Engineered orthogonal cd3 cells

Abstract

An orthogonal variant of human CD3e is provided, which orthoCD3e does not bind to an antibody of interest, e.g. antibodies having a binding specificity of OKT3, but which retains binding to cellular proteins CD3d and CD3g. T cells that are engineered to express the orthoCD3e express normal levels of the T cell receptor complex on their surface and have a functional T cell receptor complex. These engineered T cells are useful in therapeutic methods where it is desirable to lymphodeplete resident T cells from an individual while sparing transferred therapeutic T cells.

Description

ENGINEERED ORTHOGONAL CD3 CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 528,264 filed July 21 , 2023, the disclosure of which application is herein incorporated by reference.GOVERNMENT RIGHTS

[0002] This invention was made with Government support under contract DGE-1656518 (FELLOWSHIP) awarded by the National Science Foundation and under contract DK132549 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Different subsets with various functions of Treg cells exist. Tregs can be usually identified by flow cytometry. The most specific marker for these cells is FoxP3, which is localized intra cellularly. Dysregulation in Treg cell frequency or functions may lead to the development of autoimmune disease. Therapeutic Treg modulation is being developed to treat certain immune disorders, for example to prevent graft v host disease, or allograft rejection.

[0004] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a form of regenerative therapy in which a patient’s damaged or defective bone marrow or immune system is replaced with that of a healthy donor. It is a life-saving procedure used to widely treat immunodeficiency disorders, genetic disorders, and cancer. However, as the transplanted graft is often from an unrelated donor and hence immunologically incompatible, most patients who receive allo-HSCT develop graft-versus-host disease (GvHD) or dysregulated immunity.

[0005] In GvHD, donor-derived immune cells (“graft”) recognize the recipient tissue (“host”) as foreign and attack the recipient body’s cells, resulting in toxicities in several organs. Despite optimization of conditioning regimen and phenotyping of donor graft to minimize the severity of GvHD, 50-80% of patients who receive allo-HSCT develop moderate to severe GvHD, leading to mortality as high as 50%. Current standard of care treatment for GvHD is glucocorticoid steroids, which suppress alloreactive, inflammatory donor immune cells to make them more tolerogenic(immunologically more tolerant). However, around 50% of GvHD cases fail to respond to first- line steroid treatment.

[0006] GvHD is associated with diminished levels of immunosuppressive cells, in particular regulatory T cells (Tregs). Thus, a promising therapy against GvHD is adoptive transfer of Tregs. Numerous pre-clinical and now clinical studies have demonstrated that adoptively transferred Tregs ameliorate GvHD. Efficacy of these therapeutic Tregs scales with their ability to engraft and proliferate in the recipient’s body to generate tolerogenic environments. However, the presence of inflammatory donor immune cells undermines engraftment and proliferation of therapeutic Tregs by occupying the physical space and expending nutrients, as well as releasing pro-inflammatory cytokines. Thus, a method to robustly, safely, and selectively deplete donor T cells, while sparing therapeutic Tregs is desirable, in order to maximize Treg engraftment and proliferation, and in turn its therapeutic potential.

[0007] As part of hematopoietic stem cell transplantation, chemotherapy or immunotherapy approaches are used to lymphodeplete recipients to increase the chance of donor hematopoietic engraftment but this may also increase the risk of GVHD and infections. Increasing donor Treg numbers can facilitate engraftment and also reduce GVHD risk.SUMMARY

[0008] Compositions and methods are provided for an orthogonal variant of human CD3e, which orthoCD3e does not bind to an antibody of interest, e.g. antibodies having a binding specificity of OKT3, including OKT3 and variants thereof, which bind to wild-type CD3e. Using site-directed mutagenesis and yeast surface display, a variant (ortho) CD3e has been engineered that has substantially no detectable binding to OKT3, but which retains binding to cellular proteins CD38 and CD3y. T cells that are engineered to express the orthoCD3e express normal levels of the T cell receptor complex on their surface and have a functional T cell receptor complex. These engineered T cells are useful in therapeutic methods where it is desirable to lymphodeplete resident T cells from an individual with antibodies such as OKT3.

[0009] The process for engineering an orthogonal binding pair may comprise the steps of (a) engineering amino acid changes into a wild-type CD3c protein, e.g. human CD3e, to disrupt binding to the antibody of interest; (b) selecting for ortho- CD3c proteins that bind to polypeptides of the CD3 complex; but lack binding to the antibody of interest. In some embodiments, knowledge of the structure of the antibody / CD3E complex is used to select amino acid positions for site- directed or error prone mutagenesis. Conveniently a yeast display system can be used for the selection process, although other display and selection methods are also useful.

[0010] In some embodiments an orthogonal human CD3E protein is provided, which protein retains binding affinity for cellular components of the T cell receptor complex, e.g. CD3y, CD35, but which substantially lacks detectable binding to an anti-CD3 antibody of interest. In some embodiments, the anti-CD3 antibody of interest has the binding specificity of OKT3, e.g. an antibody or fragment thereof comprising the 6 CDR sequences of OKT3. T cells expressing the ortho-CD3e in the absence of wild-type CD3E retain T cell receptor function.

[0011] In some embodiments an ortho CD3e protein comprises an amino acid substitution at one or more residues selected from E56, G68, E70, P100, R101 , G102, S103, K104, P105, and D107, where the substitution comprises an amino acid other than the wild-type, for example E56A, G68R, E70A, P100A, R101A, G102R, S103A, K104A, P105A, and D107A. In an embodiment an ortho CD3e protein comprises an amino acid substitution at E56, P100, R101 , G102, or K104, including without limitation E56A, P100A, R101 A, G102A, and K104A.

[0012] In some embodiments, the orthoCD3e comprises an amino acid substitution at position K104 (numbering relative to the human CD3e protein, provided herein as SEQ ID NO:1 ). In some embodiments the amino acid substitution comprises an amino acid other than a positively- charged amino acid, as shown in FIG. 3. In some embodiments the amino acid substitution is selected from K104D; K104E; K104S; K104T; K104N; K104Q; K104G; K104P; K104A; K104V; K104I; K104L; K104M; K104F; K104Y; and K104W. In some embodiments the amino acid substitution is K104E or K104D. In some embodiments the orthoCDSe comprises a combination of amino acid substitutions. The combination of amino acid substitutions may be any combination of the amino acid substitutions listed above.

[0013] In some embodiments a mouse orthoCD3e is of interest, e.g. in drug screening, preclinical assessment, etc. In some embodiments an ortho mCD3£ is selected to substantially lack detectable binding to an anti-CD3 antibody of interest, where the antibody has a binding specificity of 2C11 antibody, e.g. 2C1 1 or a binding fragment thereof. In some embodiments an ortho mCD3e protein comprises an amino acid substitution at one or more residues selected from D22, D23, A24, E25, N26, D45, S46, D47, E48, N49, K51 , Y84, T85, P86, A87, S88, N89, K90, and N91 , where the substitution comprises an amino acid other than the wild-type, e.g. D22A, D22R, D23A, A24R, E25A, E25R, N26A, N26R, D45A, D45F, S46A, D47A, D47R, D47L, E48A, E48L, N49A, N49E, K51 A, Y84A, T85A, P86A, A87R, A87E, A87L, S88A, S88L, N89A, N89R, N89E, N89L, K90A, N91A and N91 L. In some embodiments an ortho-mCD3E comprises one or more amino acid substitutions at residue D45, D45, D47, D47, D47, N49, T85, P86, A87, A87, S88, and K90.

[0014] In some embodiments, the ortho-mCD3e comprises an amino acid substitution at position P86 (numbering relative to the mouse CD3e protein, provided herein as SEQ ID NO:3). In some embodiments the amino acid substitution is selected from P86A, P86G, P86V, P86I, P86L, P86F, P86Y, P86W, P86R, P86H, P86K, P86D, P86E, P86S, P86T, P86N, and P86Q. In some embodiments the amino acid substitution is selected from P86I, P86F, P86Y, P86W, P86S, P86R, P86H, P86K, P86D, and P86E. In some embodiments the ortho-mCD3c comprises a combination of amino acid substitutions. The combination of amino acid substitutions may be any combination of the amino acid substitutions listed above.

[0015] In some embodiments a cell of interest is engineered to express an ortho CD3e and to lack expression of wild-type CD3c. A cell of interest may be a stem cell, e.g. a hematopoietic stem cell (HSC), induced pluripotent stem cells (iPSC), etc. A cell may be a hematopoietic cell, including HSC, lymphoid progenitor cells, T cells, etc. A cell of interest may be a T cell. Cells may be isolated from an individual, a cell line, etc. Various methods may be used for engineering; where, for example, CRISPR engineering provides for efficient conversion of the wild-type CD3e locus to the ortho version, or CRISPR knockout of wildtype CD3E followed by a knock-in of an expression vector, for example by viral transduction of orthoCD3c using viral transduction such as adenovirus, lentivirus, retrovirus, etc. In some embodiments the T cell of interest is a regulatory T cell (Treg). In some embodiments the T cell of interest is a chimeric antigen receptor (CAR) T cell, or T cell receptor (TCR) engineered T cell. Other engineered T effector cells, e.g. CD8+T cells, CD4+T cells may also be provided. In some embodiments the engineered T cell is a Treg cell.

[0016] Methods of activating or inducing proliferation of an engineered orthoCDSc T cells are provided, where such methods may include stimulation of the T cells with an antibody or other binding moiety that retains binding to the orthoCD3e, e.g. dynabeads, anti-human CD3e clone MEM57, anti-human CD3E cone SP34-2 allogeneic cells, etc. In other embodiments an antibody specific for the ortho CD3e is utilized for activation, etc. In some embodiments a population of engineered orthoCD3e T cells are stimulated in vitro prior to administration to a subject.

[0017] Methods of treatment are provided, where an effective dose of a population of orthoCD3e T cells are administered to an individual in need thereof, optionally after in vitro stimulation. The individual is treated with the ortho antibody, e.g. having OKT3 binding specificity, to deplete T cells other than the desired engineered orthoCD3e T cells. Antibody administration may be in combination with the T cell administration; or may follow T cell administration. This method provides (1 ) selective depletion of immunoinflammatory T cells, but not therapeutic engineered T cells, by anti-CD3, which will allow (2) enhanced engraftment and proliferation of orthoCD3c T cells.In some embodiments the orthoCD3e T cells are T reg cells. In some embodiments the orthoCD3e T cells are CAR-T cells. In some embodiments the T cells are TCR engineered T cells.

[0018] In some embodiments, a method of treating an individual with a therapeutically effective dose of orthoCD3e Treg cells is provided, comprising administering to an individual in need thereof an effective dose of a population of the engineered cells. Conditions where the cells are used may depend on the type of T cell being administered. Non-limiting examples where Treg cells are useful may include GVHD, graft tolerance / rejection, autoimmune disease such as type 1 diabetes, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, etc., wound healing, tissue regeneration, minimizing damage from ischemia or injury, etc. Non-limiting examples where CAR T cells are useful may include cancer, infection, and the like. In an embodiment, methods of treating a patient in need thereof are provided, comprising administering an effective dose of an engineered Treg cell population to the patient.

[0019] In certain embodiments, methods of reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ are provided, comprising administering an effective dose of an engineered Treg cell population to an individual in need thereof.

[0020] In certain embodiments, methods of reducing GVHD in a patient transplanted with donor hematopoietic stem cells, bone marrow cells, or a solid organ are provided, comprising administering an effective dose of an engineered Treg cell population to an individual in need thereof.

[0021] In an embodiment, methods of treating an individual with orthoCD3e CAR-T cell or engineered TCR are provided, comprising administering an effective dose of an engineered T cell population to an individual in need thereof, e.g. where the individual is treated for cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0001] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0002] FIGS1 A-1 D. OrthoCD3c engineering by yeast surface display, (a) Schematic of TCR-CD3 complex. (B) Yeast surface display of single chain CD35-CD3e. (C) OKT3 staining of yeast displayed wildtype and K104A CD3E. (D) Location of CD3E K104 in OKT3-CD3E structure, PDB 1 SY6. (E) MEM57 staining yeast displayed wildtype and K104A CD3E.

[0003] FIGS. 2A-2B. CRISPR-Cas9 mediated homology directed repair. (A) CD3e gRNA has >96% KO efficiency. (B) Post-HDR sequencing indicates high percentage of edited T cells with AAA-GCA.

[0004] FIG. 3. Back to yeast: K104 site saturation mutagenesis.

[0005] FIG. 4. K104E may be a more potent orthoCD3.

[0006] FIG. 5. CD3E K104E on T cells has no binding to OKT3.

[0007] FIG. 6. CD3E K104E is inert from teplizumab and otelixizumab.

[0008] FIG. 7. CD3E K104E maintains relative TCR expression.

[0009] FIG. 8. OrthoCD3 Teff can be readily expanded by Dynabead stimulation.

[0010] FIG. 9. OrthoCD3 T cells are inert from teplizumab-driven activation.

[0011] FIG. 10. Screening of mutants with a range of binding capabilities.

[0012] FIG. 11 A-C. Yeast surface display of human CD35E heterodimer as a proxy for anti-CD3 binding. A. Yeast surface display plasmids were cloned wherein the ectodomains of CD35 and CD3E were fused to yeast Aga2 protein with flexible amino acid linkers. Such plasmids were transformed into yeast strain EBY100, induced for surface expression, and used for binding analysis to various anti-CD3 by flow cytometry. B. Yeast-displayed CD30E has sub-nanomolar binding affinity (Kd) to Teplizumab and OKT3, consistent with previously reported values for human T cells. C. Yeast-displayed CD35E also binds to various other anti-CD3 as labeled. These anti-CD3 bind to display of CD35E heterodimer but not display of CD3E alone, consistent with previous literature that most anti-CD3 bind to conformational epitopes on CD3E that only form in CD35E or CD3yE heterodimers.

[0013] FIG. 12. Yeast surface display screen of single CD3 mutants to various anti-CD3. CD3<5E yeast surface display plasmids encoding each CD3E mutation were individually cloned and transformed into yeast. These yeast cells were induced for expression, incubated with 1 nM of anti-CD3, and analyzed for binding by flow cytometry. Mean binding fluorescence intensity was normalized to that of the wild-type CD35E binding to each anti-CD3.

[0014] FIG. 13. Yeast surface display screen of CD3E K104 site saturation mutagenesis. Using the CD30E yeast surface display plasmid, plasmids mutating CD3E K104 to all possible amino acids except cysteine were cloned and tested for binding to saturating levels (100 nM) of OKT3. Mean binding fluorescence intensity was normalized to that of the wild-type CD35E binding.

[0015] FIG. 14. Primary human T cells with mutations at CD3E K104 exhibit decreased OKT3 binding. Primary human T cells were isolated from healthy donor PBMCs and activated with anti- CD3 / 28 dynabeads prior to electroporation. Primary T cells were co-electroporated with Cas9 precomplexed to a guide RNA proximal to CD3E K104, as well as donor DNA (single-strandedoligodeoxynucleotides, ssODNs) encoding different K104 mutants. Engineered T cells were recovered and analyzed for binding to OKT3 (top) or anti-CD3 clone MEM57 (bottom) by flow cytometry. Mutants K104A, K104D, K104E, and K104G all exhibited decreased OKT3 binding compared to the internal control of unedited T cells. Mutant K104E showed the lowest OKT3 binding comparable to that of CD3c knock-out cells, and thus is optimal. Decrease in OKT3 binding is not at the expense of decreased CD3E expression on the T cell surface, as shown by comparable staining by anti-CD3 clone MEM57.

[0016] FIG. 15. OrthoCD3 (CD3E K104E) engineering and sorting of T cells. T cells are stimulated with anti-CD3 / 28 dynabeads for 48 hours prior to dynabead removal and subsequent electroporation of genetic engineering reagents such as Cas9 RNP and donor DNA. Electroporated T cells are recovered overnight in media containing commercially available NHEJ inhibitors. Following full media change to wash out NHEJ inhibitors, fresh dynabeads are added to culture expand the T cells. 7 days after electroporation, T cells are co-stained with OKT3 as well as anti-CD3 clones that maintain binding to orthoCD3 such as clones SP34-2 and MEM57. This co-stain allows separation of CD3 KO cells (OKT3 SP34-2 ) and wild-type CD3 cells (OKT3+SP34-2+) from orthoCD3 cells (OKT3 SP34-2+). OrthoCD3 T cells (OKT3 SP34-2+) can be sorted by FACS for downstream studies.

[0017] FIG. 16. OrthoCD3 T cells are functionally inert to teplizumab while maintaining activation and expansion by stimulation from other anti-CD3 reagents. FACS-sorted mock and orthoCD3 T cells were rested and plated in wells coated with anti-CD28 and various anti-CD3: teplizumab, MEM57, or SP34-2. 48 hours later, T cells were analyzed for their activation by markers LAG-3, CD69, and CD25. Mock T cells activate by teplizumab, while orthoCD3 T cells stay minimally activated comparable to anti-CD28 only control, demonstrating that orthoCD3 T cells are completely inert to teplizumab. OrthoCD3 T cells can however still be activated by MEM57 and SP34-2, as well as commercially available Dynabeads reagent. In a proliferation assay, T cells were labeled with a proliferation dye prior to plating. 120 hours later, proliferation of T cells was analyzed by dilution of the proliferation dye. Consistent with the activation data, orthoCD3 T cells do not proliferate with teplizumab, but does proliferate with stimulation from MEM57, SP34-2, and Dynabeads, identifying alternative reagents for culturing engineered orthoCD3 T cells for downstream applications.

[0018] FIG. 17. OrthoCD3 regulatory T cells engineered by homology-directed repair maintain intracellular FoxP3 levels. Regulatory T cells (Tregs) were engineered with orthoCD3 and sorted, alongside a mock T reg control as well as a mock shocked control (T regs that were electroporated in absence of genetic engineering reagents). These cells were fixed and permeabilized to stainfor intracellular FoxP3 levels. FoxP3 levels remain unchanged, demonstrating that orthoCD3 engineering does not impact FoxP3, a classic transcription factor for Tregs.

[0019] FIG. 18. Alternative orthoCD3 T cell engineering by retroviral transduction. Isolated Tregs are stimulated with anti-CD3 / 28 dynabeads for 48 hours prior to dynabead removal and subsequent electroporation of Cas9 RNP targeting the CD3E locus, achieving near 100% CD3E knock out. Following recovery in media for 6 hours, CD3E KO Tregs are split into two and transduced with retrovirus encoding wild-type CD3 (“WT TD”) or orthoCD3 (“OrthoCD3 TD”). Following transduction, CD3E co-stain shows the expected wild-type CD3 cells (OKT3+SP34-2+) in WT TD, and orthoCD3 cells (OKT3 SP34-2+) in OrthoCD3 TD. Transduced cells may be sorted for downstream studies; alternatively, the contaminating cells, which are mostly CD3E knock-out cells, will not expand with anti-CD3 stimulation and hence will be diluted with further cell expansion. “WT TD” cells serve as a better control than mock cells for comparisons to “OrthoCD3 TD” cells.

[0020] FIG. 19. OrthoCD3 regulatory T cells maintain comparable phenotype as wtCD3 regulatory T cells. Following retroviral engineering, Treg cells were cultured using dynabeads and their phenotypes examined by a high-dimension flow cytometry panel. This panel includes the TOR, classical Treg markers (CD4, CD25, CD127, FoxP3), and activation markers.

[0021] FIG. 20. OrthoCD3 regulatory T cells maintain comparable suppressive function as wtCD3 regulatory T cells. In a Teff:Treg co-culture assay, plates are coated with anti-CD3 clone SP34-2 and anti-CD28. Autologous CD4+CD25_effector T cells are stained with a proliferation dye and plated, from which they expand from anti-CD3 / 28 stimulation. Then, following retroviral engineering, WT TD and orthoCD3 TD Tregs are introduced at titrating levels to suppress the proliferation of CD4+CD25_effector T cells. % inhibition is calculated based on the percentage of effector T cells that proliferate. The suppression curves of WT TD and OrthoCD3 TD Tregs overlap, demonstrating that suppressive function is not impacted by orthoCD3 engineering.

[0022] FIG. 21. OrthoCD3 T cells have reduced binding to otelixizumab and visilizumab. This highlights that anti-CD3 besides teplizumab, such as otelixizumab and visilizumab, may be utilized to preferentially deplete wtCD3 T cells over orthoCD3 T cells.

[0023] FIG. 22A-C. Yeast surface display binding curves of mouse CD3E wild-type and mutant P86A to antibodies (A) 2C11 , (B) 17A2, and (C) 500A2. Mutant P86A shows greatly reduced 2C11 binding while maintaining comparable binding kinetics to antibodies 17A2 and 500A2 as wild-type mouse CD3. Irrelevant control are yeast cells displaying an irrelevant protein and controls for any non-specific binding at very high antibody concentrations.

[0024] FIG. 23. Yeast surface display screen of mouse CD3E P86 site saturation mutagenesis. Using the mouse CD3yE yeast surface display plasmid, plasmids mutating mouse CD3E P86 toall possible amino acids except cysteine were cloned and tested for binding to a very high concentration (1 ,000 nM) of 2C1 1 . Mean binding fluorescence intensity was normalized to that of the wild-type mouse CD3y£ binding.

[0025] FIG. 24A-B. Yeast surface display binding curves of top mouse CD3e P86 mutants to antibodies (A) 17A2 and (B) 500A2. All mutants tested showed similar binding affinity (Kd) to 17A2. Binding to 500A2 is more variable, with some mutants showing similar binding affinity.

[0026] FIG. 25. Mouse regulatory T cells with mutations at mouse CD3E P86 exhibit decreased binding to 2C11 while maintaining TOR expression. Mouse Tregs were isolated from mouse spleens and activated with mouse anti-CD3 / 28 dynabeads prior to electroporation of Cas9 RNP and donor DNA encoding the P86 mutations. Edited cells were FACS sorted by gating on 2C11 TCRp+population, isolating them from CD3 knock-out and unedited cells. Sorted cells were culture expanded and later stained for 2C11 , 17A2, 500A2, and TCR antibodies individually and analyzed by flow cytometry.

[0027] FIGS. 26A-B. Mouse regulatory T cells with mutations at mouse CD3E P86 are functionally inert to 2C1 1 while maintaining activation by 17A2 stimulation. FACS-sorted mouse Tregs with CD3E P86 mutations were plated in wells coated with or without 2C1 1 and 17A2 antibodies. 48 hours later, T cells were analyzed by flow cytometry for their activation by markers (A) PD-1 and (B) LAG3.DETAILED DESCRIPTION

[0028] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0029] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or bothof the limits, ranges excluding either or both of those included limits are also included in the invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0031] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0032] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0033] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0034] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0035] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progenyof a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0036] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0037] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0038] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” is the dose of a cell population that achieves a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0039] A “therapeutically effective amount” of a cell population may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0040] An “increased” or “enhanced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include an increase that is1.1 , 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0041] A “decrease” or “reduced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include an decrease that is 1 .1 ,1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1.5, 1.6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0042] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / cell composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0043] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0022] The CD3 protein, also known as the T-cell receptor complex, is a crucial component of the T-cell receptor (TCR) signaling pathway, and plays a critical role in transmitting signals from the TCR to the interior of the T cell, triggering a series of intracellular events that lead to T-cell activation. The CD3 protein complex consists of five distinct polypeptide chains, designated as CD3y, CD35, CD3s, and CD3 . These chains assemble together to form two functional dimers: CD3ys and CD35s. These dimers, along with the CD3^ chain, are associated with the TCR complex. The CD3y, CD35, and CD3c chains are immunoglobulin-like molecules, while the CD3 chain is more structurally diverse.

[0023] The extracellular region of CD3 proteins contains immunoglobulin-like domains, responsible for interacting with the TCRa|3 heterodimer. These interactions contribute to the stabilization of the TCR complex and facilitate antigen recognition. The cytoplasmic tail of the CD3 subchains contain immunoreceptor tyrosine-based activation motifs (ITAMs), which are phosphorylated upon TCR engagement, initiating downstream signaling events.

[0024] The reference sequence of human wild-type CD3e protein is provided as SEQ ID NO:1. The mature form of the protein starts at residue 22, modified residues in bold. MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHN DKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCME MDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD YEPIRKGQRDLYSGLNQRRI ortho K104E (SEQ ID NO:2)MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSEPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIThe refseq for mouse CD3E has the sequence (SEQ ID NO:3) MRWNTFWGILCLSLLAVGTCQDDAENIEYKVSISGTSVELTCPLDSDENLKWEKNGQELPQKH DKHLVLQDFSEVEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVDLTAVAIIIIVDICITLGLLMVI YYWSKNRKAKAKPVTRGTGAGSRPRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRAV

[0044] An established method of lymphodepletion utilizes treatment with a class of monoclonal antibodies that bind the CD3E subchain of the TCR-CD3 complex on T cells, which leads to T cell anergy and apoptosis. Anti-CD3 clone OKT3 and its humanized format teplizumab (henceforth referred to interchangeably as OKT3) have gained FDA approval for use in organ transplant rejection and prevention of type 1 diabetes (T1 D), demonstrating their immunosuppressive efficacy, safety in patients, as well as potential for facilitated repurposing for clinical GvHD. Muromonab (murine OKT3) has also been used successfully to treat GvHD with very high response rates to treatment. Recent FDA approval of Teplizumab (humanized OKT3) for T1 D, which has improved side effect profile from minimized immunogenicity, allows easy integration with Treg therapy. The binding specificity of OKT3 is shared with muromonab, and teplizumab. Ortho-CD3 also has reduced binding to otelixizumab and visilizumab. This highlights that anti- CD3 antibodies apart from teplizumab, such as otelixizumab and visilizumab, may be utilized to preferentially deplete wtCD3 T cells over orthoCD3 T cells. Antibodies shown to maintain binding to ortho-CD3e include, without limitation, MEM57 and SP34-2.

[0045] An “ortho”, or “orthogonal binding pair” refers to a genetically engineered pair of proteins that are modified by amino acid changes to (a) substantially lack binding to the antibody of interest; and (b) to retain binding to cellular components involved in CD3 activity, particularly CD3y, and CD35. In some embodiments, the affinity of the orthogonal CD3 for cellular components is comparable to the affinity of the native CD3 protein, e.g. having an affinity that is least about 1% of the native cytokine receptor pair affinity, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, and may be higher, e.g. 2X, 3X, 4X, 5X, 10X or more of the affinity of the native CD3 for the native cellular components.

[0046] As used herein, “substantially reduced binding” or “substantially lack binding” refers to no detectable binding, or an insignificant binding, i.e., having a binding affinity much lower than that of the natural ligand, e.g. reduced by at least about 10%, at least about 25%, at least about 50%,and may be reduced 2X, 3X, 4X, 5X, 10X or more. The affinity can be determined with competitive binding experiments that measure the binding of a receptor with a single concentration of labeled ligand in the presence of various concentrations of unlabeled ligand. Typically, the concentration of unlabeled ligand varies over at least six orders of magnitude. Through competitive binding experiments, IC50 can be determined. As used herein, "IC50" refers to the concentration of the unlabeled ligand that is required for 50% inhibition of the association between receptor and the labeled ligand. IC50 is an indicator of the ligand-receptor binding affinity. Low IC50 represents high affinity, while high IC50 represents low affinity.

[0047] Regulatory T cells (“Tregs”) are a specialized subpopulation of T cells that can suppress activation of the immune system and thereby maintain immune tolerance. Tregs are share the phenotype of being CD4+CD25+FOXP3+. There are various types of Tregs, for example, TCRap+CD4+ regulatory T cells, which include natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs). nTregs are T cells produced in the thymus and delivered to the periphery as a long-lived lineage of self-antigen-specific lymphocytes. iTregs are recruited from circulating lymphocytes and acquire regulatory properties under particular conditions of stimulation in the periphery. nTregs and iTregs are CD4+CD25+; both can inhibit proliferation of CD4+CD25- T cells in a dose-dependent manner, and both are anergic and do not proliferate upon TCR stimulation. Tregs can be identified or selected based on various marker expression profiles. Non-limiting examples of marker expression profiles that can be used to select Tregs include (1 ) CD4+CD25+CD127dim, (2) CD4+FOXP3+, (3) CD3+CD4+CD25+, (5) CD3+ CD4+ CD25+ CD127dim, (6) CD3+ CD4+ CD25+ CD127dim FOXP3+, (7) CD3+FOXP3+, (8) CD3+CD4+FOXP3+, (9) CD3+ CD4+CD25+FOXP3+, (10) CD3+CD25+FOXP3+, (11 ) CD3+CD25+CD127dim, (12) CD4+CD25+, (13) CD4+CD25+CD127dimFOXP3+, (14) FOXP3+, CD4+FOXP3+, (15) CD4+CD25+FOXP3+, (16) CD25+FOXP3+, or (17) CD25+ CD127dim. In some embodiments, the Treg cells are CD4+CD25+CD127loFoxP3+Treg. Selection based on certain expression profiles can be achieved based on extracellular markers and without requiring cell permeabilization, for example, selection based on CD4+CD25+CD12710phenotype.

[0048] In some embodiments a T cell, e.g. a CD4+ T cell, is engineered to express FoxP3 to achieve a Treg phenotype. Foxp3 plays a crucial role in development and function of Treg cells. Constructs and methods from expressing Foxp3 in T cells are described in, for example, WO 2007 / 065957, which is incorporated herein in its entirety. In some embodiments the Foxp3 is wildtype (WT) Foxp3. Exemplary wild type human Foxp3 sequences are described in NP 054728.2, the contents of which are incorporated herein by reference. In some embodiments, the Foxp3 isa minimal Foxp3. A “minimal Foxp3” is engineered to mimic N-terminally, C-terminally, or N-and C-terminally cleaved Foxp3 forms. Minimal Foxp3 is, in some contexts, more active than wildtype (WT) Foxp3. In some embodiments, the minimal Foxp3 comprises a Foxp3 polypeptide that has been N-terminally truncated, C-terminally truncated, or N- and C-terminally truncated. In some embodiments, the Foxp3 is constitutively active.

[0049] Other T cells of interest include CAR-T cells, which are modified to surface express a chimeric antigen receptor. As used herein, the terms “chimeric antigen receptor T-cell” and “CAR- T cell” are used interchangeably to refer to a T-cell that has been recombinantly modified to express a CAR. As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably to refer to a polyprotein comprising multiple functional domains arranged from amino to carboxy terminus in the sequence: (a) an antigen binding domain (ABD), (b) a transmembrane domain (TD); (c) one or more cytoplasmic signaling domains (CSDs) wherein the foregoing domains (a) - (c) may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence which is conventionally removed during post-translational processing and presentation of the CAR on the cell surface. CARs useful in the practice of the present invention are prepared in accordance with principles well known in the art.

[0050] Engineering cells to express CAR constructs is commonly accomplished through viral vector systems such as lentivirus, Gamma-retroviral, and adeno-associated viral (AAV) vectors. Additionally, viral-free systems such as the Sleeping Beauty (SB), or piggyBac transposon have been used to integrate CAR encoding DNA with favorable integration into the target genome. Furthermore, Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein 9 (Cas9) gene-editing technology, which allows for the insertion of DNA at specific locations directed by RNA, has also been used to engineer CAR T cells. CRISPR- engineered CAR T cells, which express CAR from an endogenous TCR locus remain active for longer periods than their virus-transduced counterparts.

[0051] As used herein, the term antigen binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. The ABD may be any polypeptide that specifically binds to one or more antigens expressed on the surface of a target cell.

[0052] In one embodiment, the ABD is a single chain Fv (ScFv). An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061 . Thegeneration of ScFvs based on monoclonal antibody sequences is well known in the art. See, e.g. The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 “Bacterial Expression, Purification and Characterization of Single-Chain Antibodies” Kipriyanov, S. Antibodies used in the preparation of scFvs may be optimized to select for those molecules which possess particular desirable characteristics (e.g. enhanced affinity) through techniques well known in the art such as phage display and directed evolution. In some embodiments, the ABD comprises an anti-CD19 scFv, an anti-PSA scFv, an anti-HER2 scFv, an anti-CEA scFv, an anti- EGFR scFv, an anti-EGFRvlll scFv, an anti-NY-ESO-1 scFv, an anti-MAGE scFv, an anti-5T4 scFv, or an anti-Wnt1 scFv. In another embodiment, the ABD is a single domain antibody obtained through immunization of a camel or llama with a target cell derived antigen, in particular a tumor antigen. See, e.g. Muyldermans, S. (2001 ) Reviews in Molecular Biotechnology 74: 277-302. Alternatively, the ABD may be generated wholly synthetically through the generation of peptide libraries and isolating compounds having the desired target cell antigen binding properties in substantial accordance with the teachings or Wigler, et al. United States Patent No. 6303313 B1 issued November 12, 1999; Knappik, et al., United States Patent No 6,696,248 B1 issued February 24, 2004, Binz, et al. (2005) Nature Biotechnology 23:1257-1268, and Bradbury, et al. (2011 ) Nature Biotechnology 29:245-254.

[0053] In some embodiments, the CARs comprise a target-specific binding element otherwise referred to as an antigen binding moiety. The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen moiety domain in the CAR of the invention include those associated with autoimmune disease, transplant rejection, and the like.

[0054] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to facilitate antigen binding. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. In one embodiment, the linker is the hinge region from an immunoglobulin, e.g. the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In those instances where the ABD is an scFv, an IgG hinge is effective. In some embodiments the linker comprises the amino acid sequence (G4S)nwhere n is 1 , 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0055] CARs useful in the practice of the present invention further comprise a transmembrane domain joining the ABD (or linker, if employed) to the intracellular cytoplasmic domain of the CAR.The transmembrane domain is comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membrane spanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e.g Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3 , CD4, CD8, CD28, etc.

[0056] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor ^-chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1 BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), p1 10a, |3, or 0 catalytic subunit of PI3 kinase, the human CD3 <- chain, cytoplasmic domain of CD134 (also referred to as 0X40 and TNFRSF4), FccRIy and |3 chains, MB1 (Igo) chain, B29 (Igp) chain, etc.), CD3 polypeptides (5, A and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.

[0057] In some embodiments, the CAR may also provide a co-stimulatory domain. The term “costimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cellreceptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of members of the TNFR superfamily, CD28, CD137 (4-1 BB), CD134 (0X40), Dap10, CD27, CD2, CD5, ICAM-1 , LFA-1 (CD11 a / CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.Source Cells

[0058] Typically, the engineered T cells are T-cells or stem cells, e.g. hematopoietic stem cells, which have been recombinantly modified by to introduce an amino acid substitution in both alleles of the CD3c locus. Cells for engineering as described above are collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation.

[0059] For example, a population of cells comprising Tregs can be isolated from whole blood. A population of cells comprising Tregs can be isolated from a peripheral blood apheresis product. A population of cells comprising Tregs can be isolated from a population of cells previously enriched and / or depleted for one or more other cell types, e.g., isolated from a population of cells depleted of CD34+ cells. In some embodiments, Tregs are isolated from the flow-through fraction of a CD34+ MACS selection.

[0060] The number of Tregs in a population of cells can be determined, for example, by flow cytometry, where Tregs can be identified as, for example, CD4+CD25+CD127l0or CD4+FOXP3+. Dose calculations can be adjusted based on measures of cell viability measurements, e.g., viability determined via flow cytometry with propidium iodide or 7-AAD, or via trypan blue exclusion.

[0061] An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, efc. Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedancechannels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide- MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0062] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS). The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0063] The Treg cells may be cultured in vitro under various culture conditions, as disclosed herein. The cell population may be conveniently suspended in an appropriate nutrient medium, such as TexMACS, Iscove's modified DMEM, RPMI-1640, etc., and as disclosed herein are supplemented with human serum. The culture may contain activation agent factors to which the cells are responsive.

[0064] Treg cells are usually obtained from a single donor, for example, obtained from mobilized peripheral blood apheresis of a single donor, although can be from combined donors. A donor and a recipient of the disclosure can be allogenic. A donor and a recipient of the cryopreserved cells can be HLA matched. A donor and a recipient of the of the cryopreserved cells can be HLA mismatched, e.g. mismatched at 1 , 2, 3, 4, 5, or 6 of the major HLA alleles. A donor and a recipient of the cryopreserved cells can be haploidentical.

[0065] In some embodiments, a cell population of the disclosure can be obtained from whole blood. A cell population of the disclosure can be obtained from a peripheral blood apheresis product, for example, a mobilized peripheral blood apheresis product. A cell population of the disclosure can be obtained from at least one apheresis product, two apheresis products, three apheresis products, four apheresis products, five apheresis products, six apheresis products, or more.Cell Engineering

[0066] Conveniently, gene editing, or genome editing, is used to introduce the amino acid substitution into the CD3e locus. In some embodiments a cell of interest is engineered to express an ortho CD3e and to lack expression of wild-type CD3e. A cell of interest may be a stem cell, e.g. a hematopoietic stem cell (HSC), induced pluripotent stem cells (iPSC), etc. A cell of interest may be a T cell. Cells may be isolated from an individual, a cell line, etc. Various methods may be used for engineering; where, for example, CRISPR engineering provides for efficient conversion of the wild-type CD3e locus to the ortho version, or CRISPR knockout of wildtype CD3E followed by a knock-in of an expression vector, for example by viral transduction of orthoCD3c using viral transduction such as adenovirus, lentivirus, retrovirus, etc.

[0067] Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using nucleases. The nucleases may be artificially engineered. Alternately, the nucleases may be found in nature. The nucleases create specific double-stranded breaks (DSBs) at desired locations in the genome. The cell's endogenous repair mechanisms subsequently repairs the induced break(s) by natural processes, such as homologous recombination (HR) and non-homologous end-joining (NHEJ). Nucleases include, for example, Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), CRISPR, (e.g., the CRISPR / Cas system), and engineered meganuclease re-engineered homing endonucleases. CRISPR nucleases include for example a Cas nuclease, a Cpf 1 nuclease, a C2c1 nuclease, a C2c3 nuclease, and a C2c3 nuclease.

[0068] In an embodiment, the nuclease comprises a CRISPR / Cas system. The CRISPR (clustered regularly interspaced short palindromic repeats) locus, which encodes RNA components of the system, and the Cas (CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002. Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res. 30: 482-496; Makarova et aL, 2006. Biol. Direct 1 : 7; Haft et al., 2005. PLoS Comput. Biol. 1 : e60) make up the gene sequences of the CRISPR / Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0069] The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNAs, the pre- crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additionalrequirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer. Activity of the CRISPR / Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called “adaptation”, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Thus, in the bacterial cell, several of the so-called “Cas” proteins are involved with the natural function of the CRISPR / Cas system and serve roles in functions such as insertion of the alien DNA.

[0070] In certain embodiments, Cas protein may be a “functional derivative” of a naturally occurring Cas protein. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or produced in vitro or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which encodes a Cas that is the same as or different from the endogenous Cas. In some cases, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.

[0071] The method also includes introducing single-guide RNAs (sgRNAs) into the cell or the organism. The guide RNAs (sgRNAs) include nucleotide sequences that are complementary to the target chromosomal DNA. The sgRNAs can be, for example, engineered single chain guide RNAs that comprise a crRNA sequence (complementary to the target DNA sequence) and a common tracrRNA sequence, or as crRNA-tracrRNA hybrids. The sgRNAs can be introduced into the cell or the organism as a DNA (with an appropriate promoter), as an in vitro transcribed RNA, or as a synthesized RNA.

[0072] To achieve expression of the recombinant CD3£, a nucleic acid encoding the orthogonal protein may alternatively be inserted into a replicable vector for expression following knock-out of the wild-type alleles. Many such vectors are available. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like.

[0073] Expression vectors for expression of the orthogonal protein may be viral vectors or non- viral vectors. Plasmids are examples of non-viral vectors. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non-viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).

[0074] In one embodiment, a non-viral vector may be provided in a non-viral delivery system. Non-viral delivery systems are typically complexes to facilitate transduction of the target cell with a nucleic acid cargo wherein the nucleic acid is complexed with agents such as cationic lipids (DOTAP, DOTMA), surfactants, biologicals (gelatin, chitosan), metals (gold, magnetic iron) and synthetic polymers (PLG, PEI, PAMAM). Numerous embodiments of non-viral delivery systems are well known in the art including lipidic vector systems (Lee et al. (1997) Crit Rev Ther Drug Carrier Syst. 14:173-206); polymer coated liposomes (Marin et al., U.S. Pat. No. 5,213,804, issued May 25, 1993; Woodie, et al., U.S. Pat. No. 5,013,556, issued May 7, 1991 ); cationic liposomes (Epand et al., U.S. Pat. No. 5,283,185, issued Feb. 1 , 1994; Jessee, J. A., U.S. Pat. No. 5,578,475, issued Nov. 26, 1996; Rose et al, U.S. Pat. No. 5,279,833, issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No. 5,334,761 , issued Aug. 2, 1994).

[0075] In another embodiment, the expression vector may be a viral vector. When a viral vector system is to be employed for CAR and expression of the orthogonal, retroviral or lentiviral expression vectors are preferred. In particular, the viral vector is a gamma retrovirus, selfinactivating lentiviral vectors, and retroviral vectors.

[0076] Transduction of T-cells with an expression vector may be accomplished using techniques well known in the art including but not limited co-incubation with host T-cells with viral vectors, electroporation, and / or chemically enhanced delivery.

[0077] The vector may be used to introduce a “suicide switch” into the cell. Several suicide gene systems, or safety switches, have been developed to improve the safety of cell transfer by allowing quick elimination of engineered cells if needed. A suicide gene can use a transgenicenzyme to activate a prodrug system, such as cytosine deaminase or herpes simplex virus thymidine kinase (HSV-TK). Cytosine deaminase activates 5-fluorocytosine, inducing 5- fluorocytosine conversion into 5-fluorouracil, which is then transformed into potent pyrimidine antimetabolites. This system induces cell proliferation inhibition and cell death. HSV-TK activates ganciclovir and can be used as a suicide gene after hematopoietic stem cell transplantation. Ganciclovir is converted into a monophosphorylated molecule and then metabolized into toxic triphosphate, inhibiting DNA synthesis in dividing cells and ultimately inducing cell death. A suicide gene system involving inducible Caspase 9 (iCaspase9) has also been tested in the clinic, and its utility has widely been proven. iCaspase9 is a fusion of a mutated 12-kDa FK506 binding protein (FKBP12) with the catalytic domain of caspase 9. FKBP12 is mutated to allow for the docking of AP1903, which is a small-molecule chemical inducer of dimerization (CID). CID administration induces cross-linking and activation of proapoptotic target molecules, leading to cell apoptosis.Activation and proliferation

[0078] Following genomic editing the population of orthoCD3e T cells may be activated in vitro to enhance activity and / or increase the number of cells. The population of cells in vitro is stimulated with an effective dose of an agent that activates, but which does not bind to the binding site of OKT3. Suitable agents include, without limitation, anti-CD3 antibodies with a specificity other than that of OKT3, dynabeads, allogeneic cells, and the like as known in the art. The cells are incubated for a period of time sufficient for activation, e.g. 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, up to 10 days, up to 2 weeks, or more.Administration

[0079] A population of cells comprising orthoCD3e T cells can be administered to a subject at a dose greater than at least about 1 x 104, 1 x 105, 2 x 105, 3 x 105, 4 x 105, 5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 10®, 1.9 x 106, 2 x 106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 10®, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106, 3.9 x 106, 4 x 106, 4.1 x 10®, 4.2 x 10®, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x 106, 4.9 x 106, 5 x 106, 5.1 x 106, 5.2 x 106, 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x106, 5.7 x 106, 5.8 x 106, 5.9 x 106, 6 x 106, 6.5 x 106, 7 x 106, 7.5 x 10s, 8 x 106, 8.5 x 106, 9 x 106, 9.5 x 106, 1 x 107, 1 .5 x 107, 2 x 107, 2.5 x 107, 3 x 107, 3.5 x 107, 4 x 107, 4.5 x 107, 5 x 107, 5.5 x107, 6 x 107, 6.5 x 107, 7 x 107, 7.5 x 107, 8 x 107, 8.5 x 107, 9 x 107, 9.5 x 107, 1 x 10s, 1 x 10s, 1 .5x 10s, 2 x 108, 2.5 x 108, 3 x 108, 3.5 x 108, 4 x 107, 4.5 x 108, 5 x 108, 5.5 x 108, 6 x 108, 6.5 x 108, 7 x 108, 7.5 x 108, 8 x 108, 8.5 x 108, 9 x 108, 9.5 x 108, 1 x 109, or more cells per kg of recipient body weight.

[0080] In some embodiments, a population of cells comprising orthoCD3e T cells administered to a subject can be administered at a dose of at most about 1 x 104, 1 x 105, 2 x 105, 3 x 105, 4 x 105,5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 1.1 x 106, 1.2 x 10s, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1 .8 x 106, 1 .9 x 106, 2 x 106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106,3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106, 3.9 x 106, 4 x 106, 4.1 x 106, 4.2 x 106, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x 106, 4.9 x 106, 5 x 106, 5.1 x 106, 5.2 x 106, 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x 106, 5.7 x 106, 5.8 x 106, 5.9 x 106, 6 x 106, 6.5 x 106, 7 x 106, 7.5 x 106, 8 x106, 8.5 x 106, 9 x 106, 9.5 x 106, 1 x 107, 1.5 x 107, 2 x 107, 2.5 x 107, 3 x 107, 3.5 x 107, 4 x 107,4.5 x 107, 5 x 107, 5.5 x 107, 6 x 107, 6.5 x 107, 7 x 107, 7.5 x 107, 8 x 107, 8.5 x 107, 9 x 107, 9.5 x107, 1 x 108, 1 x 108, 1.5 x 108, 2 x 108, 2.5 x 108, 3 x 108, 3.5 x 108, 4 x 107, 4.5 x 108, 5 x 108, 5.5 x 108, 6 x 108, 6.5 x 108, 7 x 108, 7.5 x 108, 8 x 108, 8.5 x 108, 9 x 108, 9.5 x 108, 1 x 109, or less cells per kg of recipient body weight, where Tregs are defined as CD4+CD25+CD127dim, CD3+CD4+CD25+, CD3+ CD4+ CD25+ CD127dim, CD3+ CD4+ CD25+ CD127dim F0XP3+, CD3+FOXP3+, CD3+CD4+FOXP3+, CD3+ CD4+CD25+FOXP3+, CD3+CD25+FOXP3+, CD3+CD25+CD127dim, CD4+CD25+, CD4+CD25+CD127dimFOXP3+, F0XP3+, CD4+FOXP3+, CD4+CD25+FOXP3+, CD25+FOXP3+, or CD25+ CD127dim.

[0081] A population of cells comprising orthoCDSe T cells of the disclosure can comprise, for example, greater than at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more FOXP3+ cells as a percentage of total cells, nucleated cells, or CD45+ cells, or CD4+CD25+CD127dim cells as a percentage of total cells, nucleated cells, or CD45+ cells, or as a percentage of total cells, nucleated cells, or CD45+ cells.

[0082] A population of cells comprising orthoCD3e T cells of the disclosure can have a defined level of contaminating non-T reg cells. For example, greater than at least about 1 x 102, 2 x 102, 3 x 102, 4 x 102, 5 x 102, 6 x 102, 7 x 102, 8 x 102, 9 x 102, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103,6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 105, or more non-Treg cells per kg of recipient body weight can be present in population of cells comprising Tregs of the disclosure, where non-Treg cells are FOXP3- or CD127+ / bright.

[0083] In some embodiments, at most about 1 x 102, 2 x 102, 3 x 102, 4 x 102, 5 x 102, 6 x 102, 7 x 102, 8 x 102, 9 x 102, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 105, or less noncomprising orthoCD3e T cells per kg of recipient body weight are present in a population of cells comprising Tregs of the disclosure, where non-Treg cells are FOXP3- or CD127+ / bright.

[0084] A population of cells comprising orthoCD3e T cells of the disclosure can comprise, for example, greater than at least about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008% 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1 %, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1 .8%, 1 .9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more non-Treg cells as a percentage of total cells, nucleated cells, or CD45+ cells, where non-Treg cells are FOXP3- or CD127+ / bright.

[0085] In some embodiments, a population of cells comprising orthoCD3E T cells of the disclosure comprises at most about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008% 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1 .9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or less non-Treg cells as a percentage of total cells, nucleated cells, or CD45+ cells, where non-Treg cells are FOXP3- or CD127+ / bright.Conditions for treatment

[0086] OrthoCD3e T cells, e.g. T reg cells, are useful in a variety of conditions, including, for example, treatment of cancer, in association with hematopoietic stem cell transplantation (HCT), for example to reduce graft v host disease, to reduce allograft rejection, and the like. In some embodiments a condition to be treated is an autoimmune disease, allergic disease, or inflammatory disease. For example, a population of cells comprising orthoCD3E T cells can be administered to an HCT recipient subject, and the population of orthoCD3e T cells can serve to reduce or prevent GVHD in the HCT recipient subject. In some embodiments, e.g. with engineered TCR and CART cells, a condition to be treated is cancer.

[0087] The endogenous or contaminating transplanted T cells can be lymphodepleted by administering an effective dose of an antibody with the specificity of OKT3. The dosing and regimen for depleting T cells are typically tailored to the individual's specific condition andtreatment goals and typically follow clinical guidelines. The dosing regimen usually consists of a series of intravenous infusions of OKT3 over a specified duration. The frequency and duration of treatment can vary depending on factors such as the patient's immune status, the underlying disease being treated, and the specific medical procedure or therapy being pursued. Regular monitoring of T cell counts and other relevant biomarkers helps guide the treatment course, ensuring optimal outcomes while managing any potential adverse effects associated with OKT3 therapy. The recommended dose of OKT3 for the treatment of allograft rejection, for example, is 5 mg per day in a single (bolus) intravenous injection in less than one minute for 10 to 14 days. Dosage for the purposes of the present disclosure may range from about 0.5 mg to 10 mg / day.

[0088] Graft-versus-host disease (GVHD) is an inflammatory disease that can occur in the allogenic transplant setting. GVHD involves donor cells (graft) attacking recipient cells (host). GVHD can be classified into acute GVHD (aGVHD) and chronic GVHD (cGVHD). aGVHD typically occurs in the first 3 months after transplantation. aGVHD can be life-threatening and can involve, for example, the skin, the intestines, and / or the liver. cGVHD typically occurs after the first 3 months following transplant. cGVHD is a major source of late treatment-related complications, and can be life-threatening. In addition to inflammation, cGVHD can lead to the development of fibrosis, which can result in functional disability.

[0089] The early morbidity and mortality associated with acute graft versus host disease (aGVHD) is a major factor limiting the success of HOT, as is the long-term morbidity associated with chronic GVHD (cGVHD). The incidence of aGVHD following allogeneic HOT from an HLA-matched sibling donor (MSD) is 20 to 60%, despite the use of various immunosuppressive agents such as tacrolimus, cyclosporine, methotrexate, mycophenolate, anti-thymocyte globulin and corticosteroids. Approximately one-third of patients who undergo allogeneic HOT using a MSD and a T cell replete graft will develop chronic GVHD.

[0090] GVHD severity can be graded, for example, using the Glucksberg grade (l-IV) or the International Bone Marrow Transplant Registry (IBMTR) grading system (A-D). The severity of acute GVHD is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined with (Glucksberg) or without (IBMTR) the patient’s performance status to produce an overall grade, which can have prognostic significance. Grading is important in terms of assessing the response to prophylaxis or treatment, impact upon survival, and association with graft-versus-leukemia effect.

[0091] Grade l(A) GVHD is characterized as mild disease, grade I l(B) GVHD as moderate, grade lll(C) as severe, and grade IV(D) life-threatening. The IBMTR grading system defines the severityof acute GVHD as follows: Grade A: stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement; Grade B: stage 2 skin involvement, stage 1 to 2 gut or liver involvement; Grade C: stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day); Grade D: stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day OR pain OR ileus). Patients with moderate to severe GVHD have a significantly higher mortality rate compared with those with milder disease, for example, estimated five year survival for patients with grade III (C) aGVHD is 25%, while for patients with grade IV (D) estimated five year survival is 5%.

[0092] Management of GVHD may require immunosuppressive therapy (for example, high dose corticosteroids, prolonged administration of immunosuppressants) or cytotoxic mediations, all of which are associated with toxicity. In many cases, immunosuppressive therapies can fail to effectively treat GVHD, or can result in increased susceptibility to infection, or blunted anti-tumor immunity.

[0093] In some embodiments, administration of the OrthoCD3e T cells prevent or reduce GVHD in an HOT recipient subject. For example, the methods disclosed herein can prevent any manifestation of GVHD in a subject receiving HCT.

[0094] The methods disclosed herein can prevent, for example, any GVHD of stage 1 or above, any GVHD of stage 2 or above, any GVHD of stage 3 or above, or any GVHD of stage 4 in subjects receiving HCT.

[0095] Conditions for which the cells described herein find use in treatment include a number of inflammatory conditions. Many diseases have an underlying inflammatory component that contributes to disease initiation and / or progression. Thus, the spectrum of inflammatory diseases and diseases associated with inflammation is broad and includes autoimmune diseases such rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and autoimmune hepatitis; degenerative diseases such as osteoarthritis (OA), Alzheimer's disease (AD), and macular degeneration; chronic infections such as human immunodeficiency virus (HIV), chronic hepatitis C virus (HCV) infection, chronic hepatitis B virus (HBV), chronic cytomegalovirus (CMV) infection, tuberculosis (TB) infection, as well as other chronic viral and bacterial infections; metabolic diseases including type II diabetes, metabolic syndrome, non-alcoholic steatohepatitis (NASH), and alcoholic steatohepatitis; cardiovascular diseases such as atherosclerosis; cancers which can arise from and induce inflammation; as well as other diseases with an inflammatory component.

[0096] Type 1 diabetes (T 1 D) is an autoimmune disorder characterized by insulin deficiency due to the destruction of pancreatic (3 cells. Studies have shown reduced immune suppressive functionality of Tregs in patients with T1 D. This observation and the success of Treg transplantation in maintaining immunologic tolerance has led to the application of Treg infusion in T1 D patients to rescue remaining p cells. Infusion of expanded antigen-specific Tregs showed promising results in animal models in blocking and reversing diabetes. In some embodiments, administration of the orthoCD3e T cells prevent or reduce T1 D in a recipient. For example, the methods disclosed herein can prevent any manifestation of T1 D in a subject.

[0097] Rheumatoid Arthritis (RA) is a chronic syndrome characterized usually by symmetric inflammation of the peripheral joints, potentially resulting in progressive destruction of articular and periarticular structures, with or without generalized manifestations (Firestein (2003) Nature 423(6937) :356-61 ; Mclnnes and Schett. (2011 ) N Engl J Med. 365(23):2205-19). The cause is unknown. A genetic predisposition has been identified, and, in some populations, localized to a pentapeptide in the HLA-DR betal locus of class II histocompatibility genes. Environmental factors may also play a role. For example, cigarette smoking places individuals possessing HLA- DR4 containing the "shared epitope" polymorphism at approximately 10-20 fold increased risk of developing RA. Cigarette smoking is thought to induce anti-citrullinated protein antibody (ACPA) responses, which are measured using the commercial cyclic-citrullinated peptide (CCP) assay (Klareskog et al. (2006) Arthritis Rheum. 54(1 ):38-46). In addition, periodontitis and infection with P. gingivalis might also play a role in the initiation of autoimmune responses that result in development of RA (Rutger and Persson. 2012, J Oral Microbiol. 4). Immunologic changes may be initiated by multiple factors. About 0.6% of all populations are affected, women two to three times more often than men. Onset may be at any age, most often between 25 and 50 yr.

[0098] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by malar rashes, oral ulcers, photosensitivity, serositis, seizures, low white blood cell counts, low platelet counts, seizures, a positive anti-nuclear antibody (ANA) test, and other positive autoantibodies. SLE is an autoimmune disease characterized by polyclonal B cell activation, which results in a variety of anti-protein and non-protein autoantibodies that result in immune complexes and inflammation which contributes to tissue damage (see, e.g., Kotzin et aL, 1996, Cell 85:303-06 for a review of the disease). SLE has a variable course characterized by exacerbations and remissions and is difficult to study. For example, some patients may demonstrate predominantly skin rash and joint pain, show spontaneous remissions, and require little medication. The other end of the spectrum includes patients who demonstrate severe and progressive kidney involvement (glomerulonephritis and cerebritis) that requires therapy with highdoses of steroids and cytotoxic drugs such as cyclophosphamide. Hydroxychloroquine slows SLE progression, and is a mainstay therapeutic for the management of SLE.

[0099] Multiple sclerosis (MS) is a debilitating, inflammatory, neurological illness characterized by demyelination of the central nervous system. The disease primarily affects young adults with a higher incidence in females. Symptoms of the disease include fatigue, numbness, tremor, tingling, dysesthesias, visual disturbances, dizziness, cognitive impairment, urological dysfunction, decreased mobility, and depression. Four types classify the clinical patterns of the disease: relapsing-remitting, secondary progressive, primary-progressive and progressiverelapsing (S. L. Hauser and D. E. Goodkin, Multiple Sclerosis and Other Demyelinating Diseases in Harrison's Principles of Internal Medicine 14th Edition, vol. 2, Me Graw-Hill, 1998, pp. 2409-19).

[0100] Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, involve autoimmune attack of the bowel. These diseases cause chronic diarrhea, frequently bloody, as well as symptoms of colonic dysfunction.

[0101] Systemic sclerosis (SSc, or scleroderma) is an autoimmune disease characterized by fibrosis of the skin and internal organs and widespread vasculopathy. Patients with SSc are classified according to the extent of cutaneous sclerosis: patients with limited SSc have skin thickening of the face, neck, and distal extremities, while those with diffuse SSc have involvement of the trunk, abdomen, and proximal extremities as well. Internal organ involvement tends to occur earlier in the course of disease in patients with diffuse compared with limited disease (Laing et al. (1997) Arthritis. Rheum. 40:734-42). The majority of patients with diffuse SSc who develop severe internal organ involvement will do so within the first three years after diagnosis at the same time the skin becomes progressively fibrotic (Steen and Medsger (2000) Arthritis Rheum. 43:2437-44.). Common manifestations of diffuse SSc that are responsible for substantial morbidity and mortality include interstitial lung disease (ILD), Raynaud's phenomenon and digital ulcerations, pulmonary arterial hypertension (PAH) (Trad et al. (2006) Arthritis. Rheum. 54:184-91.), musculoskeletal symptoms, and heart and kidney involvement (Ostojic and Damjanov (2006) Clin. Rheumatol. 25:453-7). Current therapies focus on treating specific symptoms, but disease-modifying agents targeting the underlying pathogenesis are lacking.

[0102] Autoimmune hepatitis is a disease in which the body's immune system attacks liver cells. This immune response causes inflammation of the liver, also called hepatitis. Researchers think a genetic factor may make some people more susceptible to autoimmune diseases. About 70 percent of those with autoimmune hepatitis are female. The disease is usually quite serious and, if not treated, gets worse over time. Autoimmune hepatitis is typically chronic, meaning it can lastfor years, and can lead to cirrhosis-scarring and hardening-of the liver. Eventually, liver failure can result.

[0103] The presence of inflammation can be detected by a variety of approaches, including clinical history, physical examination, laboratory testing, histologic analysis of tissue, analysis of biomarkers, and imaging. Clinical features and physical exam markers of inflammation include swelling, effusions, edema, redness, warmth, pain, or associated pathologically with the influx of inflammatory cells or production of inflammatory mediators. Laboratory testing and / or histologic markers are abnormal when increased numbers of inflammatory cells are demonstrated. Markers of inflammation can include a molecular marker(s), and examples of a molecular marker(s) include C-reactive protein, a cytokine, an antibody, a DNA sequence, an RNA sequence, a cartilage marker, a metabolic marker, a bone marker, or combinations thereof. Imaging can reveal findings including enhancement of tissues, edema and swelling of tissues, and other findings indicative of inflammation. Examples of imaging markers of inflammation can include imaging markers measured using magnetic resonance imaging, ultrasound, computed tomography, angiography, and combinations thereof.

[0104] The presence of low-grade inflammation is characterized by an elevation(s) in the local or systemic concentrations of cytokines such as TNF-. alpha., IL-6, and c-reactive protein (CRP), and occurs in adiposity, osteoarthritis, Alzheimer's disease, type II diabetes, metabolic syndrome, coronary artery disease, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis, and many chronic and degenerative diseases. Low-grade inflammation is manifest by inflammation present at a level below the "high-grade" inflammation detected in active autoimmune diseases (such as active rheumatoid arthritis, psoriasis, Crohn's disease, systemic lupus erythematous, autoimmune hepatitis, and other autoimmune states) and in certain viral and bacterial infections during which humans experience clinical symptoms (such as influenza virus infection, Staphylococcus aureus infection, and other infections).

[0105] The reduction or amelioration of inflammation is indicated by dissipation of inflammation, a reduction in number of inflammatory cells or in levels of inflammatory mediators as evidenced by symptomatic relief (including but not limited to pain relief), radiographic changes, biochemical changes, pathologic / histologic changes, decreased progression of such markers of inflammation, decreased development of findings indicative of tissue or organ damage, decreased development of symptoms or signs of disease, or decreased development of disease.

[0106] In other embodiments, the types of cancer that can be treated using the subject methods of the present invention include but are not limited to adrenal cortical cancer, anal cancer, aplasticanemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, non-melanoma skin cancers, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, or Waldenstrom's macroglobulinemia.

[0107] Peptide ligands of the TCR are peptide antigens against which an immune response involving T lymphocyte antigen specific response can be generated, or against which a CART or engineered TCR can be directed. Such antigens include antigens associated with autoimmune disease, infection, foodstuffs such as gluten, etc., allergy or tissue transplant rejection. Antigens also include various microbial antigens, e.g. as found in infection, in vaccination, etc., including but not limited to antigens derived from virus, bacteria, fungi, protozoans, parasites and tumor cells. Tumor antigens include tumor specific antigens, e.g. immunoglobulin idiotypes and T cell antigen receptors; oncogenes, such as p21 / ras, p53, p210 / bcr-abl fusion product; etc.; developmental antigens, e.g. MART-1 / Melan A; MAGE-1 , MAGE-3; GAGE family; telomerase; etc.; viral antigens, e.g. human papilloma virus, Epstein Barr virus, etc.; tissue specific selfantigens, e.g. tyrosinase; gp100; prostatic acid phosphatase, prostate specific antigen, prostate specific membrane antigen; thyroglobulin, a-fetoprotein; etc. and self-antigens, e.g. her-2 / neu; carcinoembryonic antigen, muc-1 , and the like.

[0108] In some embodiments, the autoimmune disease, allergic disease, or inflammatory disease is transplant rejection. In some embodiments, the transplant rejection occurs in response totransplanted blood, bone marrow, bone, skin, heart, kidney, lung, muscle, heart or liver. In some embodiments, the transplant rejection is hyperacute rejection. In some embodiments, the transplant rejection is acute rejection. In some embodiments, the transplant rejection is chronic rejection. In some embodiments the transplant is allogeneic islet transplantation.

[0109] In some embodiments, the method increases CD45+ cell engraftment, increases overall survival, decreases serum inflammatory cytokines or a combination thereof. In some embodiments, the method decreases spleen inflammation, liver inflammation, lung inflammation, inflammation of the central nervous system (CNS), inflammation of the skin, inflammation of the pancreas, inflammation of the kidney, inflammation of the pleural cavity, inflammation of the gastrointestinal tract, inflammation of the genitourinary tract, or inflammation of the pelvis. In some embodiments, the disclosure provides a method for reducing transplant rejection in a patient transplanted with hematopoietic stem cells, bone marrow cells, or a solid organ, comprising providing administering the OrthoCD3c T cells to the patient.

[0110] In some cases, the method prevents allograft rejection, improving upon concepts described in Noyan et al. (2017) Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor. Am. J. Transplantation 2017; 17: 917-930.EXPERIMENTALExample 1Engineering OKT3-orthogonal CD3E (orthoCDSc) regulatory T cells.

[0111] Yeast surface display (YSD) allows the display of heterologous proteins on the yeast surface by fusion to the native yeast protein Aga2p. Due to the platform technology’s versatility and myriad of other attractive attributes including ease of mutagenesis and handling, compatibility with flow cytometry and sorting methods, and quick turnaround times, YSD has been widely used to study, characterize, and engineer protein-protein interactions, including many found on human T cells. YSD provides an efficient method for engineering orthoCD3c, a mutant version of CD3E that is inert to OKT3.

[0112] Results: Due to the highly multimeric assembly of the TCR-CD3 complex (FIG. 1 A), minimal units of TCR-CD3 necessary to replicate OKT3 binding on yeast were tested. Yeast display of single chain CD35-CD3E (Fig. 1 B) shows strong binding to OKT3 with binding affinity ( d) of 1.4 nM (Fig. 1C, red), comparable toof 0.83 nM reported for primary T cells. Yeast display of CD3E alone did not result in detectable OKT3 binding, consistent with previous literature that OKT3 binds CD3E only when in complex with CD36 or CD3y. These results demonstrate that yeast displayed CD3E mimics CD3E on T cells.

[0113] Using the published co-crystal structure of CD3E and OKT3, 10 residues of CD3E pivotal for OKT3 binding were identified and mutated to amino acids with vastly different physiochemical properties. Following screening, CD3E mutant K104A was identified, with >1 ,000-fold reduced affinity for OKT3 compared to wildtype (Fig. 1 C). Indeed, residue K104 makes prominent contact with OKT3 (Fig. 1 D). As control, it was shown that another conformational anti-CD3, clone MEM57, stains both wildtype and K104A CD3E equally (Fig. 1 E). Thus, ortho mutant K104A preserves the native fold of CD3E, minimizing any potential perturbations to TCR-CD3 complex.Example 2Genetically engineer regulatory T cells with orthoCD3e by homology-directed repair.

[0114] Cell surface expression of TCR-CD3 requires a complete multimer assembly, as lack of a single sub- chain leads to trapping of the incomplete complex in the endoplasmic reticulum. Thus, impact on CD3E expression level must be minimized when installing orthoCDSc (K104A). The simplicity of the mutation, as well as the availability of an adjacent NGG PAM site, facilitates the use of CRISPR-Cas9-mediated homology-directed repair (HDR). Compared to traditional approaches like viral transduction, HDR allows highly specific and controlled editing of CD3E at its native locus, preventing alterations to the expression level of TCR-CD3 complex and hence Treg function.

[0115] HDR efficiency scales with gRNA cleavage efficiency. CD3E K104-proximal gRNA, when co- electroporated with Cas9 into primary T cells, showed -96% CD3E knockout (Fig. 2A). With high efficiency cleavage confirmed, a single-stranded donor DNA was designed with the orthoCDSc K104A mutation (AAA^GCA), and co-electroporated with gRNA-Cas9 into primary T cells. Sequencing from the initial HDR experiment shows that a majority of cells contain the K104A mutation, indicating high HDR efficiency (Fig. 2B).

[0116] To demonstrate that orthoCD3E-expressing T cells are inert to OKT3, HDR-engineered primary T cells are stained, as well as control mock-transfected T cells, with OKT3, MEM57, and anti-TCRap for flow cytometry. OKT3 staining demonstrates the knockout of OKT3 binding in the context of T cells. MEM57 and anti-TCRap staining demonstrate comparable expression level of TCR-CD3 on mock and orthoCD3£ T cells, indicating that HDR engineering has no impact on TCR-CD3 expression, as desired. Finally, TCR+and OKT3‘ gating are used to sort engineered T cells from unedited cells, which are TCR+OKT3+.Example 3Evaluate orthoCD3E regulatory T cells for maintained TCR and immunosuppressive functions

[0025] For efficacy against GvHD, it is essential for orthoCD3£ Tregs to maintain their native immunosuppressive function as well as native TCR signaling. The latter is critical for two reasons: (1 ) to ex vivo culture and expand orthoCD3£ Tregs by TCR-CD3 stimulation and (2) to maximize engraftment and proliferation in patients following adoptive transfer.

[0026] Treg cells are cultured and expanded ex vivo by bead- or plate-coated anti-CD3 and anti- CD28, where the anti-CD3 clone used is often OKT3. OKT3-inert orthoCD3£ Tregs are cultured with other mitogenic anti-CD3 clones. OrthoCD3£ mutant K104A maintains wildtype-like binding to clone MEM57 (Fig. 1 E), which similarly to OKT3 has potent mitogenic properties. OrthoCD3£ or wildtype Tregs are cultured in plates coated with OKT3 or MEM57. Cell proliferation is tracked by CFSE dye, and cell activation by staining surface markers CD69, CD25, and PD-1 . OrthoCD3£ Tregs are expected to neither proliferate nor activate when cultured with OKT3, while proliferating and activating similarly to wildtype Tregs with MEM57.

[0027] In a more direct demonstration, orthoCD3£ or wildtype T regs are activated by co-culturing PBMCs isolated from another donor’s blood and characterizing the resulting alloreactive TCR- CD3 signaling. Signaling molecules downstream of TCR such as ZAP70 and LAT are analyzed by quantifying their phosphorylation by intracellular phospho-staining or western blot. OrthoCD3£ and wildtype Tregs are expected to have comparable TCR downstream signaling patterns.

[0028] To demonstrate wildtype-like immunosuppressive capacity of orthoCD3£ Tregs, orthoCD3£ or wildtype Tregs are cultured with conventional CD4+or CD8+T cells (Tcons). Tcons are pre-activated with anti-CD3 / 28 treatment and co-cultured with Tregs, then examined for suppression of Tcons’ proliferation, activation, and cytokine secretion. Proliferation and activation by CFSE and surface marker staining are quantitated, and secreted inflammatory cytokines like IL-2, IFN-y, and TNFa are measured through Cytometric Bead Array (CBA) assays. Tregs are evaluated in established Treg suppression assays involving co-culture with immune effector cells. These immune effector cells co-cultured with orthoCD3£ or wildtype Tregs are expected to have comparable immunosuppression, indicating maintained function despite orthoCD3£ engineering.Example 4Evaluate efficacy of 0KT3 and orthoCD3£ Treg combination therapy in xenograft GvHD models.

[0029] A key in vivo experiment to highlight the synergistic mechanism of OKT3 and orthoCD3£Treg combination therapy is to demonstrate selective depletion of wildtype T cells, but not orthoCD3£ Tregs, by OKT3 infusion. By doing so, the benefits of this combination therapy over monotherapy can be fully realized. Xenograft models are necessary for this study as OKT3 is nonspecies cross-reactive with mouse CD3E.

[0030] OrthoCD3£ Tregs and wildtype primary T cells are lentivirally transduced for constitutive expression of luciferases, such as P. pyralis and R. reniformis luciferases (PLuc and RLuc), allowing non-invasive quantification of cells in vivo by bioluminescence imaging (BLI). PLuc+orthoCD3e Tregs and PLuc+wildtype T cells are co-infused into immunodeficient NSG mice that have been irradiated to rid its immune system. The mice are infused with OKT3. Cell counts of PLuc+orthoCD3£ Tregs and RLuc+wildtype T cells are monitored over time by BLI. Signal from RLuc+wildtype T cells is expected to decrease to near background, while signal from PLuc+orthoCD3e Tregs is maintained or even increased.

[0031] The efficacy of OKT3 and orthoCD3e Treg combination therapy is evaluated along with monotherapy controls. Irradiated NSG mice are implanted with human PBMCs to induce GvHD, and the GvHD severity score and survival is monitored (n = 7 mice per group, three experiments). Combination treatment arm receives co-infusion of OKT3 and orthoCD3e T regs. Control treatment arms receive OKT3 only, wildtype Treg only, orthoCD3£ Treg only, or combination of OKT3 and wildtype Tregs. Statistically insignificant differences are expected in survival and GvHD score for groups treated with wildtype or orthoCD3E Tregs.Example 5Saturation mutagenesis at position 104 of CD3E

[0032] As shown in FIGS. 3-9, saturation mutagenesis and screening at residue 104 of CD3E revealed a range of binding affinities, shown in FIG. 3. In particular, K104E was selected for further screening. Shown in FIG. 4, the binding of OKT3 to the K104E mutant was lower than the K104A mutant, and there was no detectable binding to OKT3 (FIG. 5). There was no binding to teplizumab or otelixizumab (FIG. 6), while relative TCR expression was maintained (FIG. 7). OrthoCD3 T effector cells were readily expanded with Dynabeads (FIG. 8) but inert to teplizumab driven activation (FIG. 9).Example 6

[0033] Using the published co-crystal structure of CD3£ and OKT3, 10 residues of CD3E pivotal for OKT3 binding were identified and mutated to amino acids with different physiochemical properties. The residues tested with substitutions were E56, G68, E70, P100, R101 , G102, S103, K104, P105, and D107. For the mutation screen, we devised a yeast surface display strategy to display human CD35E heterodimer, shown in FIG 1 1 A-C, as a proxy for CD3 molecules on human T cells.

[0034] Shown in FIG 12 are the results of mutant CD3 displaying yeast cells stained with antibodies Teplizumab, which have the binding specificity of OKT3; Otelixizumab; and antibodies Clone MEM57, Clone HIT3a, Clone UCHT1 and Clone G19-4, which have a binding specificity other than that of OKT3. The mutants tested were E56A, G68R, E70A, P100A, R101A, G102R, S103A, K104A, P105A, and D107A. Mutant D107A had non-detectable surface expression and henceforth removed from the mutant screen. A strong differential in binding was observed for residues E56, P100 and K104.

[0035] K104 was selected for further analysis, results shown in FIG. 13. Amino acid substitutionsK104R, K104H, K104D, K104E, K104S, K104T, K104N, K104Q, K104G, K104P, K104A, K104V, K104I, K104L, K104M, K104F, K104Y, K104W were made and tested, results shown in FIG. N1C. With the exception of K104R, each amino acid substitution resulted in significantly decreased OKT3 binding relative to the wild-type CDRe.Example 7

[0036] As shown in FIG. 14, primary human T cells with mutations at CD3E K104 exhibit decreased OKT3 binding. Primary human T cells were isolated from healthy donor PBMCs and activated with anti-CD3 / 28 dynabeads prior to electroporation. Primary T cells were coelectroporated with Cas9 pre-complexed to a guide RNA proximal to CD3E K104, as well as donor DNA (single-stranded oligodeoxynucleotides, ssODNs) encoding different K104 mutants. Engineered T cells were recovered and analyzed for binding to OKT3 (top) or anti-CD3 clone MEM57 (bottom) by flow cytometry. Mutants K104A, K104D, K104E, and K104G all exhibited decreased OKT3 binding compared to the internal control of unedited T cells. Mutant K104E showed the lowest OKT3 binding comparable to that of CD3E knock-out cells, and thus is optimal. Decrease in OKT3 binding is not at the expense of decreased CD3E expression on the T cell surface, as shown by comparable staining by anti-CD3 clone MEM57.

[0037] OrthoCD3 (CD3E K104E) engineering and sorting of T cells are shown in FIG. 15. T cells are stimulated with anti-CD3 / 28 dynabeads for 48 hours prior to dynabead removal and subsequent electroporation of genetic engineering reagents such as Cas9 RNP and donor DNA. Electroporated T cells are recovered overnight in media containing commercially available NHEJ inhibitors. Following full media change to wash out NHEJ inhibitors, fresh dynabeads are added to culture expand the T cells. 7 days after electroporation, T cells are co-stained with OKT3 as well as anti-CD3 clones that maintain binding to orthoCD3 such as clones SP34-2 and MEM57. This co-stain allows separation of CD3 KO cells (OKT3 SP34-2 ) and wild-type CD3 cells(OKT3+SP34-2+) from orthoCD3 cells (OKT3 SP34-2+). OrthoCD3 T cells (OKT3 SP34-2+) can be sorted by FACS for downstream studies.

[0038] OrthoCD3 T cells are functionally inert to teplizumab while maintaining activation and expansion by stimulation from other anti-CD3 reagents, as shown in FIG. 16. FACS-sorted mock and orthoCD3 T cells were rested and plated in wells coated with anti-CD28 and various anti- CD3: teplizumab, MEM57, or SP34-2. 48 hours later, T cells were analyzed for their activation by markers LAG-3, CD69, and CD25. Mock T cells activate by teplizumab, while orthoCD3 T cells stay minimally activated comparable to anti-CD28 only control, demonstrating that orthoCD3 T cells are completely inert to teplizumab. OrthoCD3 T cells can however still be activated by MEM57 and SP34-2, as well as commercially available Dynabeads reagent. In a proliferation assay, T cells were labeled with a proliferation dye prior to plating. 120 hours later, proliferation of T cells was analyzed by dilution of the proliferation dye. Consistent with the activation data, orthoCD3 T cells do not proliferate with teplizumab, but does proliferate with stimulation from MEM57, SP34-2, and Dynabeads, identifying alternative reagents for culturing engineered orthoCD3 T cells for downstream applications.

[0039] OrthoCD3 regulatory T cells engineered by homology-directed repair maintain intracellular FoxP3 levels, shown in FIG. 17. Regulatory T cells (Tregs) were engineered with orthoCD3 and sorted, alongside a mock Treg control as well as a mock shocked control (Tregs that were electroporated in absence of genetic engineering reagents). These cells were fixed and permeabilized to stain for intracellular FoxP3 levels. FoxP3 levels remain unchanged, demonstrating that orthoCD3 engineering does not impact FoxP3, a classic transcription factor for Tregs.

[0040] Alternative orthoCD3 T cell engineering by retroviral transduction, shown in FIG. 18. Isolated Tregs are stimulated with anti-CD3 / 28 dynabeads for 48 hours prior to dynabead removal and subsequent electroporation of Cas9 RNP targeting the CD3E locus, achieving near 100% CD3E knock out. Following recovery in media for 6 hours, CD3E KO Tregs are split into two and transduced with retrovirus encoding wild-type CD3 (“WT TD”) or orthoCD3 (“OrthoCD3 TD”). Following transduction, CD3E co-stain shows the expected wild-type CD3 cells (OKT3+SP34-2+) in WT TD, and orthoCD3 cells (OKT3 SP34-2+) in OrthoCD3 TD. Transduced cells may be sorted for downstream studies; alternatively, the contaminating cells, which are mostly CD3E knock-out cells, will not expand with anti-CD3 stimulation and hence will be diluted with further cell expansion. “WT TD” cells serve as a better control than mock cells for comparisons to “OrthoCD3 TD” cells.

[0041] OrthoCD3 regulatory T cells maintain comparable phenotype as wtCD3 regulatory T cells, shown in FIG. 19. Following retroviral engineering, Treg cells were cultured using dynabeads andtheir phenotypes examined by a high-dimension flow cytometry panel. This panel includes the TCR, classical Treg markers (CD4, CD25, CD127, FoxP3), and activation markers.

[0042] OrthoCD3 regulatory T cells maintain comparable suppressive function as wtCD3 regulatory T cells, shown in FIG. 20. In a Teff:Treg co-culture assay, plates are coated with anti- CDS clone SP34-2 and anti-CD28. Autologous CD4+CD25_effector T cells are stained with a proliferation dye and plated, from which they expand from anti-CD3 / 28 stimulation. Then, following retroviral engineering, WT TD and orthoCD3 TD Tregs are introduced at titrating levels to suppress the proliferation of CD4+CD25_effector T cells. % inhibition is calculated based on the percentage of effector T cells that proliferate. The suppression curves of WT TD and OrthoCD3 TD Tregs overlap, demonstrating that suppressive function is not impacted by orthoCD3 engineering.

[0043] Shown in FIG. 21 , orthoCD3 T cells have reduced binding to otelixizumab and visilizumab. This highlights that anti-CD3 antibodies apart from teplizumab, such as otelixizumab and visilizumab, may be utilized to preferentially deplete wtCD3 T cells over orthoCD3 T cells.Example 8

[0044] Using the methodology established for engineering a human ortho-CD3 protein, mutations in the mouse CD3 counterpart protein were developed that would differentially bind to anti-mCD3e protein, while maintaining function of the protein, e.g. with respect to expression, activation, and binding to other members of the CD3 complex.

[0045] mCD3 was examined for solvent accessible residues that are involved in binding to antibodies such as 2C1 1 . 2C11 is commercially available from multiple sources, including Thermo Fisher as 145-2C1 1. The 145-2C1 1 antibody is a widely used phenotypic marker for mouse T cells. In addition, binding of 145-2C1 1 antibody to CD3e can induce cell activation. The crystal structure provides further insight, see Fernandes, R.A. et al. 2012. J. Biol. Chem. 287: 13324- 13335. As with the human system, there is interest in differential binding to CD3 specific antibodies, exemplified by the 17A2 antibody, and the 500A2 antibody, which are also widely available from commercial sources.

[0046] Similar to the human system, a yeast surface display screening platform was devised where single mutants of mouse CD3y£ heterodimer were displayed and binding to anti-CD3 characterized by flow cytometry. Residues selected for mutagenesis and screening included D22, D23, A24, E25, N26, D45, S46, D47, E48, N49, K51 , Y84, T85, P86, A87, S88, N89, K90, and N91. Mutations tested included D22A, D22R, D23A, A24R, E25A, E25R, N26A, N26R, D45A, D45F, S46A, D47A, D47R, D47L, E48A, E48L, N49A, N49E, K51 A, Y84A, T85A, P86A, A87R,A87E, A87L, S88A, S88L, N89A, N89R, N89E, N89L, K90A, N91A and N91 L. Mutagens with little to no detectable binding at a saturating concentration (10 nM) of 2C11 antibody included D45A, D45F, D47A, D47R, D47L, N49A, T85A, P86A, A87R, A87L, S88L, and K90A.

[0047] Binding curves of yeast-displayed mouse CD3E P86A to antibodies 2C11 , 17A2 and 500A2 are shown in FIG 22.

[0048] The P86 residue was further mutated to P86A, P86G, P86V, P86I, P86L, P86M, P86F, P86Y, P86W, P86R, P86H, P86K, P86D, P86E, P86S, P86T, P86N, and P86Q. Binding to 2C11 antibody is shown in FIG. YA. With the exception of P86M, all of the substitutions substantially reduced binding to 2C11. In particular, mutants P86I, P86F, P86Y, P86W, P86S, P86R, P86H, P86K, P86D, and P86E had less binding to 2C11 than P86A. Binding curves of these mutants to antibodies 17A2 and 500A2 are shown in FIG. 23.

[0049] CRISPR-Cas9-mediated homology-directed repair (HDR) was used to introduce selected mutations — P86A, P86I, P86E, P86Y, and P86K — into the genome of mouse Treg cells. To demonstrate that m-orthoCD3£-expressing T cells maintain differential binding, wild-type and HDR-engineered Treg cells were stained with 2C1 1 , 17A2, 500A2 and TCR|3 antibodies as shown in FIG. 24. Antibody staining demonstrates comparable expression level of TCR-CD3 on wild-type and m-orthoCD3£ T cells, indicating that HDR engineering has no impact on TCR-CD3 expression, as desired.

[0050] The results of activation of the wild-type and P86 mutant T cells with plate-coated 2C1 1 and 17A2 antibodies are shown in FIG. 25. As expected, mouse Tregs with wild-type CD3£ activate with either 2C11 or 17A2 stimulation. CD3c mutants P86E, P86Y, and P86K however do not activate with 2C11 , while activating with 17A2. This demonstrates that these P86 mutants are functionally inert from 2C11 as desired, while highlighting the 17A2 antibody as a culturing reagent for these T cells.References

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[0058] Zeiser, R., R. Marks, H. Bertz, and J. Finke. 2004. Annals of Hematology. 83: 551 -565.

[0059] Chert, G. M., and J. R. Cochran. 2015. Methods in Molecular Biology. 1319: 155-175.

[0060] Cole, M. S., C. Anasetti, and J. Y. Tso. 1997. Journal of Immunology. 159: 3613-3621.

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[0062] Lars, K. N., M. A. Dunstone, L. Kostenko, L. K. Ely, T. Beddoe, N. A. Mifsud, A. W. Purcell, A. G.

[0063] Brooks, J. McCluskey, and J. Rossjohn. 2004. PNAS. 101 : 7675-7680.

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[0066] lliopoulou, B. P., K. Hsu, M. Perez-Cruz, S. Tang, W. W. Pang, T. Erkers, N.Kambham, G. J.

[0067] Freeman, R. H. Dekruyff, and E. H. Meyer. 2019. Blood Advances. 3: 3419-3431 .

[0068] Fernandes, R.A., D.A. Shore, M. T. Vuong, C. Yu, X. Zhu, S. Pereira-Lopes, H.Brouwer, J. A.

[0069] Fennelly, C. M. Jessup, E. J. Evans, I. A. Wilson, and S. J. Davis. 2012. Journal of Biological Chemistry. 287: 13324-13335.

[0117] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An orthogonal CD3e protein comprising at least one amino acid substitution sufficient to substantially reduce binding to an antibody of interest; but retain binding to cellular proteins of the T cell receptor complex.

2. The orthogonal CD3e protein of claim 1 , wherein the function of the T cell receptor complex is maintained in T cells expressing the orthogonal CD3E protein.

3. The orthogonal CD3E protein of claim 1 or claim 2, wherein the protein is human CD3E.

4. The orthogonal CD3E protein of claim 3, wherein the amino acid substitution is at a residue selected from E56, G68, E70, P100, R101 , G102, S103, K104, P105, and D107 relative to SEQ ID NO:1 , where the substitution comprises an amino acid other than the wild-type.

5. The orthogonal CD3E protein of claim 4, wherein the amino acid substitution is at a residue selected from E56, P100, R101 , G102, and K104.

6. The orthogonal CD3E protein of any of claims 3-5, wherein the amino acid substitution is at residue K104.

7. The orthogonal CD3E protein of any of claims 3-6, wherein the amino acid substitution is at a residue selected from K104D; K104E; K104S; K104T; K104N; K104Q; K104G; K104P; K104A; K104V; K104I; K104L; K104M; K104F; K104Y; and K104W.

8. The orthogonal CD3E protein of claim 7, wherein the amino acid substitution is K104E.

9. The orthogonal CD3E protein of any of claims 1 -8, wherein the antibody of interest has the binding specificity of OKT3.

10. The orthogonal CD3E protein of claim 1 or claim 2, wherein the protein is mouse CD3E.1 1. The orthogonal CD3E protein of claim 10, wherein the amino acid substitution is at a residue selected from D22, D23, A24, E25, N26, D45, S46, D47, E48, N49, K51 , Y84, T85, P86,A87, S88, N89, K90, and N91 relative to SEQ ID NO:3, where the substitution comprises an amino acid other than the wild-type.

12. The orthogonal CD3e protein of claim 11 , wherein the amino acid substitution is at a residue selected from D45, D45, D47, D47, D47, N49, T85, P86, A87, A87, S88, and K90.

13. The orthogonal CD3E protein of any of claims 3-5, wherein the amino acid substitution is at residue P86.

14. The orthogonal CD3E protein of claim 13, wherein the amino acid substitution is selected from P86I, P86F, P86Y, P86W, P86S, P86R, P86H, P86K, P86D, and P86E.

15. The orthogonal CD3E protein of any of claims 10-14, wherein the antibody of interest has the binding specificity of 2C11 .

16. An engineered hematopoietic cell that comprises DNA encoding the ortho CD3E of any of claims 1 -15.

17. The engineered hematopoietic cell of claim 16, wherein the cell is a T cell.

18. The engineered hematopoietic cell of claim 16 wherein the cell is a hematopoietic stem cell.

19. The cell of any of claims 16-18, wherein the CD3E locus of both alleles of the cell are engineered to comprise only orthogonal CD3E sequences.

20. The cell of any of claims 16-19, wherein the CD3E locus of both alleles are replaced by genome engineering.21 . The cell of any of claims 16-20, wherein a vector comprising sequences encoding the ortho-CD3E are introduced into the cell.

22. The cell of any of claims 16-21 , wherein the cell is a T reg cell, a CD4+ T cell, a CD8+ T cell, a CAR-T cell, or an TCR engineered T cell.

23. The cell of claim 22, wherein the T cell is a Treg cell.

24. The cell of claim 22 wherein the T cell is specific for an antigen of interest.

25. A method of activating in vitro a T cell according to any of claims 17-24, the method comprising: contacting the T cell with an effective dose of an agent that specifically binds to the ortho- CD3e or T cell receptor present on the T cell.

26. A method of treatment, the method comprising: administering an effective dose of T cells according to any of claims 17-24, optionally stimulated by the method of claim 25, to an individual in need thereof.

27. The method of claim 26, further comprising lymphodepleting the individual with an effective dose of the antibody of interest.

28. The method of claim 26 or claim 27, wherein the T cell is a Treg cell.

29. The method of any of claims 26-28 wherein the individual is treated for graft versus host disease.

30. The method of any of claims 26-28 wherein the individual is treated for autoimmune disease.

31. The method of any of claims 26-28 wherein the individual is treated to reduce transplant rejection.

32. The method of claim 13 or claim 14, wherein the T cell is a Treg cell and the individual is treated to reduce transplant rejection.

33. The method of any of claims 26-27, wherein the T cell is a CAR-T cell or a TCR- engineered T cell.

34. The method of claim 33, wherein the individual is treated for cancer.

35. The method of claim 33, wherein the individual is treated for infection.

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