.

94. The method of any one of claims 59-93, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from at least 0.5 pg / mL, at least 0.6 pg / mL, at least 0.7 pg / mL, at least 0.8 pg / mL, at least 0.9 pg / mL, at least 1.0 pg / mL, at least 1.1 pg / mL, at least 1.2 pg / mL, at least 1.3 pg / mL, at least 1.4 pg / mL, at least 1.5 pg / mL, at least 1.6 pg / mL, at least 1.7 pg / mL, at least 1.8 pg / mL, at least 1.9 pg / mL, or at least 2.0 pg / mL human or humanized a-CD3 antibody.

95. The method of any one of claims 59-94, wherein the amount of antibody, or bivalent binding fragments thereof, used is chosen from about 0.5 pg / mL to about 5.0 pg / mL, about 1.0 pg / mL to about 4.0 pg / mL, about 1.0 pg / mL to about 3.0 pg / mL, and about 1.5 to about 2.5 pg / mL.

96. The method of either of claims 94 or 95, wherein the human or humanized a-CD3 antibody is chosen from humanized OKT3 and teplizumab.

97. The method of any one of claims 59-96, wherein the time of treating with the antibody is chosen from at least 18 hours after editing and / or transduction, at least 24 hours after editing and / or transduction, at least 36 hours after editing and / or transduction, and at least 48 hours after editing and / or transduction.

98. The method of claim 97, wherein the time of treating with the antibody is chosen from 18-48 hours after editing and / or transduction, 24-48 hours after editing and / or transduction, 36-48 hours after editing and / or transduction, and 24-36 hours after editing and / or transduction.Attorney Docket No. WGN0029-401-PC99. The method of claim 98, wherein the time of treating with the antibody is 24-48 hours after editing and / or transduction.

100. The method of any one of claims 59-99. wherein incubation time with the a-CD3 antibody is chosen from at least 3 days, at least 4 days, at least 5 days, at least 6 days, and at least 7 days.

101. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 3-11 days, 3-10 days, 3-9 days, 3-8 days, 3-7 days, 3-6 days, 3-5 days, and 3-4 days.

102. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 4-11 days, 4-10 days, 4-9 days, 4-8 days, 4-7 days, 4-6 days, and 4-5 days.

103. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 5-11 days, 5-10 days, 5-9 days, 5-8 days, 5-7 days, and 5-6 days.

104. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 6-11 days, 6-10 days, 6-9 days, 6-8 days, and 6-7 days.

105. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 7-11 days, 7-10 days, 7-9 days, and 7-8 days.

106. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 8-11 days, 8-10 days, and 8-9 days.

107. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is chosen from 9-11 days, and 9-10 days.

108. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is 10-11 days.

109. The method of any one of claims 59-100, wherein incubation time with the a-CD3 antibody is 3-11 days.

110. The method of any one of claims 59-109, wherein there are no more than 100,000 TCRa / 0 positive T cells per kilogram patient weight.

111. The method of any one of claims 59-110, wherein there are no more than 70,000 TCRa / 0 positive T cells per kilogram patient weight.

112. The method of any one of claims 59-111, wherein the method does not comprise a magnetic separation (MACS) step.Attorney Docket No. WGN0029-401-PC113. The method of any one of claims 59-111, wherein the method additionally comprises a magnetic separation (MACS) step.

114. The method of any one of claims 59-111, wherein the T cells are in the resting state.

115. The method of any one of claims 59-111, wherein the T cells are in the activated state.

116. The method of any one of claims 59-111, wherein the T cells are in the partially activated state.

117. The method of claim 59, wherein the one or more proteins bound by the CAR is / are antigen(s) overexpressed on the surface a malignant T-cell.

118. The method of claim 117 wherein the proteins are tumor-associated antigens and chosen from CD2, CD3, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD70, CD123, CD171, CD174, CD274, CD276, CD319, EGFR, EGFR vIII, GD2, Mesothelin, PSA, FLT3, CEA. HER2. TNFRSF17, IL3RA, SDC1, MS4A1, TNFRSF8, NCAM1, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EPCAM, EPHA2, ERBB2, GPC3, MUC1, PDCD1, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, R0R1, SLAMF7, PSCA, GPNMB, CSPG4, CD133, TEM1, and DLL3.

119. The method of claim 59, wherein the CAR is designed to bind to one protein.

120. The method of claim 119, wherein the CAR selectively binds one protein.

121. The method of claim 119, wherein the CAR specifically binds one protein.

122. The method of claim 121, wherein the CAR binds to CD7.

123. The method of claim 59, wherein the genome of the immune effector cell is edited to delete the protein to which the CAR binds.

124. The method of claim 123, wherein the genome of the immune effector cell is edited to delete CD7.

125. The method of any one of claims 59-124, wherein the genome is edited using a CRISPR associated protein (CRISPR / Cas), a transcription activator-like effector nuclease (TALEN), or a zinc-finger nuclease (ZFN).

126. The method of claim 125, wherein the genome is edited using a CRISPR / Cas9 associated protein.

127. The method of claim 125, wherein the genome is edited using a base editor.Attorney Docket No. WGN0029-401-PC128. The method of claim 126, wherein the Cas9 is delivered into the cell as mRNA or protein.

129. The method of claim 128, wherein the Cas9 is delivered into the cell as protein.

130. The method of claim 126, wherein the Cas9 is delivered contemporaneously with at least one guide RNA (gRNA) targeting the one or more gene(s) to be edited.

131. The method of any one of claims 59-130, wherein the population of expanded T- cells contains less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% unedited T-cells.

132. The method of any one of claims 59-131, wherein the population of genome- edited chimeric antigen receptor (CAR) T-cells undergoes a sorting step.

133. The method of any one of claims 59-131, wherein the population of genome- edited chimeric antigen receptor (CAR) T-cells does not undergo a sorting step.

134. The method of any one of claims 59-133, wherein the population of genome- edited chimeric antigen receptor (CAR) T-cells is activated for up to five days.

135. The method of any one of claims 59-134, wherein the population of genome- edited chimeric antigen receptor (CAR) T-cells is expanded for greater than or equal to about 7-14 days.

136. The method of any one of claims 59-135, wherein the method is performed at a temperature of between about 25°C and about 40°C.

137. A population of genome-edited CAR-T-cells made by the method of any one of claims 59-136.

138. A method of treating a solid organ tumor or hematologic malignancy in a patient comprising administering the population of genome-edited CAR-T-cells of claim 137.

139. The method of claim 138, wherein the hematologic malignancy is a T-cell malignancy.

140. The method of claim 139, wherein the T-cell malignancy is T-cell acute lymphoblastic leukemia (T-ALL).

141. The method of claim 139, wherein the T-cell malignancy is T-cell lymphoblastic lymphoma (T-LBL).

142. The method of claim 139, wherein the T-cell malignancy is any one of: T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1 -positiveAttorney Docket No. WGN0029-401-PC313.(HTLV-1 +) adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and various peripheral T-cell lymphomas (PTCLs), angioimmunoblastic T-cell lymphoma (AITL), ALK-positive anaplastic large cell lymphoma, or ALK-negative anaplastic large cell lymphoma.

143. The method of claim 139, wherein the T-cell malignancy is non-Hodgkin’s lymphoma.

144. The method of claim 138, wherein the hematologic malignancy is a B-cell malignancy.

145. The method of claim 144, wherein the B-cell malignancy is a B cell lymphoma.

146. The method of claim 144, wherein the B-cell malignancy is a B cell leukemia.

147. The method of claim 144, wherein the hematologic malignancy is a myeloid malignancy.

148. The method of claim 138, wherein the hematologic malignancy is acute myeloid leukemia.

149. The method of any one of claims 1-148, wherein the cells express SEQ ID NO:51.

150. The method of any one of claims 1-149, wherein the cells comprise DNA encoding SEQ ID NO:51.

151. The method of any one of claims 1-150, wherein the DNA comprises SEQ ID NO:52.

152. The method of any one of claims 1-151, wherein the CAR comprises SEQ ID NO:53.

153. The method of any one of claims 1-152, wherein the population of genome-edited immune effector cells comprise DNA encoding SEQ ID NO:53.

154. The method of any one of claims 1-153, wherein the DNA comprises SEQ ID NO:54.