WO2022098756A1

WO2022098756A1 - Chimeric antigen receptor cell therapy

Info

Publication number: WO2022098756A1
Application number: PCT/US2021/057894
Authority: WO
Inventors: Ayman Kabakibi
Original assignee: Wugen Inc
Current assignee: Wugen Inc
Priority date: 2020-11-03
Filing date: 2021-11-03
Publication date: 2022-05-12
Anticipated expiration: 2023-05-03

Abstract

Disclosed herein are pharmaceutical compositions and methods comprising chimeric antigen receptor (CAR)-bearing immune effector cells such as CAR-T cells for the treatment of cancers.

Description

CHIMERIC ANTIGEN RECEPTOR CELL THERAPY

[001] This application claims the benefit of priority of United States Provisional Application No. 63/109,076 filed November 3, 2020, the disclosure of which is incorporated by reference s if written herein in its entirety.

[002] CAR-T cells have emerged as a promising therapy for the treatment of hematological malignancies. Despite remarkable clinical efficacy against B cell malignancies, the success of CAR-T therapy has been limited by severe, life-threatening toxicities, observed in over 50% of patients. These toxicities have resulted in several deaths leading to early termination of clinical trial. Toxicities primarily manifest as cytokine release syndrome (CRS, also referred to as “cytokine storm”) characterized by high elevations of cytokines including INFγ, granulocyte-macrophage colony-stimulating factor, IL- 10, and IL- 6. These cytokine elevations result in a plethora of clinical symptoms including fever, hypotension, organ dysfunction, respiratory failure and coagulopathy. CRS can be fatal. Additionally, neurotoxicity often presents even after the initial symptoms of CRS have subsided. The pathogenesis of CRS and associated neurotoxicity is poorly understood and further understanding of the mechanism would be useful for the successful translation of CAR-T therapy. In the meanwhile, disrupting the pathogenesis of CRS by reducing the level of cytokine genes available for expression is one way to mitigate the condition.

[003] Disclosed herein are methods of gene deletion and endogenous suppression, of a particular gene in a chimeric antigen receptor (CAR)-bearing immune effector cell, such as CAR-T cell. For example, in some embodiments, a CAR-T cell as described herein (i.e., UCART7) may express a CAR that recognizes and binds (i.e., targets) to CD7 on the surface of a CD7+ tumor cell, while also itself lacking expression of CD7. In this way, the cell will target CD7+ tumor cells but not other CAR-T cells, referred to herein as fratricide. These gene deletion methods may include, but are not limited to, insertion of the CAR into a specific locus or gene thereby blocking its expression; gene editing with Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), or CRISPR/Cas9; expression of an scFv with an endoplasmic reticulum (ER) binding tether to bind the cytokine in the ER and prevent secretion; and transfection of small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs). Also disclosed herein are CAR-bearing immune effector cells modified with these described gene deletion methods, and methods of treatment of diseases with immunotherapy with a reduced incidence of cytokine release syndrome (CRS) or other toxicity associated with immunotherapy. BRIEF DESCRIPTION OF THE SEQUENCES

[004] SEQ ID NOs:l-21 - Amino acid sequences of different CAR components (Table 1). [005] SEQ ID NOs:22-23 - Amino acid sequences of variable heavy (V_H) and light (V_L) chains of the scFv (Table 2).

[006] SEQ ID NOs:24-30 - Amino acid sequences of CARs (Table 3). [007] SEQ ID NOs:31-33 - DNA sequence encoding CARs (Table 3).

BRIEF DESCRIPTION OF THE DRAWINGS

[008] FIG. 1 - Shows a Schedule of Assessments for the Phase I study. ^a Informed Consent must be obtained ≤28 days prior to the initiation of study treatment; ^b Physical examinations will include measurements of weight and vital signs (resting heart rate, blood pressure, oral temperature). No need to repeat physical exams on Cycle 1 Day 1 (CID1) if the baseline physical exam was performed ≤72 hours. ^c Triplicate 12-lead ECGs will be collected approximately 5 minutes apart at the Screening Visit. In addition, triplicate 12-lead ECGs will be taken as clinically indicated. ^d Hematological parameters include the following laboratory tests: complete blood count consisting of hematocrit, hemoglobin, total white blood count (WBC) with 5-part differential, and platelet count plus reticulocyte count. Do not repeat on C1D1 if baseline within 72 hours. CBCs are collected before treatment. ^e CMP panel includes the following: blood urea nitrogen, creatinine, sodium, potassium, calcium, chloride, carbon dioxide, magnesium, phosphorous, glucose, albumin, total protein, total bilirubin, alkaline phosphatase, AST, ALT, and lactate dehydrogenase. Do not repeat on DI if baseline within 72 hours. ^f PT/PTT, Fibrinogen and C-Reactive protein (CRP) will be assessed at screening by standard method at a local laboratory. ⁸ Urine testing dipstick will be done at baseline and D35. If abnormalities are present microscopic testing should be done. ^h A serum or urine pregnancy test will be performed at baseline (no need to repeat on CID 1 if within 72 hours). For women of child-bearing potential, the test will be repeated on Day 35. ¹ Cellular PK blood sample collection. ¹ Bone marrow aspirate and disease assessment will be done within 7 days of treatment and at the end of Month 3. ^k If relevant, tumor imaging studies will be performed at baseline (<28 days of dosing) and at the end of Month 3. ¹ All patients will undergo the DLT period treatment assessments on Day 28. Patients must be followed for AEs for 30 calendar days after the last dose of study drug. ^m All patients have blood drawn for cellular PK to assess persistence of UCART7 clone at every scheduled clinic visit until such time as the previous test returned a negative result. [009] FIG. 2 - Shows an overview of the study design of the Phase I study.

[010] FIG. 3 - Shows an overview of the dose-escalation methodology of the Phase I study.

[Oil] FIG. 4 - Shows a clinical algorithm for the evaluation of CRS per ASTCT Guidelines.

DETAILED DESCRIPTION

[012] The following disclosure will detail embodiments, alternatives, and uses of the compositions and cells as described herein, as well as the use of such compositions or cells in, for example, immunotherapy and adoptive cell transfer for the treatment of diseases. Accordingly, provided herein are the following embodiments.

[013] Embodiment 1. A single dose of a pharmaceutical composition comprising from about 1 x 10⁵ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight, wherein the immune effector cells each comprise one or more chimeric antigen receptors (CARs) targeting one or more T-cell antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), wherein the immune effector cells are deficient in cell surface expression of the at least one or more T-cell antigens to which the one or more CAR(s) specifically bind(s), and wherein the composition does not elicit persistent grade 3 or grade 4 cytokine release syndrome (CRS).

[014] Embodiment 2. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 2.0 x 10⁶ to about 18.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[015] Embodiment 3. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 2.0 x 10⁶ to about 12 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[016] Embodiment 4. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 2.0 x 10⁶ to about 6 x 10⁶ CAR-bearing immune effector cells per kg of body weight. [017] Embodiment 5. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 6.0 x 10⁶ to about 18 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[018] Embodiment 6. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 2.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[019] Embodiment 7. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 6.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[020] Embodiment 8. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 12.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[021] Embodiment 9. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 18.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[022] Embodiment 10. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 100 x 10⁶ to about 900 x 10⁶ CAR-bearing immune effector cells.

[023] Embodiment 11. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 100 x 10⁶ to about 600 x 10⁶ CAR-bearing immune effector cells.

[024] Embodiment 12. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 100 x 10⁶ to about 600 x 10⁶ CAR-bearing immune effector cells.

[025] Embodiment 13. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 300 x 10⁶ to about 900 x 10⁶ CAR-bearing immune effector cells.

[026] Embodiment 14. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 100 x 10⁶ CAR-bearing immune effector cells.

[027] Embodiment 15. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 300 x 10⁶ CAR-bearing immune effector cells. [028] Embodiment 16. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 600 x 10⁶ CAR-bearing immune effector cells.

[029] Embodiment 17. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises about 900 x 10⁶ CAR-bearing immune effector cells.

[030] Embodiment 18. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 1.0 x 10⁵ to about 5 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[031] Embodiment 19. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 1.1 x 10⁵ to about 5.4 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

[032] Embodiment 20. The single dose of a pharmaceutical composition of Embodiment 1, wherein the composition comprises from about 1.4 x 10⁵ to about 8.6 x 10⁵ CAR-bearing immune effector cells per kg of body weight.

[033] Embodiment 21. The single dose of a pharmaceutical composition of any of Embodiments 1-20, wherein the T-cell antigen targeted by the CAR, and in which the cells are deficient in cell surface expression, is CD7.

[034] Embodiment 22. The single dose of a pharmaceutical composition of Embodiment 21, wherein the CAR-bearing immune effector cells are T-cells.

[035] Embodiment 23. The single dose of a pharmaceutical composition of Embodiment 22, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is one or more of the T cell receptor complex alpha chain (TCRα/TRAC), the T cell receptor complex beta chain (TCRβ ), the T cell receptor complex delta chain (TCR5), and the T cell receptor complex gamma chain (TCRγ).

[036] Embodiment 24. The single dose of a pharmaceutical composition of Embodiment 23, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is the T cell receptor complex alpha chain (TCRα/TRAC).

[037] Embodiment 25. The single dose of a pharmaceutical composition of Embodiment 24, wherein the CAR targeting CD7 comprises: a CD28 co-stimulatory domain; or a 4- IBB co-stimulatory domain; or a CD28 co-stimulatory domain and a 4- IBB co-stimulatory domain. [038] Embodiment 26. The single dose of a pharmaceutical composition of Embodiment 25, wherein the CAR targeting CD7 comprises a 4- IBB co-stimulatory domain and a CD3-zeta effector domain.

[039] Embodiment 27. The single dose of a pharmaceutical composition of Embodiment 25 or 26, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[040] Embodiment 28. The single dose of a pharmaceutical composition of Embodiment 27, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30. [041] Embodiment 29. The single dose of a pharmaceutical composition of Embodiment 28, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30. [042] Embodiment 30. The single dose of a pharmaceutical composition of Embodiment 29, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[043] Embodiment 31. The single dose of a pharmaceutical composition of Embodiment 30, wherein the CAR comprises the amino acid sequence SEQ ID NO:24. [044] Embodiment 32. The single dose of a pharmaceutical composition of Embodiment 30, wherein the CAR comprises the amino acid sequence SEQ ID NO:30. [045] Embodiment 33. The single dose of a pharmaceutical composition of any of Embodiments 1-26, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO:22.

[046] Embodiment 34. The single dose of a pharmaceutical composition of Embodiment 33, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NOTE

[047] Embodiment 35. The single dose of a pharmaceutical composition of Embodiment 34, wherein the CAR comprises a 4- IBB costimulatory domain comprising SEQ ID NO:9.

[048] Embodiment 36. The single dose of a pharmaceutical composition of Embodiment 35, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

[049] Embodiment 37. The single dose of a pharmaceutical composition of Embodiment 36, wherein the CAR comprises: a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

[050] Embodiment 38. The single dose of a pharmaceutical composition of Embodiment 25 or 26, wherein the CAR is encoded by a nucleotide sequence encoding the amino acid sequence(s) as recited in any of Embodiments 27-37.

[051] Embodiment 39. The single dose of a pharmaceutical composition Embodiment 38, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

[052] Embodiment 40. A pharmaceutical composition comprising a dose of genetically modified T-cells transduced with one or more chimeric antigen receptors (CARs), wherein the one or more CARs targets CD7, has a CD28 costimulatory domain or a 4- 1BB costimulatory domain or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and has a CD3-zeta effector domain; wherein the T-cells are genetically modified to be deficient in cell surface expression of TCRα/TRAC, and wherein the T-cells are genetically modified to be deficient in cell surface expression of CD7, wherein the dose is effective to treat a T-cell malignancy and prevent grade 3 or grade 4 cytokine release syndrome (CRS).

[053] Embodiment 41. The pharmaceutical composition of Embodiment 40, wherein the genetically modified T cells a) are deficient in CD7 surface expression due to deletion or disruption of the CD7 gene and/or b) express a functionally disabled T-cell receptor (TCR) due to deletion of the T-cell receptor alpha chain (TCRα/TRAC) gene. [054] Embodiment 42. The pharmaceutical composition of Embodiment 41, wherein the CD7 and/or TCRα/TRAC gene(s) is/are deleted by CRISPR/Cas9 gene editing.

[055] Embodiment 43. The pharmaceutical composition of any of Embodiments 1-42, wherein the T-cell malignancy is a hematological malignancy.

[056] Embodiment 44. The pharmaceutical composition of Embodiment 43, wherein the hematological malignancy is a T-cell malignancy.

[057] Embodiment 45. The pharmaceutical composition of Embodiment 44, wherein the hematological malignancy is a CD7+ T-cell malignancy.

[058] Embodiment 46. The pharmaceutical composition of either of Embodiments 32 or 33, wherein the T-cell malignancy is AML, T-ALL, and non- Hodgkin’ s lymphoma.

[059] Embodiment The pharmaceutical composition or single dose thereof of any of Embodiments 1-46, wherein the CRS is grade 3 or grade 4 CRS.

[060] Embodiment 48. The pharmaceutical composition of any of Embodiments 40-47, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[061] Embodiment 49. The pharmaceutical composition of Embodiment 48, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[062] Embodiment 50. The pharmaceutical composition of Embodiment 49, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[063] Embodiment 51. The pharmaceutical composition of Embodiment 50, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[064] Embodiment 52. The pharmaceutical composition of Embodiment 51, wherein the CAR comprises the amino acid sequence SEQ ID NO:24.

[065] Embodiment 53. The pharmaceutical composition of Embodiment 51 , wherein the CAR comprises the amino acid sequence SEQ ID NO:30.

[066] Embodiment 54. The pharmaceutical composition of any of Embodiments 40-47, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO:22.

[067] Embodiment 55. The pharmaceutical composition of Embodiment 54, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NO: 11. [068] Embodiment 56. The pharmaceutical composition of Embodiment 55, wherein the CAR comprises a 4- IBB costimulatory domain comprising SEQ ID NO:9.

[069] Embodiment 57. The pharmaceutical composition of Embodiment 56, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

[070] Embodiment 58. The pharmaceutical composition of Embodiment 57, wherein the CAR comprises: a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

[071] Embodiment 59. The pharmaceutical composition of any of Embodiments 40-47, wherein the CAR is encoded by a nucleotide sequence encoding the amino acid sequence(s) as recited in any of Embodiments 48-58.

[072] Embodiment 60. The pharmaceutical composition Embodiment 59, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

[073] Embodiment 61. A method of treatment of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

[074] Embodiment 62. A method of achieving complete remission of one or more T- cell malignancies in a patient comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[075] Embodiment 63. A method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a dose of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells. [076] Embodiment 64. A method of treatment of T-cell malignancies in a patient comprising administration of a dose of UCART7 cells, wherein the levels of interleukin (IL)- 1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)γ, and/or fracktalkine are not elevated to a level that would correspond with or result in grade 3 or 4 CRS as a result of treatment.

[077] Embodiment 65. A method of achieving an overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[078] Embodiment 66. A method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[079] Embodiment 67. A method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[080] Embodiment 68. A method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a dose of UCART7 cells, wherein the dose of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome.

[081] Embodiment 69. A method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose of UCART7 cells to the patient; and evaluating CRS symptoms in the patient.

[082] Embodiment 70. A method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, continuing treatment with UCART7 cells.

[083] Embodiment 71. The method of any of Embodiments 62-70, wherein the T-cell malignancy is a hematological malignancy. [084] Embodiment 72. The method of Embodiment 71, wherein the hematological malignancy is a T-cell malignancy.

[085] Embodiment 73. The method of Embodiment 72, wherein the hematological malignancy is a CD7+ T-cell malignancy.

[086] Embodiment 74. The method of any of Embodiments 72 or 73, wherein the T- cell malignancy is AML, T-ALL, or lymphoma.

[087] Embodiment 75. The method of Embodiment 74, wherein the lymphoma is non-

Hodgkin’s lymphoma.

[088] Embodiment 76. The pharmaceutical composition of any of Embodiments 40-60, or the method of any of Embodiments 61-75, wherein the composition comprises: from about 1 x 10⁵ to about 6.3 x 10⁸ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 18.0 x 10⁶ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 12 x 10⁶ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 6 x 10⁶ CAR-T cells per kg of body weight; from about 6.0 x 10⁶ to about 18 x 10⁶ CAR-T cells per kg of body weight; about 2.0 x 10⁶ CAR-T cells per kg of body weight; about 6.0 x 10⁶ CAR-T cells per kg of body weight; about 12.0 x 10⁶ CAR-T cells per kg of body weight; about 18.0 x 10⁶ CAR-T cells per kg of body weight; from about 100 x 10⁶ to about 900 x 10⁶ CAR-T cells; from about 100 x 10⁶ to about 600 x 10⁶ CAR-T cells; from about 100 x 10⁶ to about 300 x 10⁶ CAR-T cells; from about 300 x 10⁶ to about 900 x 10⁶ CAR-T cells; about 100 x 10⁶ CAR-T cells; about 300 x 10⁶ CAR-T cells; about 600 x 10⁶ CAR-T cells; about 900 x 10⁶ CAR-T cells; from about 1.0 x 10⁵ to about 5 x 10⁶ CAR-T cells per kg of body weight; from about 1.1 x 10⁵ to about 5.4 x 10⁶ CAR-T cells per kg of body weight; or from about 1.4 x 10⁵ to about 8.6 x 10⁵ CAR-T cells per kg of body weight.

[089] Embodiment 77. The method of any of Embodiments 61-86, wherein the dose of

UCART7 is administered intravenously in a single dose.

[090] Embodiment 78. The method of any of Embodiments 61-76, wherein the dose of

UCART7 is administered intravenously in more than one dose. [091] Embodiment 79. The method of any of Embodiments 87-77, wherein the dose of UCART7 cells is administered in a clinical setting or a hospital.

[092] Embodiment The method of any of Embodiments 61-79 wherein determining the efficacy of a dose of UCART7 cells comprises evaluation of progression-free survival (PFS) and/or duration of response (DoR) in the patient.

[093] Embodiment 81. The method of any of Embodiments 61-79, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome (TLS) in the patient.

[094] Embodiment 82. The method of any of Embodiments 61-79, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of treatment- related toxicity in the patient.

[095] Embodiment 83. The method of any of Embodiments 61-79, wherein toxicity of a dose of UCART7 cells is classified as: any grade 3 or higher neurotoxicity within 28 days of administration of UCART7 cells; any grade 4 CRS of any duration; grade 3 CRS that does not improve to grade 2 within 72 hours; grade 3 or greater infusion reaction; grade 3 or greater vital organ toxicity; grade 2 or greater GVHD; renal failure requiring dialysis for longer than 3 days; any intubation; and/or any grade 3 or 4 nonhematological toxicity lasting for more than 7 days.

[096] Embodiment 84. The method of Embodiment 70, wherein the therapy for treating CRS comprises a monoclonal antibody.

[097] Embodiment 85. The method of Embodiment 84, wherein the monoclonal antibody for treating CRS comprises an anti-interleukin- 6 receptor antibody.

[098] Embodiment 86. The method of Embodiment 85, wherein the monoclonal antibody for treating CRS comprises tocilizumab.

[099] Embodiment 87. The method of Embodiment 86, wherein the therapy for treating CRS comprises a small molecule kinase inhibitor.

[0100] Embodiment 88. The method of Embodiment 87, wherein the small molecule kinase inhibitor comprises a Janus kinase (JAK) inhibitor.

[0101] 89. The method of either of Embodiments 87 or 88, wherein the small molecule kinase inhibitor comprises ruxolitinib (Jakofi®), baricitinib, itacitinib, or dasatinib. [0102] Embodiment 90. The method of any of Embodiments 61-89, wherein the patient has not had any anti-CD7 therapy prior to treatment.

[0103] Embodiment 91. The method of any of Embodiments 61-90, wherein the patient does not have grade 3 or 4 graft-versus-host-disease (GVHD).

[0104] Embodiment 92. The method of any of Embodiments 61-91, wherein the patient has grade 2 GVHD treatable with topical therapy.

[0105] Embodiment 93. The method of any of Embodiments 61-92, wherein the patient is pre-treated with cyclophosphamide at a dose of 500 mg/m² IV daily for 3 days, and fludarabine at a dose of 30 mg/m² IV daily for 3 days before treatment with UCART7.

[0106] Embodiment 94. The method of any of Embodiments 61-93, wherein the patient does not exhibit profound and persistent T cell aplasia before administration of UCART7 cells.

[0107] Embodiment 95. The method of any of Embodiments 61-94, wherein the patient exhibits T cell aplasia as a result of chemotherapeutic therapy before administration of UCART7 cells.

[0108] Embodiment 96. The method of any of Embodiments 61-95, wherein lymphodepletion occurs on days 1-3, wherein UCART7 cells are administered 3 days after the lymphodepletion period.

[0109] Embodiment 97. The method of any of Embodiments 61-96, wherein the patient is observed twice weekly for 3 weeks after administration of UCART7 cells, then weekly for 4 weeks, then monthly for a period of 5 months, then monthly until no more UCART7 cells are detectable in the patient, then quarterly for 2 years, then annually through year 15.

[0110] Embodiment 98. The method of any of Embodiments 61-97, wherein cellular PK samples are obtained from the patient at each visit until no UCART7 cells are detectable in the patient.

[0111] Embodiment 99. The method of any of Embodiments 61-98, wherein cellular PK samples comprise one or more of AUC_{(0-∞ )}, AUC_(0-Ƭ), maximum drug concentration in the body after dosing [C_max], and T_max.

[0112] Embodiment 100. The method of any of Embodiments 61-99, wherein complete remission (CR) for a patient having ALL is classified as:

< 5% blasts in the bone marrow of the patient; normal maturation of all cellular components in the bone marrow; no extramedullary disease; absolute neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

[0113] Embodiment 101. The method of any of Embodiments 61-99, wherein complete remission (CR) for a pediatric patient having ALL is classified as: complete donor chimerism (≥ 95% donor chimerism without recipient cells detected); no extramedullary disease; neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

[0114] Embodiment 102. The method of any of Embodiments 110 or 101, wherein the extramedullary disease comprises CNS or soft tissue disease.

[0115] Embodiment 103. The method of any of Embodiments 61-99, wherein complete remission with incomplete hematologic recovery (CRi) for a patient having ALL is classified as:

< 5% blasts in the bone marrow of the patient; normal maturation of all cellular components in the bone marrow; no extramedullary disease; and transfusion independent.

[0116] Embodiment 104. The method of any of Embodiments 61-103, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[0117] Embodiment 105. The method of Embodiment 104, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[0118] Embodiment 106. The method of Embodiment 105, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[0119] Embodiment 107. The method of Embodiment 106, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

[0120] Embodiment 108. The method of Embodiment 107, wherein the CAR comprises the amino acid sequence SEQ ID NO:24.

[0121] Embodiment 109. The method of Embodiment 107, wherein the CAR comprises the amino acid sequence SEQ ID NO: 30. [0122] Embodiment 110. The method of any of Embodiments 61-103, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO:22.

[0123] Embodiment 111. The method of Embodiment 110, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NO: 11.

[0124] Embodiment 112. The method of Embodiment 111, wherein the CAR comprises a 4- IBB costimulatory domain comprising SEQ ID NO:9.

[0125] Embodiment 113. The method of Embodiment 112, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

[0126] Embodiment 114. The method of Embodiment 113, wherein the CAR comprises: a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

[0127] Embodiment 115. The method of any of Embodiments 61-103, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

[0128] Embodiment 116. The method of any of Embodiments 1-39, or the pharmaceutical composition of any of Embodiments 40-60, or the method of any of Embodiments 61-115, wherein the CAR-bearing immune effector cell or CAR-T cell or UCART7 does not comprise a membrane-bound IL-15 protein or IL-15/IL-15R fusion protein or a constitutively active IL-7R protein.

[0129] Also disclosed herein as Embodiment Pl is a single dose of a pharmaceutical composition comprising from about 1 x 10⁵ to about 6.3 x 10⁸ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight, wherein the immune effector cells each comprise one or more chimeric antigen receptors (CARs) targeting one or more T-cell antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), wherein the immune effector cells are deficient in cell surface expression of the at least one or more T-cell antigens to which the one or more CAR(s) specifically bind(s), and wherein the composition does not elicit grade 3 or grade 4 cytokine release syndrome (CRS).

[0130] Embodiment P2- The single dose of a pharmaceutical composition as recited in Embodiment Pl, wherein the composition comprises from about 1.0 x 10⁵ to about 5 x 10⁶ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0131] Embodiments P3 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P2, wherein the composition comprises from about 1.0 x 10⁶ to about 7.0 x 10⁷ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight. [0132] Embodiment P4 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P3, wherein the composition comprises from about 3.0 x 10⁶ to about 2.1 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0133] Embodiment P5 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P3, wherein the composition comprises from about 6.0 x 10⁶ to about 4.2 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight.

[0134] Embodiment P6 - The single dose of a pharmaceutical composition of any of Embodiments P1-P4, wherein the composition comprises from about 9.0 x 10⁶ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0135] Embodiment P7 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P6, wherein the composition comprises from about 1.1 x 10⁵ to about 5.4 x 10⁶ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight. [0136] Embodiment P8 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P7, wherein the composition comprises from about 1.4 x 10⁵ to about 8.6 x 10⁵ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight. [0137] Embodiment P9 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P8, wherein the T-cell antigen targeted by the CAR, and in which the cells are deficient in cell surface expression, is CD7.

[0138] Embodiment P10 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P9, wherein the CAR-bearing immune effector cells are T-cells. [0139] Embodiment Pl 1 - The single dose of a pharmaceutical composition as recited in any of Embodiments P1-P10, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is one or more of the T cell receptor complex alpha chain (TCRα, also referred to as TRAC), the T cell receptor complex beta chain (TCR[3), the T cell receptor complex delta chain (TCR5), and the T cell receptor complex gamma chain (TCRγ).

[0140] Embodiment P12 - The single dose of a pharmaceutical composition of any of Embodiments Pl -Pl 1, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is the T cell receptor complex alpha chain (TCRα/TRAC).

[0141] Embodiment P13 - The single dose of a pharmaceutical composition of any of Embodiments Pl -P12, wherein the CAR targeting CD7 comprises: a CD28 co- stimulatory domain; or a 4- IBB co-stimulatory domain; or a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0142] Embodiment P14 - The single dose of a pharmaceutical composition of any of Embodiments P1-P13, wherein the CAR targeting CD7 comprises a 4-1BB co-stimulatory domain and a CD3-zeta effector domain.

[0143] Embodiment P15 - A pharmaceutical composition comprising a dose of genetically modified T-cells transduced with one or more chimeric antigen receptors (CARs), wherein the one or more CARs targets CD7, has a CD28 costimulatory domain or a 4- 1BB costimulatory domain or both a CD28 costimulatory domain and a 4-1BB costimulatory domain, and has a CD3-zeta effector domain; wherein the T-cells are genetically modified to be deficient in cell surface expression of TCRα/TRAC, and wherein the T-cells are genetically modified to be deficient in cell surface expression of CD7, wherein the dose is effective to treat a T-cell malignancy and prevent grade 3 or grade 4 cytokine release syndrome (CRS).

[0144] Embodiment P16 - The pharmaceutical composition of any of Embodiments Pl- Pl 5, wherein the genetically modified T cells a) are deficient in CD7 surface expression due to deletion or disruption of the CD7 gene and/or b) express a functionally disabled T-cell receptor (TCR) due to deletion of the T-cell receptor alpha chain (TCRα/TRAC) gene.

[0145] Embodiment P17 - The pharmaceutical composition of any of Embodiments Pl- Pl 6, wherein the CD7 and/or TCRα/TRAC gene(s) is/are deleted by CRISPR/Cas9 gene editing.

[0146] Embodiment P18 - The pharmaceutical composition of any of Embodiments Pl- P17, wherein the T-cell malignancy is a hematological malignancy. [0147] Embodiment P19 - The pharmaceutical composition of any of Embodiments Pl- Pl 8, wherein the hematological malignancy is a T-cell malignancy.

[0148] Embodiment P20 - The pharmaceutical composition of any of Embodiments Pl- P19, wherein the hematological malignancy is a CD7+ T-cell malignancy.

[0149] Embodiment P21 - The pharmaceutical composition of any of Embodiments Pl- P20, wherein the T-cell malignancy is AML, T-ALL, and non-Hodgkin’s lymphoma.

[0150] Embodiment P22 - The pharmaceutical composition or single dose thereof of any of Embodiments P1-P21, wherein the CRS is grade 3 or grade 4 CRS.

[0151] Embodiment P23 - A method of treatment of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

[0152] Embodiment P24 - A method of achieving complete remission of one or more T- cell malignancies in a patient comprising administration of a low dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0153] Embodiment P25 - A method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a low dose of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells.

[0154] Embodiment P26 - A method of treatment of T-cell malignancies in a patient comprising administration of a low dose of UCART7 cells, wherein the levels of interleukin (IL)-l, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony- stimulating factor (GM-CSF), interferon (IFN)y, and/or fracktalkine are not elevated to a level that would correspond with or result in grade 3 or 4 CRS as a result of treatment.

[0155] Embodiment P27 - A method of achieving overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a low dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS). [0156] Embodiment P28 - A method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a low dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS). [0157] Embodiment P29 - A method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a low dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS). [0158] Embodiment P30 - A method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a low dose of UCART7 cells, wherein the low dose of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome.

[0159] Embodiment P31 - A method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a low dose of UCART7 cells to the patient; and evaluating CRS symptoms in the patient.

[0160] Embodiment P32 - A method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a low dose of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, continuing treatment with UCART7 cells.

[0161] Embodiment P33 - The method of any of Embodiments P23-P32, wherein the T- cell malignancy is a hematological malignancy.

[0162] Embodiment P34 - The method of any of Embodiments P23-P33, wherein the hematological malignancy is a T-cell malignancy.

[0163] Embodiment P35 - The method of any of Embodiments P23-P34, wherein the hematological malignancy is a CD7+ T-cell malignancy.

[0164] Embodiment P36 - The method of any of Embodiments P23-P35, wherein the T- cell malignancy is AML, T-ALL, or lymphoma.

[0165] Embodiment P37 - The method of any of Embodiments P23-P36, wherein the lymphoma is non- Hodgkin’ s lymphoma.

[0166] Embodiment P38 - The pharmaceutical composition of any of Embodiments P15-P22, or the method of any of Embodiments P23-P37, wherein the composition comprises from about 1.0 x 10⁶ to about 7.0 x 10⁷ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight.

[0167] Embodiment P39 - The pharmaceutical composition of any of Embodiments P15-P22, or the method of any of Embodiments P23-P38, wherein the composition comprises from about 3.0 x 10⁶ to about 2.1 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0168] Embodiment P40 - The pharmaceutical composition of any of Embodiments P15-P22, or the method of any of Embodiments P23-P39, wherein the composition comprises from about 6.0 x 10⁶ to about 4.2 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight.

[0169] Embodiment P41 - The pharmaceutical composition of any of Embodiments P15-P22, or the method of any of Embodiments P23-P40, wherein the composition comprises from about 9.0 x 10⁶ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight.

[0170] Embodiment P42 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P41, wherein the dosage of UCART7 comprises from about 1 x 10⁵ cells per kg to about 5 x 10⁶ cells per kg.

[0171] Embodiment P43 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P42, wherein the dosage comprises from about 1.4 x 10⁵ cells per kg to about 8.6 x 10⁵ cells per kg.

[0172] Embodiment P44 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P43, wherein the dosage comprises administration of about 1.4 x 10⁵ cells per kg.

[0173] Embodiment P45 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P44, wherein the dosage comprises administration of about 8.6 x 10⁵ cells per kg.

[0174] Embodiment P46 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P45, wherein the dosage comprises administration of about 10⁵ cells per kg.

[0175] Embodiment P47 - The pharmaceutical composition of any of Embodiments P15- P22, or the method of any of Embodiments P23-P46, wherein the dosage comprises administration of fewer than 10⁶ cells per kg.

[0176] Embodiment P48 - The method of any of Embodiments P23-P47, wherein the low dose of UCART7 is administered intravenously in a single dose.

[0177] Embodiment P49 - The method of any of Embodiments P23-P48, wherein the low dose of UCART7 is administered intravenously in more than one dose.

[0178] Embodiment P50 - The method of Embodiment P36, wherein the acute lymphocytic leukemia (T-ALL) comprises overexpression of CD7 and one or more of CD2 and CD5. [0179] Embodiment P51 - The method of any of Embodiments P23-P50, wherein the low dose of UCART7 cells is administered in a clinical setting or a hospital

[0180] Embodiment P52 - The method of any of Embodiments P23-P51, wherein determining the efficacy of a dose of UCART7 cells comprises evaluation of progression-free survival (PFS) and/or duration of response (DoR) in the patient.

[0181] Embodiment P53 - The method of any of Embodiments P23-P52, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome (TLS) in the patient.

[0182] Embodiment P54 - The method of any of Embodiments P23-P53, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of treatment- related toxicity in the patient.

[0183] Embodiment P55 - The method of any of Embodiments P23-P54, wherein toxicity of a dose of UCART7 cells is classified as: any grade 3 or higher neurotoxicity within 28 days of administration of UCART7 cells; any grade 4 CRS of any duration; grade 3 CRS that does not improve to grade 2 within 72 hours; grade 3 or greater infusion reaction; grade 3 or greater vital organ toxicity; grade 2 or greater GVHD; renal failure requiring dialysis for longer than 3 days; any intubation; and/or any grade 3 or 4 nonhematological toxicity lasting for more than 7 days.

[0184] Embodiment P56 - The method of Embodiment P32, wherein the therapy for treating CRS comprises a monoclonal antibody.

[0185] Embodiment P57 - The method of Embodiment P56, wherein the monoclonal antibody for treating CRS comprises an anti-interleukin-6 receptor antibody.

[0186] Embodiment P58 - The method of Embodiment P57, wherein the monoclonal antibody for treating CRS comprises tocilizumab.

[0187] Embodiment P59 - The method of Embodiment P58, wherein the therapy for treating CRS comprises a small molecule kinase inhibitor.

[0188] Embodiment P60 - The method of Embodiment P59, wherein the small molecule kinase inhibitor comprises a Janus kinase (JAK) inhibitor. [0189] Embodiment P61 - The method of either of Embodiments P59 or P60, wherein the small molecule kinase inhibitor comprises Ruxolitinib (Jakofi®), Baricitinib, itacitinib, or Dasatinib.

[0190] Embodiment P62 - The method of any of Embodiments P23-P61, wherein the patient has not had any anti-CD7 therapy prior to treatment.

[0191] Embodiment P63 - The method of any of Embodiments P23-P62, wherein the patient does not have grade 3 or 4 graft-versus-host-disease (GVHD).

[0192] Embodiment P64 - The method of any of Embodiments P16-P52, wherein the patient has grade 2 GVHD treatable with topical therapy.

[0193] Embodiment P65 - The method of any of Embodiments P16-P53, wherein the patient is pre-treated with cyclophosphamide at a dose of 500 mg/m² IV daily for 3 days, and fludarabine at a dose of 30 mg/m² IV daily for 3 days before treatment with UCART7.

[0194] Embodiment P66 - The method of any of Embodiments P23-P65, wherein the patient does not exhibit profound and persistent T cell aplasia before administration of UCART7 cells.

[0195] Embodiment P67 - The method of any of Embodiments P23-P66, wherein the patient exhibits T cell aplasia as a result of chemotherapeutic therapy before administration of UCART7 cells.

[0196] Embodiment P68 - The method of any of Embodiments P23-P67, wherein lymphodepletion occurs on days 1-3, wherein UCART7 cells are administered 3 days after the lymphodepletion period.

[0197] Embodiment P69 - The method of any of Embodiments P23-P68, wherein the patient is observed twice weekly for 3 weeks after administration of UCART7 cells, then weekly for 4 weeks, then monthly for a period of 5 months, then monthly until no more UCART7 cells are detectable in the patient, then quarterly for 2 years, then annually through year 15.

[0198] Embodiment P70. The method of any of Embodiments P23-P69, wherein cellular PK samples are obtained from the patient at each visit until no UCART7 cells are detectable in the patient.

[0199] Embodiment P71 - The method of any of Embodiments P23-P70, wherein cellular PK samples comprise one or more of AUC_{(0-∞ )}, AUC_(0-Ƭ), maximum drug concentration in the body after dosing [C_max], and T_max.

[0200] Embodiment P72 - The method of any of Embodiments P23-P71 wherein complete remission (CR) for a patient having ALL is classified as: < 5% blasts in the bone marrow of the patient; normal maturation of all cellular components in the bone marrow; no extramedullary disease; absolute neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

[0201] Embodiment P73 - The method of any of Embodiments P23-P72, wherein complete remission (CR) for a pediatric patient having ALL is classified as: complete donor chimerism (≥ 95% donor chimerism without recipient cells detected); no extramedullary disease; neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

[0202] Embodiment P74 - The method of any of Embodiments P72 or P73, wherein the extramedullary disease comprises CNS or soft tissue disease.

[0203] Embodiment P75 - The method of any of Embodiments P23-P74, wherein complete remission with incomplete hematologic recovery (CRi) for a patient having ALL is classified as:

[0204] In one aspect, the disclosure provides a single dose of a pharmaceutical composition comprising from about 1 x 10⁵ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight for treatment of T-cell malignancies, wherein the immune effector cells each comprise one or more chimeric antigen receptors (CARs) targeting one or more antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR) and/or are deficient in cell surface expression of at least one or more antigens to which the one or more CAR(s) specifically binds, wherein the composition does not elicit cytokine release syndrome (CRS). In one embodiment, the composition comprises from about 1.0 x 10⁶ to about 7.0 x 10⁷ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 3.0 x 10⁶ to about 2.1 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 6.0 x 10⁶ to about 4.2 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 9.0 x 10⁶ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0205] In one embodiment, the composition comprises from about 1.1 x 10⁵ to about 5.4 x 10⁶ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 1.4 x 10⁵ to about 8.6 x 10⁵ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. [0206] Also provided herein is a pharmaceutical composition comprising a dose of genetically modified T-cells transduced with a chimeric antigen receptor (CAR), wherein the CAR targets CD7, has a CD28 costimulatory domain or a 4- IBB costimulatory domain or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and has a CD3-zeta effector domain, wherein the genetically modified T-cells are deficient in TCRα (TRAC) expression, and wherein the genetically modified T-cells are deficient in CD7 surface expression, wherein the dose is effective to treat a T-cell malignancy and prevent cytokine release syndrome (CRS).

[0207] Also provided herein is a method of treatment of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

[0208] Also provided is a method of achieving complete remission of one or more T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0209] Also provided is a method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells.

[0210] Also provided herein is a method of treatment of T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the levels of interleukin (IL)- 1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)y, and/or fracktalkine are not elevated as a result of treatment.

[0211] Also provided herein is a method of achieving an overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0212] Also provided herein is a method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0213] Also provided herein is a method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0214] Also provide herein is a method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the dose (including a low dose) of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome.

[0215] Also provided herein is a method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising:

[0216] administering a dose (including a low dose) of UCART7 cells to the patient; and [0217] evaluating CRS symptoms in the patient.

[0218] Also provided herein is a method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose (including a low dose) of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, resuming treatment with UCART7 cells.

[0219] In one aspect, the disclosure provides a single dose of a pharmaceutical composition comprising from about 1 x 10⁵ to about 5 x 10⁶ chimeric antigen receptor (CAR) -bearing immune effector cells per kg of body weight, wherein the immune effector cells each comprise one or more chimeric antigen receptors (CARs) targeting one or more T-cell antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), wherein the immune effector cells are deficient in cell surface expression of the at least one or more T-cell antigens to which the one or more CAR(s) specifically bind(s), and wherein the composition does not elicit grade 3 or grade 4 cytokine release syndrome (CRS). In one embodiment, the T-cell antigen targeted by the CAR, and in which the cells are deficient in cell surface expression, is CD7. In another embodiment, the CAR-bearing immune effector cells are T-cells. In another embodiment, the subunit of the TCR in which the cells are deficient (in cell surface expression) is one or more of the T cell receptor complex alpha chain (TCRα/TRAC), the T cell receptor complex beta chain (TCRβ ), the T cell receptor complex delta chain (TCR5), and the T cell receptor complex gamma chain (TCRγ). In another embodiment, the subunit of the TCR in which the cells are deficient (in cell surface expression) is the T cell receptor complex alpha chain (TCRα/TRAC). In another embodiment, the CAR targeting CD7 comprises: a CD28 co- stimulatory domain; or a 4- IBB co-stimulatory domain; or a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain.

[0220] In another embodiment, the CAR targeting CD7 comprises a 4- IBB co-stimulatory domain and a CD3-zeta effector domain.

[0221] In another aspect, the disclosure provides a pharmaceutical composition comprising a dose of genetically modified T-cells transduced with one or more chimeric antigen receptors (CARs), wherein the one or more CARs targets CD7, has a CD28 costimulatory domain or a 4- 1BB costimulatory domain or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and has a CD3-zeta effector domain; wherein the T-cells are genetically modified to be deficient in cell surface expression of TCRα/TRAC, and wherein the T-cells are genetically modified to be deficient in cell surface expression of CD7, wherein the dose is effective to treat a T-cell malignancy and prevent grade 3 or grade 4 cytokine release syndrome (CRS).

[0222] In one embodiment, the genetically modified T cells a) are deficient in CD7 surface expression due to deletion or disruption of the CD7 gene and/or b) express a functionally disabled T-cell receptor (TCR) due to deletion of the T-cell receptor alpha chain (TCRα/TRAC) gene. [0223] In one embodiment, the CD7 and/or TCRα/TRAC gene(s) is/are deleted by CRISPR/Cas9 gene editing. In another embodiment, the T-cell malignancy is a hematological malignancy. In another embodiment, the hematological malignancy is a T-cell malignancy. In another embodiment, the hematological malignancy is a CD7+ T-cell malignancy. In another embodiment, the T-cell malignancy is AML, T-ALL, and non-Hodgkin’s lymphoma. In another embodiment, the CRS is grade 3 or grade 4 CRS.

[0224] In another aspect, the disclosure provides a method of treatment of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

[0225] In another aspect, the disclosure provides a method of achieving complete remission of one or more T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0226] In another aspect, the disclosure provides a method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells. [0227] In another aspect, the disclosure provides a method of treatment of T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the levels of interleukin (IL)- 1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)γ, and/or fracktalkine are not elevated to a level that would correspond with or result in grade 3 or 4 CRS as a result of treatment.

[0228] In another aspect, the disclosure provides a method of achieving an overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0229] In another aspect, the disclosure provides a method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0230] In another aspect, the disclosure provides a method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0231] In another aspect, the disclosure provides a method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the dose (including a low dose) of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome. [0232] In another aspect, the disclosure provides a method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose (including a low dose) of UCART7 cells to the patient; and evaluating CRS symptoms in the patient.

[0233] In another aspect, the disclosure provides a method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose (including a low dose) of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, resuming treatment with UCART7 cells.

In one embodiment, the T-cell malignancy is a hematological malignancy. In another embodiment, the hematological malignancy is a T-cell malignancy. In another embodiment, the hematological malignancy is a CD7+ T-cell malignancy. In another embodiment, the T- cell malignancy is AML, T-ALL, or lymphoma. In another embodiment, the lymphoma is non- Hodgkin’ s lymphoma. In another embodiment, the composition comprises from about 1.0 x 10⁶ to about 7.0 x 10⁷ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 3.0 x 10⁶ to about 2.1 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 6.0 x 10⁶ to about 4.2 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the composition comprises from about 9.0 x 10⁶ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In another embodiment, the dosage of UCART7 comprises from about 1 x 10⁵ cells per kg to about 5 x 10⁶ cells per kg. In another embodiment, the dosage comprises from about 1.4 x 10⁵ cells per kg to about 8.6 x 10⁵ cells per kg. In another embodiment, the dosage comprises administration of about 1.4 x 10⁵ cells per kg. In another embodiment, the dosage comprises administration of about 8.6 x 10⁵ cells per kg. In another embodiment, the dosage comprises administration of about 10⁵ cells per kg. In another embodiment, the dosage comprises administration of fewer than 10⁶ cells per kg. In another embodiment, the dose (including a low dose) of UCART7 is administered intravenously in a single dose. In another embodiment, the dose (including a low dose) of UCART7 is administered intravenously in more than one dose. In another embodiment, the acute lymphocytic leukemia (T-ALL) comprises overexpression of CD7 and one or more of CD2 and CD5. In another embodiment, the dose (including a low dose) of UCART7 cells is administered in a clinical setting or a hospital. In another embodiment, determining the efficacy of a dose of UCART7 cells comprises evaluation of progression-free survival (PFS) and/or duration of response (DoR) in the patient. In another embodiment, determination of the safety of a dose of UCART7 cells comprises evaluation of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome (TLS) in the patient. In another embodiment, determination of the safety of a dose of UCART7 cells comprises evaluation of treatment-related toxicity in the patient. In another embodiment, toxicity of a dose of UCART7 cells is classified as: any grade 3 or higher neurotoxicity within 28 days of administration of UCART7 cells; any grade 4 CRS of any duration; grade 3 CRS that does not improve to grade 2 within 72 hours; grade 3 or greater infusion reaction; grade 3 or greater vital organ toxicity; grade 2 or greater GVHD; renal failure requiring dialysis for longer than 3 days; any intubation; and/or any grade 3 or 4 nonhematological toxicity lasting for more than 7 days. [0234] In one embodiment, the therapy for treating CRS comprises a monoclonal antibody. In another embodiment, the monoclonal antibody for treating CRS comprises an anti- interleukin-6 receptor antibody. In another embodiment, the monoclonal antibody for treating CRS comprises tocilizumab. In another embodiment, the therapy for treating CRS comprises a small molecule kinase inhibitor. In another embodiment, the small molecule kinase inhibitor comprises a Janus kinase (JAK) inhibitor. In another embodiment, the small molecule kinase inhibitor comprises Ruxolitinib (Jakofi®), Baricitinib, itacitinib, or Dasatinib. In another embodiment, the patient has not had any anti-CD7 therapy prior to treatment. In another embodiment, the patient does not have grade 3 or 4 graft-versus-host- disease (GVHD). In another embodiment, the patient has grade 2 GVHD treatable with topical therapy. In another embodiment, the patient is pre-treated with cyclophosphamide at a dose of 500 mg/m² IV daily for 3 days, and fludarabine at a dose of 30 mg/m² IV daily for 3 days before treatment with UCART7. In another embodiment, the patient does not exhibit profound and persistent T cell aplasia before administration of UCART7 cells. In another embodiment, the patient exhibits T cell aplasia as a result of chemotherapeutic therapy before administration of UCART7 cells. In another embodiment, lymphodepletion occurs on days 1- 3, wherein UCART7 cells are administered 3 days after the lymphodepletion period. In another embodiment, the patient is observed twice weekly for 3 weeks after administration of UCART7 cells, then weekly for 4 weeks, then monthly for a period of 5 months, then monthly until no more UCART7 cells are detectable in the patient, then quarterly for 2 years, then annually through year 15. In another embodiment, cellular PK samples are obtained from the patient at each visit until no UCART7 cells are detectable in the patient. In another embodiment, cellular PK samples comprise one or more of AUC_{(0-∞ )}, AUC_(0-Ƭ), maximum drug concentration in the body after dosing [C_max], and T_max. In another embodiment, complete remission (CR) for a patient having ALL is classified as:

[0235] In another embodiment, complete remission (CR) for a pediatric patient having ALL is classified as: complete donor chimerism (≥ 95% donor chimerism without recipient cells detected); no extramedullary disease; neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

[0236] In another embodiment, the extramedullary disease comprises CNS or soft tissue disease. In another embodiment, complete remission with incomplete hematologic recovery (CRi) for a patient having ALL is classified as: < 5% blasts in the bone marrow of the patient; normal maturation of all cellular components in the bone marrow; no extramedullary disease; and transfusion independent.

[0237] In some embodiments, the dose (including a low dose) is a dose appropriate for treatment of acute myeloid leukemia (AML).

[0238] In some embodiments, the dose (including a low dose) is a dose appropriate for treatment of acute lymphocytic leukemia (ALL).

[0239] In some embodiments, the dose (including a low dose) is a dose appropriate for treatment of lymphoma.

Cytokine Release Syndrome (CRS)

[0240] As described herein, cytokine release syndrome (CRS) or cytokine storm refers to a toxicity or condition that may occur after treatment with some types of immunotherapy, such as monoclonal antibodies and CAR-T or other CAR-bearing immune effector cells in response to immunotherapy (or other immunological stimulus). CRS clinically manifests when large numbers of lymphocytes (B cells, T cells, and/or natural killer cells) and/or myeloid cells (macrophages, dendritic cells, and monocytes) become activated and release inflammatory cytokines. CRS has classically been associated with therapeutic mAb infusions, most notably anti-CD3 (OKT3), anti-CD52 (alemtuzumab), anti-CD20 (rituximab), and the CD28 super-agonist, TGN1412. Symptom onset can occur within minutes to hours after the infusion begins. CRS has also been reported following administration of bi-specific antibodies for leukemia, infusion of haploidentical mononuclear cells to patients with refractory leukemia, and adoptive immunotherapies for cancer, most notably T cells engineered to express CARs.

[0241] CRS is caused by a large, rapid release of cytokines into the blood from immune cells in response to the immunotherapy. Symptoms of CRS include fever, fatigue, loss of appetite, muscle and joint pain, nausea, vomiting, diarrhea, rashes, fast breathing, rapid heartbeat, low blood pressure, seizures, headache, confusion, delirium, hallucinations, tremor, and loss of coordination. CRS can manifest along a spectrum of mild to fatal, and can be ranked by severity as follows:

• Grade 1: Mild reaction, infusion interruption not indicated; intervention not indicated • Grade 2: Therapy or infusion interruption indicated but responds promptly to symptomatic treatment (e.g., antihistamines, NSAIDS, narcotics, IV fluids); prophylactic medications indicated for <=24 hrs

• Grade 3: Prolonged (e.g., not rapidly responsive to symptomatic medication and/or brief interruption of infusion); recurrence of symptoms following initial improvement; hospitalization indicated for clinical sequelae (e.g., renal impairment, pulmonary infiltrates)

• Grade 4: Life-threatening consequences; pressor or ventilatory support indicated

• Grade 5: Death

See, e.g., “Common Terminology Criteria for Adverse Events (CTCAE) Version v4.03,” National Institutes of Health and National Cancer Institute, June 14, 2010; Lee DW et al., “Current concepts in the diagnosis and management of cytokine release syndrome,” Blood 2014 124(2): 188-95.

[0242] CRS is associated with elevated circulating levels of several cytokines, which can include, but are not limited to, interleukin (IL)-l, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)γ, and/or fracktalkine. In some embodiments, a treatment such as tocilizumab, which is an anti-IL-6 receptor antibody, may be administered to treat CRS.

[0243] Timing of symptom onset and CRS severity depends on a number of factors, such as the inducing agent (i.e., the specific immunotherapy) and the magnitude of immune cell activation. For example, CRS following rituximab for CD20+ malignancies typically occurs within minutes to hours, and patients with >50 x 10⁹/L circulating lymphocytes have increased rates of CRS symptoms. In recent reports of CRS following adoptive T-cell therapy for cancer, the incidence and severity of the syndrome also appears greater when patients have large tumor burdens, presumably because this leads higher levels of T-cell activation. In contrast, symptom onset typically occurs days (cases 1 and 3) to occasionally weeks (case 2) after the T-cell infusion, coinciding with maximal in vivo T-cell expansion. A clear cell dose:response relationship for CRS related to adoptive T-cell therapies has been difficult to define, but higher doses of T cells administered to a patient with a T-cell malignancy may result in earlier onset or more severe symptoms.

[0244] Thus, in some embodiments, preventing or reducing the occurrence of CRS or other toxicity described herein in a patient having a T-cell malignancy may be effected by lowering the dose of UCART7 cells administered to the patient. Administration of a low dose of UCART7 immune effector cells may refer to a dose of UCART7 described herein, such as a dose of about 1 x 10⁵ to about 5 x 10⁶ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight. In other embodiments, a low dose of UCART7 immune effector cells may refer to a dose of from about 1 x 10⁵ to about 9 x 10⁵ cells per kg. In still further embodiments, a dose of a UCART7 immune effector cells may refer to a dose of from about 1.1 x 10⁵ to about 5.4 x 10⁶ cells per kg of body weight, or a dose of from about 1.4 x 10⁵ to about 8.6 x 10⁵ cells per kg of body weight.

[0245] In other embodiments, a low dose of UCART7 immune effector cells may refer to a dose of from about 1.4 x 10⁵ cells per kg to about 8.6 x 10⁵ cells per kg. In other embodiments, a low dose of UCART7 immune effector cells may refer to a dose of about 1.4 x 10⁵ cells per kg, or a dose of about 8.6 x 10⁵ cells per kg, or a dose of about 10⁵ cells per kg, or a dose of fewer than about 10⁶ cells per kg.

[0246] Graft-vs-Host Disease

[0247] As used herein, “graft- vs-host disease” or “GVHD” or “GvHD” refers to a condition that can occur when transplanted tissues or cells elicit an immune response in the recipient. White blood cells present in the transplanted tissue or cells recognize the recipient cells as foreign and attack them, resulting in skin involvement (i.e., rash) and liver and gastrointestinal tract damage. Grades 1 through 4 GVHD refer to the severity of GVHD, and are as follows:

• Grade 1: predominantly affects the skin, with skin rash affecting <25% of the body, bilirubin levels of 26-60 μmol/L, and gut fluid loss of 500-1000 ml/day;

• Grade 2: skin rash affecting 25-50% of the body, bilirubin levels of 61-137 μmol/L, and gut fluid loss of 1000-1500 ml/day;

• Grade 3: skin rash affecting >50% of the body, or erythroderma, bilirubin levels of 138-257 μmol/L, and gut fluid loss of >1500 ml/day; and

• Grade 4: bullae desquamation of the skin, bilirubin levels >257 μmol/L, and gut fluid loss of >2500 ml/day, or ileus.

[0248] In some embodiments of the present disclosure, a patient or subject as described herein may develop GVHD and may, as a result, require treatment. Typically, Grade 1 or 2 GVHD is treated using topical therapy, such as steroid creams, including topical treatments such as triamcinolone. In some cases, tacrolimus may be given for resistant skin involvement. One of skill in the art will recognize that any standard topical therapy for treating GVHD may be given to a patient as described herein. In some embodiments, patients with Grades 3 or 4 GVHD are excluded from the study. In some embodiments, patients with Grades 1 or 2 GVHD may be treated with topical therapy and remain in the study. In some embodiments, a patient may remain in the study if the patient develops Grade 2 GVHD that improves to Grade 1 GVHD as a result of topical therapy. Likewise, a patient may be excluded from the study if the patient develops Grade 2 GVHD that does not improve to Grade 1 GVHD as a result of topical therapy.

Neurotoxicity

[0249] Immunotherapy can have significant effects on the central or peripheral nervous systems that can limit the course of treatment. With the development of biological and immunotherapeutic agents to treat cancer, there are new patterns of neurotoxicity that are less well-described. Neurotoxicity is one of the most common toxicities related to immunotherapy. The occurrence of nervous system toxicity depends on a variety of factors, including the dose of treatment delivered, route of administration, interactions with other agents, the presence of underlying structural nervous system disease, and individual patient vulnerability, most of which are poorly understood. Toxicity can occur by direct damage to neurons or glia, or indirectly by altering the surrounding microenvironment, such as localized vascular injury. Dose adjustment or discontinuation may be required to prevent further symptoms.

Tumor lysis syndrome

[0250] Tumor lysis syndrome (TLS) refers to the occurrence of metabolic abnormalities as a complication during treatment of cancer, where large amounts of tumor cells are killed off (lysed) at the same time by the treatment, releasing their contents into the bloodstream. This occurs most commonly after the treatment of lymphomas and leukemias. In oncology and hematology, this can be fatal, and patients at increased risk for TLS should be closely monitored before, during, and after their course of chemotherapy.

[0251] Tumor lysis syndrome is characterized by high blood potassium (hyperkalemia), high blood phosphate (hyperphosphatemia), low blood calcium (hypocalcemia), high blood uric acid (hyperuricemia), and higher than normal levels of blood urea nitrogen (BUN) and other nitrogen-containing compounds (azotemia). These changes in blood electrolytes and metabolites are a result of the release of cellular contents of dying cells into the bloodstream from breakdown of cells. In this respect, TLS is analogous to rhabdomyolysis, with comparable mechanism and blood chemistry effects but with different cause. In TLS, the breakdown occurs after cytotoxic therapy or from cancers with high cell turnover and tumor proliferation rates. The metabolic abnormalities seen in tumor lysis syndrome can ultimately result in nausea and vomiting, but more seriously acute uric acid nephropathy, acute kidney failure, seizures, cardiac arrhythmias, and death.

[0252] Tumors with a high cell turnover rate, rapid growth rate, and high tumor bulk tend to be more associated with the development of tumor lysis syndrome. The most common tumors associated with this syndrome are poorly differentiated lymphomas (such as Burkitt’ s lymphoma), other Non-Hodgkin’ s Lymphomas (NHL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML). Other cancers (such as melanoma) have also been associated with TLS but are less common. Certain patient-related factors can also affect the development of clinical tumor lysis syndrome, such as elevated baseline serum creatinine, kidney failure, dehydration, and other issues affecting urinary flow or the acidity of urine. Thus, in some embodiments, blood samples maybe taken from patients receiving UCART7 to treat a T-cell malignancy as described herein in order to assess the health of the kidneys in order to prevent or reduce the occurrence of TLS.

[0253] In addition, chemo-sensitive tumors, such as lymphomas, carry a higher risk for the development of tumor lysis syndrome. Those tumors that are more responsive to a chemotherapy agent carry a higher TLS risk. Usually, the precipitating medication regimen includes combination chemotherapy, but TLS can be triggered in cancer patients by steroid treatment alone, and sometimes without any treatment, referred to as “spontaneous tumor lysis syndrome.”

Cluster of Differentiation 7 (CD7)

[0254] CD7 (Cluster of Differentiation 7) is a transmembrane glycoprotein that, in humans, is encoded by the CD7 gene. CD7 is expressed by T cells and natural killer (NK) cells and their immediate precursors; it is also expressed in 95% of lymphoblastic T-cell leukemias and lymphomas and in a subset of peripheral T-cell lymphomas (PTCL). CD7 plays a costimulatory role in T-cell activation upon binding to its ligand K12/SECTM1. However, it appears not to make a pivotal contribution to T-cell development or function because genomic disruption of CD7 in murine T-cell progenitors permits normal T-cell development and homeostasis and only minor alterations in T-cell effector function. CD7 expression is restricted to immune cells (T and NK cells).

[0255] CD7 is the earliest T-cell-associated molecule to appear in prethymic stages and extends its expression all the way to the mature T-cell stages. CD7, however, does not seem to be expressed in early bone marrow hematopoietic progenitor cells: hematopoietic stem cells, multipotent progenitor cells, and common myeloid progenitor cells. Expression is first documented in the common lymphoid progenitor cells before they exit the bone marrow, but some reports show that CD7 expression appears in early T-cell progenitor cells in the thymus. CD7 is also present on most NK cells. However, it is noted that some reports mention the possibility of CD7 expression on pluripotent stem cells that are capable of differentiating to T cells as well as on erythroid, megakaryocytic, and myeloid precursors. CD7 expression is maintained in all stages of T-cell development in the thymus and on T-cells in peripheral blood.

[0256] Recent studies in CD7-deficient mice have provided new insights into CD7 function and have demonstrated key roles for CD7 in regulating peripheral T- and NK-cell cytokine production and sensitivity to LPS-induced shock syndromes.

[0257] The human protein atlas shows the presence of CD7 primarily located in the reticulo-endothelial system. Specifically, and as may be expected by the distribution of T cells and NK cells, CD7 is found in immunocompetent or immune processing tissues including the tonsils, appendix, spleen, bone marrow, and lymph nodes. CD7 is not found in, or expressed by, other tissues.

[0258] As CD7 is not expressed in tissues other than the marrow and hematopoietic cell lines, off-target toxicity should be minimal; given the expression of CD7 in a significant subset of leukemia and lymphoma patients, it is an attractive target for a therapeutic agent. Thus, in some embodiments, a CAR-T cell as described herein may target CD7-positive (CD7+ or CD7+ve) tumor cells. This may be accomplished as described herein by expressing one or more chimeric antigen receptors (CARs) in a T-cell such that at least one of the CARs recognizes and binds to CD7 in tumor cells. In addition, in order to prevent fratricide, expression of CD7 in the CAR-T cells may be reduced or eliminated. These particular aspects are described herein.

Clinical Application in CD7- Associated Tumors

[0259] CD7 can be aberrantly expressed in refractory anemia with excess blasts and may confer a worse prognosis in this condition. A lack of CD7 expression is also associated with mycosis fungoides (or cutaneous T-cell lymphoma) and Sezary syndrome (commonly considered a more advanced form of cutaneous T-cell lymphoma). The presence of CD7 on other leukemia and lymphomas does not appear to have a specific prognostic or causal relevance; the expression of CD7 can be accompanied by the expression of other CD antigens. In other embodiments, expression of CD7 is accompanied by the expression of other CD antigens. In some embodiments, changes in expression of CD7 may be accompanied by changes in the expression of other CD antigens.

[0260] The CD7 glycoprotein has been extensively documented in T-cell lineage disease, being expressed in approximately 95% of T-cell acute lymphocytic leukemia (T-ALL) and in approximately 50% of PTCL. While less frequently expressed in non-T-cell disease, it has been reported to be expressed in between 5% and 7.5% of diffuse large B-cell lymphoma (DLBCL) patients, and in approximately 30 % of acute myeloid leukemia (AML) patients. UCART7

[0261] UCART7 is a genetically modified T-cell which is transduced with a chimeric antigen receptor (CAR) targeting CD7, which is genome-edited to be deficient in cell surface expression of CD7, which is genome edited to be deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), which has a CD28 costimulatory domain, a 4- IBB costimulatory domain, or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and which has a CD3-zeta effector domain. To prevent fratricide, the target antigen CD7, which is expressed on both malignant and healthy T cells, is deleted in T cells as described herein that are derived from a healthy donor by CRISPR/Cas9 gene editing. To prevent alloreactivity of T cells derived from allogeneic healthy donor T cells, the T-cell receptor alpha chain is genetically altered allowing the use of allogeneic donor T cells with a decreased risk of graft-versus-host disease (GvHD). Thus, UCART7 is a GvHD-deficient, fratricide-resistant CAR-T for the treatment of CD7+ve T-cell malignancies. The use of healthy allogenic donor T cells for UCART7 mitigates the risk of inadvertently generating therapy-resistant malignant clones during cellular manufacturing processes.

[0262] In some embodiments, UCART7 refers to a T cell with a CAR comprising a CD7- targeting scFv, a linker, a hinge region, and a transmembrane region, a 4-1BB costimulatory domain, and a CD3z effector domain; and deletion of both the TCRα/TRAC gene and the CD7 gene.

Dosage of UCART7 Immune Effector Cells

[0263] It some embodiments, as described herein, UCART7 immune effector cells may be administered alone as a treatment for a hematologic malignancy, such as a T-cell malignancy. As described herein, medications deemed appropriate by a physician, clinician, or study director, may be administered simultaneously with, concurrent with, or at any specific time period or interval as necessary. In some embodiments, UCART7 may be administered as a single-dose treatment or therapy for a hematologic malignancy. In other embodiments, UCART7 may be administered to a patient in one or more subsequent doses, i.e., a second dose, a third dose, a fourth dose, or the like.

[0264] CAR-T cells may be autologous, meaning that they are engineered from a subject’s own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft- vs-host reaction. As would be understood by one of skill in the art, immune therapy using autologous vs allogeneic cells may determine the numbers or ranges of cells that may be most beneficial to administer to a patient. For example, autologous cells are T-cells that were taken from the patient himself, modified as described herein to produce an immune effector cell that targets a malignant cell, and administered back to the patient. As would be expected, this type of therapy or treatment would typically be administered in a single dose, as each administration would require an additional period of time for collection, culture, and genetic modification of the cells. On the other hand, allogeneic cells are not patient-specific, but rather are cells taken from a healthy donor and then modified to possess elements for use as an immunotherapy as described herein. Therefore, allogeneic cells may provide a uniquely “universal” therapy for hematologic cancers, with the ability to tailor or “customize” the cells to target a desired cancer type or cell surface protein, such as CD7.

[0265] In some embodiments, an immune effector cell as described herein may refer to a CAR-T cell having one or more chimeric antigen receptors (CARs) targeting one or more antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR) and/or are deficient in cell surface expression of at least one or more antigens to which the one or more CAR(s) specifically binds, wherein the composition does not elicit cytokine release syndrome (CRS). Specific components of the cells are described herein below in detail. Prevention of CRS or another toxicity described herein is a specific embodiment of the present disclosure.

[0266] Also disclosed herein is pharmaceutical composition comprising a dose of genetically modified T-cells transduced with one or more chimeric antigen receptor (CAR), targeting CD7, having a CD28 costimulatory domain or a 4-1BB costimulatory domain or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and having a CD3- zeta effector domain, wherein the genetically modified T-cells are deficient in TCRα/TRAC expression, and wherein the genetically modified T-cells are deficient in CD7 surface expression, wherein the dose is effective to treat a T-cell malignancy and prevent cytokine release syndrome (CRS). Dosages of CAR-T cells and/or compositions comprising these as described herein may be administered at a particular dosage. A dosage as used herein refers to the number of CAR-T cells given to a patient based on the body weight of the patient. Determination of the particular dosage can be determined experimentally in order to prevent the occurrence of CRS or another toxicity as described herein.

[0267] In some embodiments, UCART7 may be administered to a patient having a hematologic or T-cell malignancy at a dose of from about 1 x 10⁵ cells per kilogram (kg) of body weight to about 6.3 x 10⁸ cells per kg of body weight. For examples, UCART7 may be administered to a patient having a hematological malignancy at any dose, dose range, or sub- range of the dosages described herein, such as including, but not limited to, a dose range of from about 1.0 x 10⁵ cells/kg to about 6.3 x 10⁸ cells/kg, such as including, but not limited to, any dose of about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about

4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 9.6, about 9.7, about 9.8, or about 9.9 x 10⁵ cells/kg; any dose of about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2.0, about 2.5, about

2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, or about 9.5 x 10⁶ cells/kg; any dose of about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5 x 10⁷ cells/kg; or any dose of about 1.0, about 1.5, about 2.0, about 2.1, about 2.2, about 2.5, about 3.0, about 3.5, about 4.0, about 4.1, about 4.2, about 4.3, about 4.5, about 5.0, about 5.5, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5 x 10⁸ cells/kg.

[0268] It has been demonstrated in the literature that high doses of immunotherapy can result in higher rates of toxicity (e.g., CRS, neurotoxicity, TLS, or the like) in some patients. In order to evaluate this, the present study may administer an initial dose (including a low dose) of UCART7 immune effector cells to patients in a study group as described herein, and the safety and efficacy evaluated in those patients. Following this patient population, a higher dose of UCART7 may be administered to a second patient population, and the safety and efficacy of the second dose evaluated in the second patient population. Additional, higher or lower doses of UCART7 immune effector calls may be administered to a patient population as set forth herein or in the Examples, if deemed appropriate by a clinician or study director. In some embodiments, a dose or dose range of UCART7 may be escalated in a patient or patient population to determine the most beneficial dose. For example, as described herein, a patient or patient population may be administered an initial dose (including a low dose) of UCART7, and the safety and efficacy of the dose evaluated in the population as described herein. For example, in some embodiments, a patient population may be administered UCART7 at a dose of 1.4 x 10⁵ cells/kg, the safety and efficacy evaluated in each patient, followed by administration of a higher dose, for example, 8.6 x 10⁵ cells/kg, in a second patient population. As described herein, higher doses of UCART7 may be associated with higher incidence of toxicity, such as CRS. One of skill in the art will recognize and be able to determine an appropriate “low” dose of UCART7 using the compositions and methods and related disclosure as described herein.

[0269] As used herein to refer to dosages of immune effector cells, “cells/kg” refers to a recited number of immune effector cells per kg of body weight of the patient. Thus, in some embodiments, a composition or method of the present disclosure may provide any number of cells required to produce the desired therapeutic effect, i.e., treatment of hematological malignancies. Such a desired therapeutic effect may be a reduction in the size of a tumor, a reduction in the number of malignant cells, a reduction in the number or percentage of non- native CD-I- cells, or any other descriptor demonstrating an improvement in prognosis for a patient having a hematologic malignancy as described herein.

[0270] In some embodiments, UCART7 immunotherapy may be administered to patients at a particular dose or dose range in order to prevent toxicity in the patient. “Toxicity” as described herein may refer to any biochemical or biological condition that occurs in the patient as a result of the immunotherapy. For example, a toxicity as described herein may refer to cytokine release syndrome (CRS), neurotoxicity, tumor lysis syndrome (TLS), or graft- vs-host disease (GVHD). Development of any of these conditions may result in additional illness in the patient, thereby causing loss of a drug response in the patient, or even death. One of skill in the art will understand that dosages of drugs as described herein may be altered as needed for a patient or subject without deviating from the scope of the disclosure. [0271] In some embodiments, UCART7 may be administered with one or more additional drugs or treatments. For example, as described herein, one or more drugs for lymphodepletion may be administered prior to administration of UCART7. As used herein, “lymphodepletion” refers to administration of a therapy to deplete normal lymphocytes prior to administration of an immunotherapy, such as CAR T cells. Any drugs appropriate for lymphodepletion may be used as described herein, such as including, but not limited to, cyclophosphamide at a dosage of 500 mg/m², administered by intravenous injection (IV) daily for 3 days, and fludarabine 30 mg/m² IV daily for 3 days. In other embodiments, additional drugs may be administered to reduce or eliminate symptoms associated with an adverse event (AE) as described herein. Adverse events will be monitored and treatments for such adverse events are at the discretion of the study director. For example, steroids such as prednisone or variations thereof, may be administered to a patient as deemed appropriate. [0272] In some embodiments, patients enrolled in the study may be given bridging therapy while awaiting collection of bone marrow by the donor. For example, bridging therapy for T-ALL and NHL patients may include, but is not limited to, steroids, hydroxyurea, and vincristine, alone or in combination. In addition, bridging therapy for AML patients may include, but is not limited to, hydroxyurea, low-dose cytarabine, or either decitabine or azacitidine.

[0273] In some embodiments, patients receiving UCART7 may be administered a drug or therapy to control or treat CRS, or other toxicity related to immunotherapy. The most common treatment for CRS currently is tocilizumab (atlizumab), which is a monoclonal antibody that targets the IL-6 receptor (IL-6R). In other embodiments, other monoclonal antibody drug treatments may be administered as necessary or deemed appropriate, such as including, but not limited to, Abrezekimab, Adalimumab, Afelimomab, Afasevikumab, Anifrolumab, Anrukinzumab (=IMA-638), asiliximab, Bermekimab, Bimekizumab, Brazikumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cergutuzumab, amunaleukin, Certolizumab pegol, Clazakizumab, Daclizumab, Dectrekumab, Dupilumab, Eldelumab, Elsilimomab, Emapalumab, Enokizumab, Faralimomab, Fezakinumab, Fletikumab, Flotetuzumab, Fontolizumab, Gevokizumab, Golimumab, Guselkumab, Infliximab, Inolimomab, Ixekizumab, Lebrikizumab, Lutikizumab, Mavrilimumab, Mepolizumab, Mirikizumab, Nemolizumab, Nerelimomab, Netakimab, Olokizumab, Otilimab, Ozoralizumab, Pascolizumab, Pateclizumab, Perakizumab, Placulumab, Pogalizumab, Prezalumab, Remtolumab, Reslizumab, Risankizumab, Romilkimab, Rontalizumab, SA237, Sarilumab, Satralizumab, Secukinumab, Sifalimumab, Siltuximab, Sirukumab, Tildrakizumab, TNX-650, Tralokinumab, Ustekinumab, Vobarilizumab, Vunakizumab. In other embodiments, any other drug or therapy may be used without deviating from the scope of the present disclosure.

[0274] In some embodiments, small molecule inhibitors of CRS may be used, including, but not limited to, Janus kinase (JAK) inhibitors. Exemplary JAK inhibitors include, but are not limited to, Ruxolitinib (Jakofi®), S-Ruxolitinib (INCB018424),

Tofacitinib (Xeljanz®/Jakvinus®, formerly known as tasocitinib and CP-690550), Oclacitinib (Apoquel®), Baricitinib (Olumiant®, INCB028050)), Itacitianib, Peficitinib (ASP015K, JNJ-54781532; Smyraf®), Fedratinib (SAR302503; Inrebic®), Upadacitinib (Rinvoq®; ABT-494), Filgotinib (G- 146034, GLPG-0634), Cerdulatinib (PRT062070), Gandotinib (LY-2784544), Lestaurtinib (CEP-701), Momelotinib (GS-0387, CYT-387), Pacritinib (SB1518), PF-06700841, Abrocitinib (PF- 04965842), Cucurbitacin I (JSI-124), CHZ868, AZD1480, AT9283, WP1066, TG101209, NVP-BSK805 2HC1, AZ 960, CEP-33779, WHI-P154, XL019, ZM 39923 HC1, WHI-P258, Selective JAK3 inhibitor 1, Solcitinib, PF-06651600, FM-381, Decemotinib (VX-509), FLLL32, B MS-911543, WHI-P97, Itacitinib (INCB39110), GLPG0634 analogue, Go6976, and Curcumol. As would be understood by one of skill in the art, other kinase inhibitors can be used as described herein, such as including, but not limited to, Gefitinib, Sorafenib, Erlotinib, Imatinib, Dasatinib, Lapatinib, Temsirolimus, Axitinib, Crizotinib, Ponatinib, Trametinib, Dabrafenib, Ceritinib, Palbociclib, Lenvatinib, Masitinib, Cediranib, Genistein, 7-Hydroxy staurosporine, Alvocidib, Vatalanib, AT9283, CYC116, Alisertib, MKC-1, SNS- 314, Seliciclib, Semaxanib, Midostaurin, Tofacitinib,, Osimertinib, Alectinib, Rabusertib, Barasertib, Spebrutinib, Danusertib, Bafetinib, Flumatinib, Vistusertib, Orantinib, Losmapimod, Indirubin, Merestinib, MK-5108, TAK-901, PF-03814735, Sunitinib, Nilotinib, Vandetanib, Ridaforolimus, Pazopanib, Bosutinib, Cabozantinib, Ruxolitinib, Vemurafenib, Regorafenib, Afatinib, Ibrutinib,, Nintedanib, Tivozanib, Wortmannin, Everolimus,, Mangostin, Sirolimus, Emodin, Fasudil, MLN8054, obimetinib, Encorafenib, Ribociclib, Icotinib, Neratinib, Rociletinib, Dacomitinib, Binimetinib, Abemaciclib, Lorlatinib Brigatinib, Olmutinib, Pimasertib, RO-5126766 free base, Pelitinib, Motesanib, AV-412, Ilorasertib, Momelotinib, GSK-2636771, GSK-2636771, Gedatolisib, Tesevatinib, Entrectinib, Naquotinib, Rigosertib, Rimacalib, Rebastinib, AZD-0424, X-396, Rivoceranib, Ensartinib, PF-06700841, Zanubrutinib, Upadacitinib, Voruciclib, Abivertinib, Avapritinib, H-89, Doramapimod, AKI-001, Reversine, Dorsomorphin, AZD-8330, (Rp)-cAMPS, Ripretinib.

[0275] One of skill in the art will understand small molecule inhibitors and their mechanisms, and be able to identify a small molecule inhibitor, such as a JAK kinase inhibitor, useful for the present disclosure.

[0276] In some embodiments, reducing the level of cytokines released in CRS can prevent or reduce the development and/or maintenance of CRS. This can be accomplished by modifying, disrupting, or deleting one or more cytokine/chemokine/transcription factor genes that encode a nucleic acid or polypeptide involved in CRS. In some embodiments, such modification, disruption, or deletion can be performed for any combination of genes involved in CRS, all of which are encompassed within the scope of the present disclosure.

[0277] One method to accomplish this is genetic ablation (gene silencing) in which gene expression is abolished through the alteration or deletion of genetic sequence information. This can be accomplished using known genetic engineering tools in the art such as Transcription Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases (ZFNs), CRISPR, and also by transfection of small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs). In some embodiments, transient suppression thru technologies such as protein expression blockers (PEBL).

[0278] For example, in some embodiments, deletion, mutation, or inactivation can be performed for a cytokine gene, a chemokine gene, or a transcription factor gene involved in CRS including, but not limited to, AHR, BCL6, XCL1, XCL2, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CD28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX3CL1, FOXP3, GATA3, IFNy, IL-1α, IL-1β, IL-1RA, IL- 18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-a, IFN-β , IFN-γ, CD154, LT-β , TNF- α,TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, A2M, ACKR1, ACKR2, ACKR3, ACVR1, ACVR2B, ACVRL1, ADIPOQ, AGER, AGRN, AHR, AIMP1, AREG, BCL6, BMP1, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR2, C10orf99, C1QQNF4, C5, CCL28, CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CD109, CD36, CD4, CD40LG, CD74, CER1, CHRD, CKLF, CLCF1, CMTM1, CMTM2, CMTM3, CMTM4, CMTM5, CMTM6, CMTM7, CMTM8, CNTF, CNTFR, COPS5, CRLF1, CSF1, CSF1R, CSF2, CSF3, CSF3R, CTF1, CX3CR1, CXCL16, CXCL17, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, EBI3, EDN1, ELANE, ENG, FAM3B, FAM3C, FAM3D, FAS, FASLG, FGF2, FLT3LG, FZD4, GBP1, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF5, GDF6, GDF7, GDF9, GM- CSF, G-CSF, GPI, GREM1, GREM2, GRN, HAX1, HFE2, HMGB1, HYAL2, IFNA10, IFNA14, IFNA16, IFNA2, IFNA5, IFNA6, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNK, IFNL1, IFNL3, IFNW1, IL10RA, IL11RA, IL12A, IL12B, IL12RB1, IL17A, IL17B, IL17C, IL17D, IL17F, IL18BP, IL-19, IL1F10, IL1R1, IL1R2, IL1RAPL1, IL1RL1, IL1RN, IL20RA, IL20RB, IL21, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL25, IL26, IL27, IL2RA, IL2RB, IL2RG, IL31, IL31RA, IL32, IL33, IL34, IL36A, IL36B, IL36G, IL36RN, IL37, IL6R, IL6ST, INHA, INHBA, INHBB, INHBC, INHBE, ITGA4, ITGAV, ITGB1, ITGB3, KIT, KITLG, KLHL20, LEFTY1, LEFTY2, LIFR, LTA, LTB, LTBP1, LTBP3, LTBP4, MAF, MCP1 (CCL2), MCP-2, M-CSF, MIF, MINOS1-, MSTN, NAMPT, NBL1, NDP, NLRP7, NODAL, NOG, NRG1, NRP1, NRP2, OSMR, PARK7, PDPN, PF4, PF4V1, PGLYRP1, PLP2, PPBP, PXDN, RORC, SCG2, SCGB3A1, SECTM1, SLURP1, SOSTDC1, SP100, SPI1, SPP1, TBX21, TCAP, TGFBR1, TGFBR2, TGFBR3, THBS1, THNSL2, THPO, TIMP1, TNF, TNFRSF11, TNFRSF1A, TNFRSF9, TNFRSF10, TNFSF11, TNFSF12, TNFSF12-, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18, TNFSF4, TNFSF8, TNFSF9, TRIM16, TSLP, TWSG1, TXLNA, VASN, VEGFA, VSTM1, WFIKKN1, WFIKKN2, WNT1, WNT2, WNT5A, WNT7A, and ZFP36.

[0279] In some embodiments, UCART7 may be administered to the patient intravenously. In other embodiments, UCART7 may be administered to the patient in any form, and at any dose or dose range deemed appropriate by a clinician or practitioner. As described herein, one of skill in the art will understand that different doses or dose ranges, or different routes of administration, may be used without deviating from the scope of the disclosure.

[0280] In some embodiments, new clinically important symptoms or signs may also affect the dosage of UCART7, or any other important medical events that might increase toxicity of the immunotherapy or pre-dispose the patient to new or worsening infection (e.g., undergoing surgery, hospitalization, being treated with antibiotics, having a clinical infection, developing new clinically significant conditions). In such cases, UCART7 may be temporarily stopped as deemed appropriate or as described herein in the Examples.

Acute Lymphocytic Leukemia

[0281] The overall incidence of ALL is estimated to be 1.6 cases per 100,000 population, or approximately 5300 new patients in the United States (US) in 2019. Of these, approximately 25% will be of the T-cell lineage (T-cell acute lymphocytic leukemia [T- ALL]) and will therefore represent approximately 1300 new patients per year.

[0282] T-cell malignancies represent a class of hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond 5 years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20% to 40% of cases. T- ALL represents a genetically diverse group of diseases, but all with universal overexpression of CD7 as well as several other T-cell markers such as CD2 and CD5. Outcomes in children are significantly worse than in children with B-cell acute lymphoblastic leukemia and relapse is often associated with death in spite of many of these patients progressing to allogeneic stem cell transplant.

[0283] Allogeneic stem cell transplant, with its associated risks and toxicities, is currently the only curative therapy for relapsed T-ALL. Over 50% of adults with T-ALL will relapse after standard-of-care chemotherapy and only -20% those will achieve a second remission with the best salvage chemotherapy. Novel agents have been tried in relapsed T-ALL, none with the exception of nelarabine (complete remission [CR] rates of only 20% to 30% for relapsed T-ALL), have been US Food and Drug Administration (FDA) approved.

Lymphoma

[0284] In 2019, it was estimated that there would be 74,200 new cases of non-Hodgkin’ s lymphoma (NHL) in the US. The number of new cases of NHL is approximately 19.6 per 100,000 men and women per year, with a death rate of approximately 5.6 per 100,000 men and women per year. The data are extrapolated from age-adjusted reported data from the period 2012 through 2016.

[0285] NHLs are a heterogeneous group of lymphoproliferative disorders originating from B-cell lymphocytes, T lymphocytes, or NK cells. While CD7 may be expressed on multiple NHL subsets, of particular interest are the PTCLs, so named based on their tendency to be found in peripheral lymph nodes. This group of diseases represent <15% of NHL cases worldwide; in the US context this translates to approximately 11,000 new patients per annum. [0286] The most common subtype is called PTCL-not-otherwise specified and is most frequently diagnosed in individuals living in North America and Europe. Anaplastic large cell lymphoma is common in North America and Europe, whereas angioimmunoblastic T-cell lymphoma, the second most common subtype, is found more often in Europe. The types known as NK-/T-cell lymphoma and adult T-cell leukemia are most common in Asia. In 2016 it was estimated that there were 1660 cases of peripheral T/NK lymphoma, corresponding to an incidence rate of 0.4 per 100,000 men and women per year.

[0287] Overall, in comparison to B-cell lymphomas, T-cell lymphomas are generally associated with lower rates of chemotherapy sensitivity, higher rates of relapse, and fewer long-term remissions. Anthracycline-containing regimens, such as a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP plus etoposide are recommended as the first-line treatments for PTCL. However, low response rates and survival benefits have been seen in clinical trials. A retrospective evaluation of clinical outcomes in common PTCL subtypes reveals a 5-year survival rate of 7% to 32% with large variations dependent on PTCL subtype. Therefore, considerable research effort in recent years has been expended on the development on novel therapies for T-cell lymphoma, and treatment is moving to a personalized medicine approach with a number of targeted, immunomodulatory, or epigenetic agents either already in the clinic or in development.

Acute Myeloid Leukemia

[0288] It is estimated that there will be 21,450 newly diagnosed AML patients in the US in 2019, with 10,920 deaths. Consequently, AML is a relatively rare malignancy and is considered an orphan disease by the FDA. Further, the CD7 antigen has been detected in 15% of AML cases, and its expression correlated with FAB M1/M2 morphology. CD7 -positive (CD7+ve) disease may be anticipated in slightly over 3200 patients annually.

[0289] The current outcomes for patients with AML are well documented. Multiple international retrospective studies show that while approximately 60% to 70% of adults with AML can be expected to attain CR status following appropriate induction therapy, only 40% of patients younger than age 60 years survive more than 5 years. More than 25% of adults with AML (about 45% of those who attain CR) can be expected to survive 3 or more years and may be cured. Remission rates in adult AML are inversely related to age, with an expected remission rate of more than 65% for those younger than 60 years.

[0290] Treatment should be sufficiently aggressive to achieve CR because partial remission offers no substantial survival benefit. Few patients who relapse after achieving a CR survive more than 5 years. Successful treatment of AML requires the control of bone marrow and systemic disease and specific treatment of central nervous system (CNS) disease, if present. The cornerstone of this strategy includes systemically administered combination chemotherapy. Because only 5% of patients with AML develop CNS disease, prophylactic treatment is not indicated.

[0291] A theoretical key advantage of non-autologous derived CAR-T therapies is the avoidance of delay in treating patients who have demonstrated progressive disease (PD). In the KYMRIAH® (Tisagenlecleucel) Biologies License Application (BLA), 29 deaths were reported between the time of informed consent to the final end-of-study data analysis. Of these deaths, 12 (41.4%) patients succumbed to their disease pre-infusion while awaiting manufacture of KYMRIAH®. The availability of an effective “off-the-shelf’ therapy has the potential to avoid or reduce the number of early patient deaths otherwise likely due to the time incurred to process autologous CAR-T cells. [0292] Both CD7+ve ALL and AML represent significant unmet medical needs, especially for those patients who relapse where no effective or targeted therapies currently exist. The availability of off-the-shelf CAR-T therapy may be of benefit and reduce early disease mortality associated with manufacturing delays of autologous CAR-T therapies.

CART -Mediated Inflammation

[0293] UCART7-mediated inflammation in normal tissues will be a function of the number of cells in that tissue expressing CD7 and the density of expression on those cells. It is anticipated that toxicity may be prolonged in these tissues due to the potential persistence of the UCART7 cell clone. Since CD7 expression is not a characteristic of normal tissues, it is anticipated that toxicity, if it occurs, will be limited to tissues with large numbers of CD7+ve T cells. Tissues impacted are all part of the reticuloendothelial system, and will include bone marrow, spleen, lymph nodes, and the tonsils. Overall, the potential for clinical toxicity driven by UCART7 persistence and tissue penetration is considered to be low.

Methods of Treating T-Cell Malignancies using UCART7

[0294] In some embodiments, the present disclosure provides methods of administration of UCART7 immune effector cells to a patient having a T-cell malignancy. As would be understood by one of the art and described in detail herein, a method useful for treating such a patient may be any method appropriate for use with UCART7 cells as described herein and in the Examples. For example, in non-limiting examples, a method of the present disclosure that may be appropriate for treatment of one or more T-cell malignancies in a patient may comprise administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

[0295] In another embodiment, the disclosure provides a method of achieving complete remission of one or more T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0296] In another embodiment, the disclosure provides a method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells.

[0297] In another embodiment, the disclosure provides a method of treatment of T-cell malignancies in a patient comprising administration of a dose (including a low dose) of UCART7 cells, wherein the levels of interleukin (IL)-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)γ, and/or fracktalkine are not elevated to a level that would correspond with or result in grade 3 or 4 CRS as a result of treatment.

[0298] In another embodiment, the disclosure provides a method of achieving an overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0299] In another embodiment, the disclosure provides a method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0300] In another embodiment, the disclosure provides a method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a dose (including a low dose) of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

[0301] In another embodiment, the disclosure provides a method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a dose (including a low dose) of UCART7 cells, wherein the dose (including a low dose) of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome.

[0302] In another embodiment, the disclosure provides a method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose (including a low dose) of UCART7 cells to the patient; and evaluating CRS symptoms in the patient.

[0303] In another embodiment, the disclosure provides a method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose (including a low dose) of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, continuing treatment with UCART7 cells. T Cell Depletion, Suppression, or Aplasia

[0304] Another toxicity occurring as a result of UCART7 administration may be the effect of target cell depletion. Existing literature provides conflicting information pertaining to the earliest documentation of CD7 expression on marrow progenitor cells. As CD7 is expressed on both T cells and NK cells and their precursors, it is anticipated that the normal lymphoid lines expressing CD7 will be depleted rapidly following the administration of UCART7. The persistence of the UCART7 clone, intended to have an ongoing effect against the malignant cells, may also lead to a prolonged reduction of aplasia of these cell lines.

[0305] However, there are 2 factors that may mitigate this toxicity:

[0306] (1) CD7 is not expressed by all peripheral T cells and therefore a CD7-ve subset may be spared, limiting the extent of any potential T-cell aplasia and potentially maintaining some level of immune defense in treated patients

[0307] (2) Lymphoid stem cells do not express CD7. Repopulation of normal cells is anticipated to occur over time, once the UCART7 clone has dissipated

[0308] One risk associated with CD7 targeted T-cell therapy is the prolonged suppression or destruction of T-cell precursors in the bone marrow, rendering normal recovery delayed or absent. Significant suppression of T cells may predispose the patient to opportunistic infections and to the reactivation of past viral infections such as cytomegalovirus (CMV), herpes simplex, and other viruses. The protocol includes mitigation strategies and clinical guidance in the event this toxicity is documented. Because prolonged aplasia may be associated with dosage of CAR-T cells, or supplementation of CAR-T cells with, for example, an enhancer molecule that may increase persistence of CAR-T cells over time (e.g., IL-7, IL-15, IL-2, analogs thereof, fusion proteins thereof, etc.), a dose (including a low dose) of UCART7 treatment disclosed herein is expected to mitigate aplasia.

Pharmacokinetic (PK) Analysis

[0309] In some embodiments, patients or subjects of the present disclosure may have blood samples taken for PK analysis during treatment with UCART7. As used herein, a cellular PK parameter (cPK parameter) may include, but is not limited to, AUC(0-∞ ), AUC(0-Ƭ ), and maximum drug concentration in body after dosing [C_max]. In some embodiments, cPK blood samples may be taken at specific timepoints as described herein. Reference may be made to particular days, which are counted from the day of UCART7 administration. One of skill in the art will understand that other parameters may be taken at any time point and at the discretion of the study director. CAR-Bearing Immune Effector Cells

[0310] A chimeric antigen receptor (CAR) is a recombinant fusion protein comprising: 1) an extracellular ligand-binding domain, i.e., an antigen-recognition domain, 2) a transmembrane domain, and 3) a signaling transducing domain.

[0311] Methods for CAR design, delivery and expression, and the manufacturing of clinical-grade CAR-T cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res., 2012, 18(10): 2780-90. An engineered chimeric antigen receptor polynucleotide that encodes for a CAR comprises: a signal peptide, an antigen recognition domain, at least one co- stimulatory domain, and a signaling domain.

[0312] The antigen- specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. An “antigen-specific extracellular domain” (or, equivalently, “antigen-binding domain”) specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 μM, preferably about 0.1 pM to about 1 μM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An antigen-specific extracellular domain suitable for use in a CAR of the present disclosure may be any antigen- binding polypeptide, a wide variety of which are known in the art. In some instances, the antigen-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains) and “camelized” antibody variable domains are suitable for use. In some instances, T-cell receptor (TCR) based recognition domains such as single chain TCR (scTv, single chain two-domain TCR containing VαVβ ) are also suitable for use.

[0313] A chimeric antigen receptor of the present disclosure also comprises an “intracellular domain” that provides an intracellular signal to the CAR-bearing immune effector cell upon antigen binding to the antigen-specific extracellular domain. The intracellular signaling domain of a chimeric antigen receptor of the present disclosure is responsible for activation of at least one of the effector functions of the immune effector cell in which the chimeric receptor is expressed. The term “effector function” refers to a specialized function of a differentiated cell, such as a T cell, NK cell, or iNKT cell. Thus, the term “intracellular domain” refers to the portion of a CAR that transduces the effector function signal upon binding of an antigen to the extracellular domain and directs the immune effector cell to perform a specialized function. Non-limiting examples of suitable intracellular domains include the zeta chain of the T-cell receptor or any of its homologs (e.g., eta, delta, gamma, or epsilon), MB 1 chain, 829, Fe Rill, Fe Rl, and combinations of signaling molecules, such as CD3ζ and CD28, CD27, 4-1 BB, DAP-1 0, 0X40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the families of activating proteins may be used, such as FcγRIII and FcεRI. While usually the entire intracellular domain will be employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0314] Typically, the antigen- specific extracellular domain is linked to the intracellular domain of the chimeric antigen receptor by a “transmembrane domain.” A transmembrane domain traverses the cell membrane, anchors the CAR to the T cell surface, and connects the extracellular domain to the intracellular signaling domain, thus impacting expression of the CAR on the T cell surface. Chimeric antigen receptors may also further comprise one or more costimulatory domain and/or one or more spacer. A “costimulatory domain” is derived from the intracellular signaling domains of costimulatory proteins that enhance cytokine production, proliferation, cytotoxicity, and/or persistence in vivo. A “peptide hinge” connects the antigen- specific extracellular domain to the transmembrane domain. The transmembrane domain is fused to the costimulatory domain, optionally a costimulatory domain is fused to a second costimulatory domain, and the costimulatory domain is fused to a signaling domain, not limited to CD3ζ. For example, inclusion of a spacer domain between the antigen-specific extracellular domain and the transmembrane domain, or between multiple scFvs, may affect flexibility of the antigen-binding domain(s) and thereby CAR function. Suitable transmembrane domains, costimulatory domains, and spacers are known in the art. For example, costimulatory domains useful for a CAR-T cell as described herein include, but is not limited to a CD28 costimulatory domain and a 4- IBB costimulatory domain. In some embodiments, a CAR-T cell as disclosed herein may have more than one costimulatory domain, for example a CD28 costimulatory domain and a 4- IBB costimulatory domain. CD3-zeta is an example of an effector domain. In some embodiments, a CAR-T cell as disclosed herein may have a CD28 costimulatory domain and/or a 4- IBB costimulatory domain, and a CD3-zeta effector domain. [0315] Engineered CARs may be introduced into CAR-bearing immune effector cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome. Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR/Cas systems (e.g., type I, type II, or type Ill systems using a suitable Cas protein such as Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl Od, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3,Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, etc.). In some embodiments, newer classes of CRISPR that result in base changes may also be used for gene editing as described herein. Base editors are known and available in the art. For example, in some embodiments, cytidine base editors (e.g., BE4) that catalyze cytidine to thymidine at specific genetic loci, or adenine base editors (e.g., ABE7.10) may be used to introduce specific targeted single-nucleotide changes in nucleic acids. In some embodiments, C-to-T base editors (CBE), A-to-G base editors (ABE), the REPAIR™ system for A-to-I editing, the RESCUE™ system for C-to-U editing, or any other gene editing systems that may be useful as described herein. Other nucleases related to or similar to the CRISPR/Cas proteins described herein may also be used, such as including MAD7 nuclease. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 Jan;20(l):l-10.

[0316] Manipulation of PI3K signaling can be used to prevent altered CAR-T cell differentiation due to constitutive CAR self-signaling and foster long-lived memory T cell development. Pharmacologic blockade of PI3K during CAR-T manufacture and ex vivo expansion can abrogate preferential effector T cell development and restore CAR-T effector/memory ratio to that observed in empty vector transduced T cells, which can improve in vivo T cell persistence and therapeutic activity. Inhibition of pl 105 PI3K can enhance efficacy and memory in tumor- specific therapeutic CD 8 T cells, while inhibition of pl 10a PI3K can increase cytokine production and antitumor response.

[0317] This is proposed to be because the presence of a CAR on a T cell’s surface can alter its activation and differentiation, even in the absence of ligand. Constitutive self- signaling through CAR, related to both the scFv framework and the signaling domains, can lead to aberrant T cell behavior, including altered differentiation and decreased survival. This is significant as the effectiveness of CAR-T cells in patients is directly associated with their in vivo longevity. The presence of the CD28 costimulatory domain increased CAR-T cell exhaustion induced by persistent CAR self-signaling; the 4- IBB costimulatory domain had a lesser effect. Furthermore, CD3-zeta significantly enhances the constitutive activation of the PI3K, AKT, mTOR, and glycolysis pathways, and fostered formation of short-lived effector cells over central/stem memory cells. See, e.g., Zhang et al., “Modulation of PI3K signaling to improve CAR T cell function,” Oncotarget, 2018 Nov 9; 9(88): 35807-35808.

[0318] CAR Antigens. Suitable antigens to be genome-edited in the immune effector cells disclosed herein, and to be recognized by the CARs of CAR-bearing immune effector cells disclosed herein, include antigens specific to hematologic malignancies. These can include T cell- specific antigens and/or antigens that are not specific to T cells. The antigen may be specifically bound by the chimeric antigen receptor of a CAR-bearing immune effector cell, and the antigen for which the CAR-bearing immune effector cell is deficient, is an antigen expressed on a malignant T cell, preferably an antigen that is overexpressed on malignant T cell (i.e., a T cell derived from a T-cell malignancy) in comparison to a nonmalignant T cell. In certain embodiments, the antigen is CD7.

[0319] Fratricide Resistance. The CAR-T, iNKT, NK and other CAR-bearing immune effector cells encompassed by the present disclosure are optionally deficient in one or more antigens to which the chimeric antigen receptor specifically binds and are therefore fratricide- resistant. In some embodiments, the one or more antigens of the cell is modified such the chimeric antigen receptor no longer specifically binds the one or more modified antigens. For example, the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen. In other embodiments, expression of the one or more antigens is reduced in the cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more. Methods for decreasing the expression of a protein are known in the art and include, but are not limited to, modifying or replacing the promoter operably linked to the nucleic acid sequence encoding the protein. In still other embodiments, the cell is modified such that the one or more antigens is not expressed, e.g., by deletion or disruption of the gene encoding the one or more antigens. In each of the above embodiments, the CAR- bearing immune effector cell may be deficient in one or preferably all the antigens to which the chimeric antigen receptor specifically binds. Methods for genetically modifying a cell to be deficient in one or more antigens are well known in art, and non- limiting examples are provided above. In an exemplary embodiment, CRISPR/cas9 gene editing can be used to modify a cell to be deficient in one or more antigens. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) may also be used. See, e.g., Shearer RF and Saunders DN, “Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives,” Genes Cells 2015 Jan;20(l):l-10.

[0320] Avoidance of Allogenicity. CAR-T, iNKT, NK and other CAR-bearing immune effector cells encompassed by the present disclosure may further be deficient in endogenous T cell receptor (TCR) signaling as a result of deleting one or more parts of the T Cell Receptor (TCR)-CD3 complex. In various embodiments it may be desirable to eliminate or suppress endogenous TCR signaling in CAR-bearing immune effector cells disclosed herein. For example, decreasing or eliminating endogenous TCR signaling in CAR-T cells may prevent or reduce graft versus host disease (GvHD) when allogenic T cells are used to produce the CAR-T cells. Methods for eliminating or suppressing endogenous TCR signaling are known in the art and include, but are not limited to, deleting a part of the TCR-CD3 receptor complex, e.g., the TCR receptor alpha chain (TCRα or TRAC), the TCR receptor beta chain (TCRβ or TRBC), CD3ε CD3γ CD35, and/or CD3ζ. Deleting a part of the TCR receptor complex may block TCR-mediated signaling and may thus permit the safe use of allogeneic T cells as the source of CAR-T cells without inducing life-threatening GvHD.

[0321] Suicide Genes. Alternatively, or in addition, CAR-bearing immune effector cells encompassed by the present disclosure may further comprise one or more suicide genes. As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a cell by standard methods known in the art that, when activated, results in the death of the cell. Suicide genes may facilitate effective tracking and elimination of the CAR-bearing immune effector cells in vivo if required. Facilitated killing by activating the suicide gene may occur by methods known in the art. Suitable suicide gene therapy systems known in the art include, but are not limited to, various the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy systems or inducible caspase 9 protein. In an exemplary embodiment, a suicide gene is a CD34/thymidine kinase chimeric suicide gene.

[0322] Components that may be included in a CAR as described herein are provided below in Tables 1 and 2.

Table 1. Amino acid sequences of different CAR components.

Table 2. Amino acid sequences of variable heavy (V_H) and light (V_L) chains of the scFvs.

CAR-T cells

[0323] In certain embodiments, the disclosure provides an engineered T cell comprising a single CAR, that specifically binds an antigen or cell surface protein, wherein the T cell is optionally deficient in that antigen or cell surface protein (e.g., CD7CARTΔCD7 cell). In non-limiting examples, the deficiency in the antigen or cell surface protein resulted from (a) modification of antigen or cell surface protein expressed by the T cell such that the chimeric antigen receptors no longer specifically binds the modified antigen or cell surface protein (e.g., the epitope of the one or more antigens recognized by the chimeric antigen receptor may be modified by one or more amino acid changes (e.g., substitutions or deletions) or the epitope may be deleted from the antigen), (b) modification of the T cell such that expression of antigen or cell surface protein is reduced in the T cell by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, or (c) modification of the T cell such that antigen or cell surface protein is not expressed (e.g., by deletion or disruption of the gene encoding antigen or cell surface protein). In each of the above embodiments, the CAR-T cell may be deficient in one or preferably all the antigens or cell surface proteins to which the chimeric antigen receptor specifically binds. The methods to genetically modify a T cell to be deficient in one or more antigens or cell surface proteins are well known in art and non-limiting examples are provided herein. In embodiments described below, the CRISPR/Cas9 system is used to modify a T cell to be deficient in one or more antigens. Any of these may be accomplished by the methods disclosed herein. In further embodiments, the T cell comprises a suicide gene.

[0324] For example, the CAR for a CD7-specific CAR-T cell may be generated by cloning a commercially synthesized anti-CD7 single chain variable fragment (scFv) into a CAR backbone with CD28 and/or 4- IBB internal signaling or costimulatory domains. In some embodiments, a CD3ζ intracellular signaling domain may also be included. An extracellular hCD34 domain may be added after a P2A peptide to enable both detection of CAR following viral transduction and purification using anti-hCD34 magnetic beads. A similar method may be followed for making CARs specific for other malignant T cell antigens.

[0325] CAR-T cells encompassed by the present disclosure may further be deficient in endogenous T cell receptor (TCR) signaling as a result of deleting a part of the T Cell Receptor (TCR)-CD3 complex. In various embodiments it may be desirable to eliminate or suppress endogenous TCR signaling in CAR-T cells disclosed herein. For example, decreasing or eliminating endogenous TCR signaling in CAR-T cells may prevent or reduce graft versus host disease (GvHD) when allogenic T cells are used to produce the CAR-T cells. Methods for eliminating or suppressing endogenous TCR signaling are known in the art and include, but are not limited to, deleting a part of the TCR-CD3 receptor complex, e.g., the TCR receptor alpha chain (TCRα or TRAC), the TCR receptor beta chain (TCRβ ) or subtypes thereof, TCR5, TCRγ, CD3∈, CD3γ, and/or CD3δ. Deleting a part of the TCR receptor complex may block TCR-mediated signaling and may thus permit the safe use of allogeneic T cells as the source of CAR-T cells without inducing life-threatening GvHD.

[0326] In addition, the CAR-T cells encompassed by the present disclosure may further comprise one or more suicide genes as described herein.

[0327] In a similar manner, other CAR-T cells may be constructed.

[0328] Disclosed are embodiments of CAR amino acid sequences that can be expressed on the surface of a genome-edited CAR-T cell derived from a cytotoxic T cell, a memory T cell, or a gamma delta (γδ) T cell. Table 3. Amino Acid Sequences of Chimeric Antigen Receptors (CARs).

Indications and Standards of Care in CAR-T Therapy

[0329] In some embodiment, the genome-edited immune effector cells and compositions disclosed herein comprising these cells, and/or generated using the methods disclosed herein, express one or more chimeric antigen receptors (CARs) and can be used as a medicament, i.e., for the treatment of disease. In many embodiments, the cells are CAR-T cells.

[0330] Cells disclosed herein, and/or generated using the methods disclosed herein, may be used in immunotherapy and adoptive cell transfer, for the treatment, or the manufacture of a medicament for treatment, of cancers, autoimmune diseases, infectious diseases, and other conditions.

[0331] A cancer that may be treated using the CAR-T cells described herein may be a hematologic malignancy or solid tumor. Hematologic malignancies include leukemias, lymphomas, multiple myeloma, and subtypes thereof. Lymphomas can be classified in various ways, often based on the underlying type of malignant cell, including Hodgkin’s lymphoma (often cancers of Reed-Sternberg cells, but also sometimes originating in B cells; all other lymphomas are non- Hodgkin’ s lymphomas), B-cell lymphomas, T-cell lymphomas, mantle cell lymphomas, Burkitt’s lymphoma, follicular lymphoma, and others as defined herein and known in the art.

[0332] B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and others as defined herein and known in the art.

[0333] T-cell lymphomas include T-cell acute lymphoblastic leukemia/lymphoma (T- ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and others as defined herein and known in the art. [0334] Leukemias include acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL) hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.

[0335] Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0336] In some embodiments, the medicament can be used for treating cancer in a patient, particularly for the treatment of solid tumors such as melanomas, neuroblastomas, gliomas or carcinomas such as tumors of the brain, head and neck, breast, lung (e.g., non small cell lung cancer, NSCLC), reproductive tract (e.g., ovary), upper digestive tract, pancreas, liver, renal system (e.g., kidneys), bladder, prostate and colorectum.

[0337] In another embodiment, the medicament can be used for treating cancer in a patient, particularly for the treatment of hematologic malignancies selected from multiple myeloma and acute myeloid leukemia (AML) and for T-cell malignancies selected from T- cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin’ s lymphoma, and T-cell chronic lymphocytic leukemia (T-CLL).

[0338] In some embodiments, the cells may be used in the treatment of autoimmune diseases such as lupus, autoimmune (rheumatoid) arthritis, multiple sclerosis, transplant rejection, Crohn’s disease, ulcerative colitis, dermatitis, and the like. In some embodiments, the cells are chimeric autoantibody receptor T-cells, or CAAR-Ts displaying antigens or fragments thereof, instead of antibody fragments; in this version of adoptive cell transfer, the B cells that cause autoimmune diseases will attempt to attack the engineered T cells, which will respond by killing them.

[0339] In some embodiments, the cells may be used in the treatment of infectious diseases such as HIV and tuberculosis.

[0340] In another embodiment, the CAR-T cells of the present disclosure can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

[0341] In some embodiments, the treatment of a patient with CAR-T cells of the present disclosure can be ameliorating, curative, preventative, or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic or allogeneic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic, is meant that the cells or population of cells used for treating patients are not originating from the patient but from a donor. [0342] The treatment of cancer with CAR-T cells of the present disclosure may be in combination with one or more therapies selected from antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, radiotherapy, laser light therapy, and radiation therapy.

[0343] The administration of CAR-T cells or a population of CAR-T cells of the present disclosure of the present disclosure be carried out by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The CAR-T cell compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present disclosure are preferably administered by intravenous injection.

[0344] The administration of CAR-T cells or a population of CAR-T cells can consist of the administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight including all integer values of cell numbers within those ranges. The CAR-T cells or a population of CAR-T cells can be administrated in one or more doses. In another embodiment, the effective amount of CAR-T cells or a population of CAR-T cells are administrated as a single dose. In another embodiment, the effective amount of cells are administered as more than one dose over a period time. Timing of administration is within the judgment of a health care provider and depends on the clinical condition of the patient. The CAR-T cells or a population of CAR-T cells may be obtained from any source, such as a blood bank or a donor. While the needs of a patient vary, determination of optimal ranges of effective amounts of a given CAR-T cell population(s) for a particular disease or conditions are within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health and weight of the patient recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0345] In another embodiment, the effective amount of CAR-T cells or a population of CAR-T cells or composition comprising those CAR-T cells are administered parenterally. The administration can be an intravenous administration. The administration of CAR-T cells or a population of CAR-T cells or composition comprising those CAR-T cells can be directly done by injection within a tumor.

[0346] In one embodiment of the present disclosure, the CAR-T cells or a population of the CAR-T cells are administered to a patient in conjunction with, e.g., before, simultaneously or following, any number of relevant treatment modalities, including but not limited to, treatment with cytokines, or expression of cytokines from within the CAR-T, that enhance T-cell proliferation and persistence and, include but not limited to, IL-2, IL-7, and IL-15.

[0347] In a second embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with agents that inhibit immunosuppressive pathways, including but not limited to, inhibitors of TGF-β , interleukin 10 (IL- 10), adenosine, VEGF, indoleamine 2,3 dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), tryptophan 2-3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2.

[0348] In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with T-cell checkpoint inhibitors, including but not limited to, anti-CTLA4 (Ipilimumab) anti-PDl (Pembrolizumab, Nivolumab, Cemiplimab), anti-PDLl (Atezolizumab, Avelumab, Durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.

[0349] In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with T cell agonists, including but not limited to, antibodies that stimulate CD28, ICOS, OX-40, CD27, 4-1BB, CD137, GITR, and HVEM [0350] In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with therapeutic oncolytic viruses, including but not limited to, retroviruses, picornaviruses, rhabdo viruses, paramyxoviruses, reoviruses, parvoviruses, adenoviruses, herpesviruses, and poxviruses.

[0351] In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with immunostimulatory therapies, such as toll-like receptors agonists, including but not limited to, TLR3, TLR4, TLR7 and TLR9 agonists.

[0352] In another embodiment, the CAR-T cells or a population of CAR-T cells of the present disclosure may be used in combination with stimulator of interferon gene (STING) agonists, such as cyclic GMP-AMP synthase (cGAS).

[0353] Immune effector cell aplasia, particularly T cell aplasia is also a concern after adoptive cell transfer therapy. When the malignancy treated is a T-cell malignancy, and CAR-T cells target a T cell antigen, normal T cells and their precursors expressing the antigen will become depleted, and the immune system will be compromised. Accordingly, methods for managing these side effects are attendant to therapy. Such methods include selecting and retaining non- malignant T cells or precursors, either autologous or allogeneic (optionally engineered not to cause rejection or be rejected), for later expansion and re- infusion into the patient, after CAR-T cells are exhausted or deactivated. Alternatively, CAR- T cells which recognize and kill subsets of TCR-bearing cells, such as normal and malignant TRBC1⁺, but not TRBC2⁺cells, or alternatively, TRBC2⁺, but not TRBCl⁺cells, may be used to eradicate a T cell malignancy while preserving sufficient normal T cells to maintain normal immune system function.

Definitions

[0354] As used herein, the terms below have the meanings indicated. Other definitions may occur throughout the specification.

[0355] When ranges of values are disclosed, and the notation “from n₁... to n₂” or “between n₁ ... and n₂” is used, where n₁ and n₂ are the numbers, then unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integral or continuous between and including the end values. By way of example, the range “from 2 to 6 carbons” is intended to include two, three, four, five, and six carbons, since carbons come in integer units. Compare, by way of example, the range “from 1 to 3 μM (micromolar),” which is intended to include 1 μM, 3 μM, and everything in between to any number of significant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

[0356] The term “about,” as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure as well, taking into account significant figures.

[0357] The term “activation” (and other conjugations thereof) in reference to cells is generally understood to be synonymous with “stimulating” and as used herein refers to treatment of cells that results in expansion of cell populations. In T cells, activation is often accomplished by exposure to CD2 and CD28 (and sometimes CD2 as well) agonists, typically antibodies, optionally coated onto magnetic beads or conjugated to a colloidal polymeric matrix.

[0358] As used herein, an “adverse event” or “AE” refers to any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related. An AE (also known as adverse experience) can be any unfavorable and unintended sign (e.g., an abnormal laboratory finding), symptom, or disease temporarily associated with the use of a drug, without any judgement about causality. An AE can arise with any use of the drug (e.g., off-label use, use in combination with another drug) and with any route of administration, formulation, dose or including overdose. As used herein, “serious adverse event” or “SAE” refers to an AE or a “suspected adverse reaction” (SAR) that is considered “serious” if it results in any of the following outcomes: Death; a life-threatening AE; inpatient hospitalization of at least 24-hours or prolongation of existing hospitalization; a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; or a congenital anomaly /birth defect. Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered serious when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in hospitalization, or development of drug dependency or drug abuse.

[0359] As used herein, an “adverse reaction” or “AR” refers to any AE caused by a drug. Adverse reactions are a subset of all SARs where there is a reason to conclude that the drug caused the event. As used herein, a SAR refers to any AE for which there is a reasonable possibility that the drug caused the AE. Reasonable possibility means that there is evidence to suggest a causal relationship between the drug and the AE. A SAR implies a lesser degree of certainty about causality than adverse reaction, which means any AE caused by a drug.

[0360] As described herein, “serious” and “severe” AE are not synonymous. Severity is a measure of intensity; however, an AE of severe intensity need not necessarily be considered serious. Seriousness serves as the guide for defining regulatory reporting obligations. “Serious” is a regulatory definition and is based on patient/event outcome or action usually associated with events that pose a threat to a patient’s life or vital functions. For example, nausea that persists for several hours may be considered severe nausea, but may not be considered an SAE. On the other hand, a stroke which results in only a limited degree of disability may be considered only a mild stroke, but would be considered an SAE. Severity and seriousness should be independently assessed when recording AEs on the eCRF screen and SAEs on the SAE Report Form.

[0361] The term "antigen" refers to a molecular entity that may be soluble or cell membrane bound in particular but not restricted to molecular entities that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to transgenic TCRs, chimeric antigen receptors (CARs), scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity. [0362] The term "cancer" is known medically as a malignant neoplasm. Cancer is a broad group of diseases involving upregulated cell growth. In cancer, cells (cancerous cells) divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream. There are over 200 different known cancers that affect humans. As used herein, cancer may refer to a hematologic malignancy, such as multiple myeloma, a T-cell malignancy, or a B cell malignancy. T cell malignancies may include, but are not limited to, T-cell acute lymphoblastic leukemia (T-ALL) or non- Hodgkin’s lymphoma. A cancer may also refer to a solid tumor, such as including, but not limited to, cervical cancer, pancreatic cancer, ovarian cancer, mesothelioma, and lung cancer. [0363] The term "chimeric antigen receptor," abbreviated "CAR," refers to engineered receptors, which graft an antigen specificity onto cells, for example T or NK cells. The CARs disclosed herein comprise an antigen binding domain also known as antigen targeting region (typically a single chain variable region comprised of antibody heavy and light chain variable regions), an extracellular spacer/linker domain or hinge region, a transmembrane domain and at least one intracellular signaling domain; it may optionally comprise other elements, such as at least one co- stimulatory domain. The extracellular domain may also comprise a signal peptide. Upon binding of the antigen- specific region to the corresponding antigen, the signaling domain mediates an effector cell function in the host cell. For example, a CAR can have an antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cellular immune activity. First-generation CARs include an extracellular ligand-binding domain and signaling transducing domain, commonly CD3ζ or FcεRIγ. Second generation CARs are built upon first generation CAR constructs by including an intracellular costimulatory domain, commonly 4- IBB or CD28. These costimulatory domains help enhance CAR-T cell cytotoxicity and proliferation compared to first generation CARs. The third generation CARs include multiple costimulatory domains, primarily to increase CAR-T cell proliferation and persistence. Chimeric antigen receptors are distinguished from other antigen binding agents by their ability both to bind MHC-independent antigens and transduce activation signals via their intracellular domain. In some embodiments, a CAR-T cell of the present disclosure may be transduced with one or more of a second generation CAR or a third generation CAR as described herein.

[0364] As used herein, a “CAR-bearing immune effector cell” is an immune effector cell which has been transduced with at least one CAR (e.g., a nucleotide sequence encoding the CAR) and/or expressing a CAR (e.g., comprising the amino acid sequence of the CAR). A “CAR-T cell” is a T cell which has been transduced with at least one CAR; similarly, the terms “CAR-iNKT cell” (equivalently, iNKT-CAR) and “CAR-NK cell” mean, respectively, an iNKT or NK cell that has been transduced with at least one CAR. CAR-T (and CAR- iNKT, and CAR-NK) cells can be mono (i.e., having a single CAR), or even dual or tandem CAR-T (or CAR-iNKT, or CAR-NK) cells, which refer to immune effector cells having more than one CAR. CAR-bearing immune effector cell can be autologous, meaning that they are engineered from a subject’s own cells, or allogeneic, meaning that the cells are sourced from a healthy donor, and in many cases, engineered so as not to provoke a host-vs-graft or graft- vs-host reaction. Donor cells may also be sourced from cord blood or generated from induced pluripotent stem cells. The CAR(s) may be expressed from a single or multiple polynucleotide sequences. In some embodiments, a “CAR-bearing immune effector cell” may be referred to interchangeably as a “CAR-positive viable T-cell.”

[0365] As used herein, “Cellular pharmacokinetic Parameters” or “cPK parameters” or “cPK analyses” refer to specific values calculated from plasma concentrations of UCART7 in each patient. Blood samples obtained from each patient will be used to calculate the cPK parameters, which include, but are not limited to, concentration-time profiles, AUCiast, AUC_inf, AUC_{(0-∞ )}, AUC_(0-Ƭ), Tmax, and maximum drug concentration in body after dosing [C_max]. These parameters will be listed by individual patient and summarized by descriptive statistics (means, medians, ranges, standard deviations, and coefficient of variation as appropriate, by treatment group/cohort).

[0366]

[0367] A “cell surface protein” as used herein is a protein (or protein complex) expressed by a cell at least in part on the surface of the cell. Examples of cell surface proteins include the TCR (and subunits thereof) and CD7.

[0368] The term “combination therapy” means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein. The term "combination therapy" may also refer to the concerted application of an immunotherapy such as the treatment with an antigen recognizing receptor and another therapy such as the transplantation of hematopoietic cells e.g. hematopoietic cells resistant to recognition by the antigen recognizing receptor. Expression of an antigen on a cell means that the antigen is sufficient present on the cell surface of the cell, so that it can be detected, bound and/or recognized by an antigen- recognizing receptor.

[0369] The term “composition” as used herein refers to an immunotherapeutic cell population combination with one or more therapeutically acceptable carriers.

[0370] As used herein, a chimeric antigen receptor natural killer (NK) cell (equivalently, NK-CAR) would have a meaning analogous to the definitions of CAR-T and iNKT-CAR. [0371] As used herein, a chimeric antigen receptor macrophage (equivalently, CAR- macrophage) would have a meaning analogous to the definitions of CAR-T, iNKT-CAR, and NK-CAR.

[0372] As used herein, the term “CAR-T associated neuropathy” means neuropathy that arises subsequent to administration of CAR-T therapy to a patient, often after intervening cytokine release syndrome has occurred and subsided. The term is relatively new, mainly because CAR-T therapy is relatively new; see, e.g., Vasthie P and Breitbart WS, “Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia,” Palliat Support Care. 2017 Aug; 15(4): 499-503. Accordingly, CAR-T associated neuropathy should be understood at this time to be equivalent to the term “CAR-bearing immune effector cell associated neuropathy,” since similar neuropathy could arise from therapy with, e.g., iNKT- CARs or NK-CARs.

[0373] As used herein, a “cytokine” is one of a class of small (-5-20 kDa), soluble signaling proteins that are that are synthesized and secreted by certain cells of the immune system at variable, and occasionally locally high, concentrations and by binding to receptors on other cells, send signals to and have an effect on those cells. A “chemokine” is a chemotactic cytokine, i.e., a subspecies of cytokine that is able to induce chemotaxis in nearby responsive cells.

[0374] As used herein, to be “deficient” in a cytokine or protein means to lack sufficient quantity of the cytokine or protein for the cytokine or protein to elicit its normal effect. A cell that is “deficient” in GM-CSF, for example, (a “GM-CSF deficient” cell) could be entirely lacking in GM-CSF, but it also could express such a negligible quantity of GM-CSF that the GM-CSF present could not contribute in any meaningful way to the development or maintenance of cytokine release syndrome.

[0375] The term “deletion” as used herein in reference to the effect of editing on a gene or its protein product, means alteration or loss of part the sequence of DNA encoding the protein so as to reduce or prevent expression of the protein product. The term “suppression” in the same context means to reduce expression of the protein product; and the term “ablation” in the same context means to knock out (KO) or prevent expression of the protein product. Deletion encompasses suppression and ablation.

[0376] The term “disease” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

[0377] A "diseased cell" refers to the state of a cell, tissue or organism that diverges from the normal or healthy state and may result from the influence of a pathogen, a toxic substance, irradiation, or cell internal deregulation. A “diseased cell” may also refer to a cell that has been infected with a pathogenic virus. Further the term “diseased cell” may refer to a malignant cell or neoplastic cell that may constitute or give rise to cancer in an individual.

[0378] The term “donor template” refers to the reference genomic material that the cell uses as a template to repair a double- stranded break through the homology-directed repair (HDR) DNA repair pathway. The donor template contains the piece of DNA to be inserted into the genome (containing the gene to be expressed, CAR, or marker) with two homology arms flanking the site of the double- stranded break. In some embodiments, a donor template may be an adeno-associated virus, a single-stranded DNA, or a double-stranded DNA.

[0379] The terms "engineered cell" and "genetically modified cell" as used herein can be used interchangeably. The terms mean containing and/or expressing a foreign gene or nucleic acid sequence, or containing a gene which has been genetically modified to deviate from its natural form or function (for example a deleted or knocked-out gene) which in turn modifies the genotype or phenotype of the cell or its progeny. Cells can be modified by recombinant methods well known in the art to express stably or transiently peptides or proteins, which are not expressed in these cells in the natural state. Methods of genetic modification of cells may include but is not restricted to transfection, electroporation, nucleofection, transduction using retroviral vectors, lentiviral vectors, non-integrating retro- or lentiviral vectors, transposons, designer nucleases including zinc finger nucleases, TALENs or CRISPR/Cas.

The term “exposing to,” as used herein, in the context of bringing compositions of matter (such as antibodies) into intimate contact with other compositions of matter (such as cells), is intended to be synonymous with “incubated with,” and no lengthier period of time in contact is intended by the use of one term instead of the other.

[0380] The term “fold selective,” as used herein, means having an affinity for one target that is at least x-fold greater than its affinity for another target, wherein x is at least 2, and may be higher, e.g., 10, 20, 50, 100, or 1000. In preferred embodiments, the fold selectivity is therapeutically meaningful, i.e., sufficient to permit cells expressing one target to be killed and cells bearing the other target to be spared.

[0381] The term “fratricide” as used herein means a process which occurs when a CAR-T cell (or other CAR-bearing immune effector cell) becomes the target of, and is killed by, another CAR-T cell comprising the same chimeric antigen receptor as the target of CAR-T cell, because the targeted cell expresses the antigen specifically recognized by the chimeric antigen receptor on both cells. CAR-T comprising a chimeric antigen receptor which are deficient in an antigen to which the chimeric antigen receptor specifically binds will be “fratricide-resistant.”

[0382] The term "genetic modification" or genetically modified" refers to the alteration of the nucleic acid content including but not restricted to the genomic DNA of a cell. This includes but is not restricted to the alteration of a cells genomic DNA sequence by introduction exchange or deletion of single nucleotides or fragments of nucleic acid sequence. The term also refers to any introduction of nucleic acid into a cell independent of whether that leads to a direct or indirect alteration of the cells genomic DNA sequence or not.

[0383] The term “genome-edited” or “gene-edited” as used herein means having a gene or potion of the genome added, deleted, or modified (e.g., disrupted) to be non-functional. Thus, in certain embodiments, a “genome-edited T cell” is a T cell that has had a gene such as a CAR recognizing at least one antigen added; and/or has had a gene such as the gene(s) to the antigen(s) that are recognized by the CAR deleted, and/or has had the gene to the TCR or a subunit thereof disrupted.

[0384] A “healthy donor,” as used herein, is one who does not have a malignancy (particularly a hematologic malignancy, e.g., a T-cell malignancy). [0001] The term “hematopoietic cells” refers to a population of cells of the hematopoietic lineage capable of hematopoiesis which include but is not limited to hematopoietic stem cells and/or hematopoietic progenitor cells (i.e., capable to proliferate and at least partially reconstitute different blood cell types, including erythroid cells, lymphocytes, and myelocytes). The term "hematopoietic cells" as used herein also includes the cells that are differentiated from the hematopoietic stem cells and/or hematopoietic progenitor cells to form blood cells (i.e. blood cell types, including erythroid cells, lymphocytes, and myelocytes). A donor hematopoietic cell resistant to recognition of an antigen by an antigen-recognizing receptor means that the cell cannot as easily be detected, bound and/or recognized by an antigen-recognizing receptor specific for the antigen or that the detection, binding and/or recognizing is impaired, so the cell is not killed during immunotherapy. [0385] As used herein, an “immature dendritic cell” or “iDC” refers to an immature dendritic cell.

[0386] The term "immune cell" or "immune effector cell" refers to a cell that may be part of the immune system and executes a particular effector function such as alpha-beta T cells, NK cells (including memory NKs, ML-NKs, and CIML-NKs), NKT cells (including iNKT cells), B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes and macrophages. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells (including memory NKs, ML-NKs, and CIML-NKs), NKT cells (including iNKT cells), ILC, CIK cells, LAK cells or gamma-delta T cells. "Effector function" means a specialized function of a cell, e.g. in an NK cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0387] The term “immunotherapy” is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response" Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based cytotoxic responses to attack cancer cells Immune cells such as T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. [0388] As used herein, the term "individual" refers to an animal. Preferentially, the individual is a mammal such as mouse, rat, cow, pig, goat, chicken dog, monkey or human. More preferentially, the individual is a human. The individual may be an individual suffering from a disease such as cancer (a patient), but the subject may be also a healthy subject.

[0389] The “intracellular signaling domain” (equivalently, cytoplasmic signalling domain or effector domain; which are part of the intracellular or endodomain) of a CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" means a specialized function of a cell, e.g. in an NK cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete or truncated part of the intracellular signaling domain of a given protein sufficient to transduce the effector function signal. Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic sequences of receptors and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement.

[0390] Generally, CAR activation of immune effector cells can be mediated by two classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the CAR (primary cytoplasmic signaling sequences) and secondly those that act in an antigen-independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences, costimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise a primary cytoplasmic signaling domain and optionally a secondary cytoplasmic signaling domain (i.e., a costimulatory or “co-stim” domain).

[0391] Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain IT AMs (immunoreceptor tyrosine-based activation motifs signaling motifs). Examples of ITAM containing primary cytoplasmic signaling sequences often used in CARs are disclosed herein and known in the art.

[0392] As used herein, a “low dose” of UCART7 refers to a dose as described herein that is intended to minimize the toxicity associated with administration of an immunotherapy to a patient, such as administration of UCART7. As described herein, a low dose may be any dose deemed appropriate and sufficient for reducing the tumor burden in a patient, while preventing or avoiding the occurrence of CRS or other toxicity associated with immunotherapy. In some embodiments, a low dose of UCART7 may be any dose described herein, such as including, but not limited to, about 1 x 10⁵ cells/kg of body weight to about 5 x 10⁶ cells/kg of body weight. One of skill in the art will recognize that a low dose of UCART7 immune effector cells may differ depending on the patient, or depending on the use, i.e., for T-ALL, NHL, and/or AML.

[0393] The term "malignant" or "malignancy" describes cells, groups of cells or tissues that constitute a neoplasm, are derived from a neoplasm or can be the origin of new neoplastic cells. The term is used to describe neoplastic cells in contrast to normal or healthy cells of a tissue. A malignant tumor contrasts with a non-cancerous benign tumor in that a malignancy is not self-limited in its growth, is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues. A benign tumor has none of those properties. Malignancy is characterized by anaplasia, invasiveness, and metastasis as well as genome instability. The term "premalignant cells" refer to cells or tissue that is not yet malignant but is poised to become malignant.

[0394] A “malignant B cell” is a B cell derived from a B-cell malignancy. B cell malignancies include, without limitation, (DLBCL), chronic lymphocytic leukemia (CLL) /small lymphocytic lymphoma (SLL), and B cell-precursor acute lymphoblastic leukemia (ALL).

[0395] A “malignant T cell” is a T cell derived from a T-cell malignancy. The term “T- cell malignancy” refers to a broad, highly heterogeneous grouping of malignancies derived from T-cell precursors, mature T cells, or natural killer cells. Non-limiting examples of T-cell malignancies include T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), human T-cell leukemia virus type 1-positive (HTLV-1 +) adult T-cell leukemia/lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), Adult T-cell lymphoma I leukemia (HTLV-1 associated), Aggressive NK-cell leukemia, Anaplastic large-cell lymphoma (ALCL), ALK positive, Anaplastic large-cell lymphoma (ALCL), ALK negative, Angioimmunoblastic T-cell lymphoma (AITL), Breast implant- associated anaplastic large-cell lymphoma, Chronic lymphoproliferative disorder of NK cells, Extra nodal NK I T-cell lymphoma, nasal type, Enteropathy-type T-cell lymphoma, Follicular T-cell lymphoma, Hepatosplenic T-cell lymphoma, Indolent T-cell lymphoproliferative disorder of the GI tract, Monomorphic epitheliotrophic intestinal T-cell lymphoma, Mycosis fungoides, Nodal peripheral T-cell lymphoma with TFH phenotype, Peripheral T-cell lymphoma (PTCL), NOS, Primary cutaneous α/β T-cell lymphoma, Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma, Primary cutaneous acral CD8+ T-cell lymphoma, Primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorders [Primary cutaneous anaplastic large-cell lymphoma (C-ALCL), lymphoid papulosis], Sezary syndrome, Subcutaneous, panniculitis-like T-cell lymphoma, Systemic EBV+ T-cell lymphoma of childhood, and T-cell large granular lymphocytic leukemia (LGL).

[0396] A “malignant plasma cell” is a plasma cell derived from a plasma cell malignancy. The term “plasma-cell malignancy” refers to a malignancy in which abnormal plasma cells are overproduced. Non- limiting examples of plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0397] As used herein, “overall response rate” or “ORR” is defined as the proportion of patients who have a partial or complete response to therapy. ORR as used herein does not include stable disease.

[0398] The term “patient” is generally synonymous with the terms “subject” and “individual” and includes all mammals including humans.

[0399] The term “persistence” as sued herein refers to the ability of cells, especially adoptively transferred into a subject, to continue to live.

[0400] As used herein, the term “pharmaceutical composition” includes a pharmaceutically acceptable excipient and/or carrier. For intravenous formulations, commonly used with adoptive cell transfer therapies, such a carrier would comprise an aqueous solution of the cells to be delivered.

[0401] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0402] As used herein, “persistent Grade 3 or Grade 4 CRS” refers to Grade 3 or Grade 4 CRS which cannot be promptly mitigated with small molecule or antibody treatment for CRS.

[0403] As used herein, “prevention” refers to keeping something (such as an illness or injury) from happening. Prevention may refer to prevention of symptoms of a disease, or may refer to prevention of the disease itself. Prevention may also refer to lessening the severity of symptoms or lessening the severity of the disease itself, as in preventing grade 3 or 4 CRS as described herein. Prevention of CRS may mean preventing or lessening the severity of certain symptoms of CRS, or may mean preventing or lessening the severity of a particular grade of CRS (e.g., grade 3 or 4 CRS). For example, in some embodiments of the present disclosure, UCART7 may be administered to a patient having a T-cell malignancy, such as T-ALL, AML, or non-Hodgkin’s lymphoma, at a particular dose in order to prevent or avoid the occurrence of CRS or other toxicity associated with immunotherapy. In some embodiments, CRS or another toxicity as described herein may be prevented by administering a low dose of UCART7 as described herein.

[0404] In general, the term "receptor" refers to a biomolecule that may be soluble or attached to the cell surface membrane and specifically binds a defined structure that may be attached to a cell surface membrane or soluble. Receptors include but are not restricted to antibodies and antibody like structures, adhesion molecules, transgenic or naturally occurring TCRs or CARs. In specific, the term "antigen-recognizing receptor" as used herein may be a membrane bound or soluble receptor such as a natural TCR, a transgenic TCR, a CAR, a scFv or multimers thereof, a Fab-fragment or multimers thereof, an antibody or multimers thereof, abi-specific T cell enhancer (BiTE), a diabody, or any other molecule that can execute specific binding with high affinity.

[0405] The term "reducing side-effects" refers to the decrease of severity of any complication, unwanted or pathological outcome of an immunotherapy with an antigen recognizing receptor such as toxicity towards an antigen-expressing non-target cell. "Reducing side-effects" also refers to measures that decrease or avoid pain, harm or the risk of death for the patient during the immunotherapy with an antigen recognizing receptor.

[0406] As used herein, a “secretable protein” is s protein secreted by a cell which has an effect on other cells. By way of example, secretable proteins include cytokines, chemokines, and transcription factors.

[0407] As used herein, a “selectable marker” refers to a marker that allows distinguishing between different cell types, such as a cell into which a CAR has been successfully inserted (i.e., a gene-edited or modified cell). Selectable markers are well known in the art and materials and methods for their use are readily available. In some embodiments, a selectable marker appropriate in accordance with the present disclosure may be a fluorescent protein gene, such as including, but not limited to, a green fluorescence (GFP) gene or a yellow fluorescent protein (YFP) gene. In some embodiments, a selectable marker may be a splice variant of a CD34 gene, such as a truncated CD34 (tCD34) gene or a truncated EGFR (tEGFR) gene. In some embodiments, a selectable marker described herein, such as GFP, or others known and available in the art, may be inserted alone into a gene as described herein (i.e., without a CAR), or may be inserted as a component of a construct comprising the selectable marker and a CAR.

[0408] As used herein, the term “sequence identity” means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods. Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTF1T, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et. al. Nucl. Acids Res. 25: 3389-3402 (1997). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm.

[0409] As used herein, a “short hairpin RNA” or “small hairpin RNA” (shRNA) is an artificial RNA molecule, often about 80 base pairs in length and with a tight hairpin turn, that can be used to silence target gene expression via processing within the cell into siRNA which in turn knocks down gene expression. ShRNAs can be incorporated into genomic DNA, and provide stable and long-lasting expression.

[0410] A "signal peptide" as used herein, in the context of a CAR, refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface.

[0411] The term “spacer” or “hinge” as used herein, in the context of a CAR, refers to the hydrophilic region which is between the antigen binding domain and the transmembrane domain. The CARs disclosed herein may comprise an extracellular spacer domain but is it also possible to pass such a spacer. The spacer may include Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8αlpha hinge.

[0412] The term "side-effects" refers to any complication, unwanted or pathological outcome of an immunotherapy with an antigen recognizing receptor that occurs in addition to the desired treatment outcome. The term "side effect" preferentially refers to on-target off-tumor toxicity, that might occur during immunotherapy in case of presence of the target antigen on a cell that is an antigen-expressing non-target cell but not a diseased cell as described herein. A side-effect of an immunotherapy may be the developing of graft versus host disease.

[0413] The terms "specifically binds" or "specific for" or "specifically recognize" with respect to an antigen-recognizing receptor refer to an antigen-binding domain of the antigen-recognizing receptor which recognizes and binds to a specific polymorphic variant of an antigen, but does not substantially recognize or bind other variants.

[0414] As used herein, “suicide gene” refers to a nucleic acid sequence introduced to a CAR-T cell by standard methods known in the art, that when activated result in the death of the CAR-T cell. If required suicide genes may facilitate the tracking and elimination, i.e., killing, of CAR-T cells in vivo. Facilitated killing of CAR-T cells by activating a suicide gene can be accomplished by standard methods known in the art. Suicide gene systems known in the art include, but are not limited to, several herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy systems and inducible caspase 9 proteins. In one embodiment, the suicide gene is a chimeric CD34/thymidine kinase.

[0415] The term "target" or "target antigen" refers to any cell surface protein, glycoprotein, glycolipid or any other structure present on the surface of the target cell. The term also refers to any other structure present on target cells in particular but not restricted to structures that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0416] The term “therapeutically acceptable” refers to substances which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and/or are effective for their intended use.

[0417] The term "therapeutically effective amount" means an amount which provides a therapeutic benefit.

[0418] As used herein, “transduction” is the process by which foreign DNA is introduced into a cell by a vims or viral vector such as a plasmid, for example by short hairpin RNAs (shRNAs); it often provides long-lasting or permanent silencing of a gene. It may be accomplished by methods known in the art, including electroporation.

[0419] Transfection is the process of deliberately introducing purified nucleic acids into eukaryotic cells, for example small interfering RNAs (siRNAs); it produces transient silencing of a gene by RNA interference with mRNA transcripts. Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector such as a plasmid, for example by short hairpin RNAs (shRNAs); it often provides long-lasting or permanent silencing of a gene. Both may be accomplished by methods known in the art, including electroporation.

[0420] The “transmembrane domain” of the CAR can be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived for example from CD8αlpha, CD28, NKG2D, or others disclosed herein or known in the art. When the key signaling and antigen recognition modules are on two (or even more) polypeptides then the CAR may have two (or more) transmembrane domains. Splitting key signaling and antigen recognition modules enables for a small molecule- dependent, titratable and reversible control over CAR cell expression (Wu et al, 2015, Science 350: 293-303) due to small molecule-dependent heterodimerizing domains in each polypeptide of the CAR.

[0421] As used herein, the term "transplant" means administering to a subject a population of donor cells, e.g. hematopoietic cells or CAR-bearing immune effector cells.

[0422] The term "treatment" as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0423] As used herein, “UCART7” means a “universal CAR-T cell targeting CD7,” and refers to a genetically modified T-cell which is transduced with a chimeric antigen receptor (CAR) targeting CD7, which is genome-edited to be deficient in cell surface expression of CD7, which is genome edited to be deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), such as the TCR alpha chain (TRAC), which has a CD28 costimulatory domain or a 4- IBB costimulatory domain or both a CD28 costimulatory domain and a 4- IBB costimulatory domain, and which has a CD3-zeta effector domain. UCART7 is “universal” in that the deficiency in cell-surface expression of CD7 and TRAC are significant enough to enable dosing of UCART7 made from allogeneic donor cells into a patient without GvHD, and without CD7-mediated fratricide; the deficiency may amount to a deletion. UCART7 is typically administered intravenously as a single dose. Examples

[0424] The disclosure is further illustrated by the following examples.

Phase 1 Dose-Escalation and Cohort Expansion Study of the Safety and Efficacy of UCART7 in Subjects with Relapsed or Refractory T-cell Acute Lymphoblastic Leukemia/Lymphoma (T-ALL/LBL) and T-cell Non-Hodgkin Lymphoma (T-NHL)

Example 1: Rationale for the Study

[0425] CD7+ve acute lymphocytic leukemia (ALL) and non- Hodgkin’ s lymphoma (T- NHL) represent a significant unmet medical need, especially for those patients who relapse where no effective or targeted therapies currently exist. The availability of off-the-shelf CAR-T therapy may be of benefit and reduce early disease mortality associated with manufacturing delays of autologous CAR-T therapies. In addition, non- autologous derived CAR-T therapies prevents any delay in treating patients who have demonstrated progressive disease (PD) and avoids or reduces the number of early patient deaths otherwise likely due to the time incurred to process autologous CAR-T cells.

[0426] The total duration of the study will be up to 60 months and will take place in two parts, Part A and Part B, described in detail below.

[0427] Up to 36 patients will be enrolled in Part A of the study. Up to 45 patients will be enrolled in Part B of the study.

[0428] The primary objectives for Part A of the study are: (1) to evaluate the safety and tolerability (incidence of adverse effects up to 28-days post infusion), and (2) to determine the recommended Phase 2 dose (RP2D) for each patient subgroup. The primary objective for Part B of the study is to determine the Objective Response Rate (up to 60 months post infusion).

[0429] Secondary Objectives of this study include (1) to evaluate the cellular kinetics of UCART7, and (2) to explore the anti-tumor activity of UCART7 in each disease subgroup: Objective Response Rate, Duration of response, and Progression-free survival (from date of UCART7 infusion until date of disease progression or death due to any cause, assessed up to 60 months.

[0430] In addition, a final objective of the study is to validate the utility of the cellular flow cytometry CD7 assay to be used to select patients for treatment. This objective will be pursued in Part B of the study only.

[0431] It is expected that lower doses of UCART7 will be effective in killing CD7+ cancer cells, and that when administered at the lower dose ranges disclosed below, will also cause or be associated with a reduced incidence of CRS compared to other CAR-T therapies, and will not cause grade 3 or 4 CRS.

Example 2: Study Drug and Mode of Administration

[0432] UCART7 is an allogeneic, fratricide-resistant, genetically modified T-cell transduced with a CAR having a CD28 costimulatory domain targeting CD7, or with a CAR having a 4- IBB costimulatory domain targeting CD7, or with a CAR having both a CD28 costimulatory domain and a 4- IBB costimulatory domain targeting CD7, or with a CAR having a CD28 costimulatory domain, and a 4-1BB costimulatory domain, and a CD3-zeta effector domain targeting CD7. It will be administered intravenously as a single dose.

Example 3: Inclusion Criteria

[0433] Patient selection criteria will include both general criteria applicable to all patients irrespective of disease subtype, as well as disease-specific criteria applicable to the phenotypic disease of each patient group. In general, all patients must meet the following criteria to be included in the study: [0434] General Inclusion Criteria

[0435] (1) Prior to treatment (i.e., administration of UCART7) of the first 5 study patients, each screened patient may be matched for potential allogeneic bone marrow transplant (BMT) in the event of profound and persistent T cell aplasia. Adequate matched marrow from a matched-related donor or haploidentical donor should be available at the clinical trial site. Patients may be considered with a matched unrelated donor if stem cells are available prior to enrollment and must be discussed with the sponsor. The need for continued access to BMT services for subsequent patients will be assessed following initial safety data on the first 5 patients.

[0436] (2) Display CD7-positive (CD7+ve) tumor cell expression demonstrated in 30% of the malignant cells in bone marrow, peripheral blood or lymph nodes within 3 months of study entry. For relapsed patients, CD7+ve tumor cell expression demonstrated in 30% of the malignant cells in bone marrow, peripheral blood, or lymph nodes within 3 months of study entry.

[0437] (3) Have adequate renal, hepatic, respiratory, and cardiovascular function, defined as follows.

[0438] Renal function: defined as calculated creatinine clearance or radioisotope glomerular filtration rate >60 mL/min/1.73 m² or normal serum creatinine based on age/gender, shown below.

[0439] Hepatic Function: defined as (a) alanine aminotransferase (ALT) ≤5 times the upper limit of normal (ULN) for age; (b) total bilirubin ≤1.5 x ULN (unless the patient has Grade 1 bilirubin elevation due to Gilbert’s disease or a similar syndrome involving slow conjugation of bilirubin).

[0440] Respiratory: must have a minimum level of pulmonary reserve defined as pulse oxygenation >91% on room air.

[0441] Cardiovascular: defined as left ventricular ejection fraction ≥45% confirmed by echocardiogram or multigated acquisition (MUGA) scan within 28 days of screening.

[0442] (4) Have a life expectancy >12 weeks.

[0443] (5) Age: lower age limit of 4 years. Patients between 4 and 18 years of age will be considered minors (pediatric patients) and will be enrolled into the pediatric disease group. Adult patients 18 and above may be enrolled into the AML, adult T-ALL, and lymphoma subgroups.

[0444] (6) Have an Eastern Cooperative Oncology Group (ECOG) Performance Status of

0 (zero) or 1 at screening (adults) or Karnofsky/Lansky Performance Status of 60 and above (children). Children may have more trouble expressing their experienced quality of life, and therefore require a somewhat more observational scoring system suggested and validated by Lansky et al., Cancer 60(7): 1651-6, 1987. See Tables 5 and 6 for relevant performance criteria.

[0445] (7) Be able to understand the nature of this study, comply with protocol requirements, and give written informed consent. For minors, legal guardian willingness to give written informed consent with patient assent, where appropriate. [0446] (8) Patients of reproductive potential: Discussed in detail below. All female study participants of reproductive potential must have a negative serum or urine pregnancy test performed within 48 hours before study entry.

[0447] Inclusion Criteria for T-ALL

[0448] (9) Relapsed or refractory ALL: (a) Primary refractory disease defined as not achieving CR after two cycles of induction chemotherapy; (b) First relapse if first remission ≤12 months; (c) Relapsed or refractory disease after two or more lines of systemic therapy; (d) Relapsed or refractory disease after allogeneic transplant and must be >3 months from SCT at the time of UCART7 infusion; or (e) Bone marrow with ≥5% lymphoblasts by morphologic assessment at screening.

[0449] (10) Patients with active central nervous system (CNS) leukemia involvement only (defined as CNS-3 by CSF findings) are eligible, but will have their UCART7 infusion delayed until CNS disease is cleared by negative cytology in 2 consecutive samples at least 1 week apart. Patients with other forms of active CNS-3 leukemic involvement, such as CNS parenchymal or ocular disease, cranial nerve involvement, or significant leptomeningeal disease are not eligible. Patients must have no acute/ongoing neurologic toxicity >Grade 1, with the exception of a history of controlled seizures or fixed neurologic deficits that have been stable/improving over the past 3 months.

[0450] Inclusion Criteria for T-NHL

[0451] (11) Relapsed or refractory lymphoma T-NHL: (a) Second or greater relapse; or

(b) Refractory as defined by not achieving a CR after 2 cycles of a standard first line chemotherapy regimen or not achieving a CR or PR following 1 cycle of a second line chemotherapy regimen; (c) Relapse after autologous stem cell transplantation.

[0452] Lymphoma patients with active CNS disease involvement (defined by Cerebrospinal fluid (CSF) cytology, magnetic resonance imaging (MRI) or computerized tomography (CT) are excluded from participation.

Example 4: Exclusion Criteria

[0453] Patients who meet any of the following criteria will be excluded from study entry: [0454] (1) Patients with concomitant genetic syndrome, such as patients with Fanconi anemia, Kostmann syndrome, Shwachman syndrome, or any other known bone marrow failure syndrome. Patients with Down syndrome will not be excluded.

[0455] (2) Previous treatment with any anti-CD7 therapy. [0456] (3) Unresolved toxicities from prior anticancer therapy, defined as having not resolved to baseline or to Common Terminology Criteria for Adverse Events (CTCAE) grade ≤1, with the exception of alopecia, or to the levels dictated in the inclusion/exclusion criteria. [0457] (4) Patient has participated in any investigational research study and is being screened for participation within a period of 5 half- lives of the last dose of the investigational therapy.

[0458] (5) Active or latent hepatitis B or active hepatitis C (test within 8 weeks of screening), or any uncontrolled infection at screening.

[0459] (6) optionally, HIV-positive test within 8 weeks of screening.

[0460] (7) Any serious active infection at the time of treatment, or another serious underlying medical condition that would impair the ability of the patient to receive protocol treatment.

[0461] (8) Presence of Grade 2 to 4 acute or extensive chronic GVHD requiring systemic immunosuppression (steroids). Grade 1 GVHD not requiring immunosuppression, and grade

2 skin GVHD, if treated with topical therapy only, is acceptable.

[0462] (9) Presence of other active cancers, or history of treatment for invasive cancer ≤

3 years. Patients with Stage I cancer who have received definitive local treatment and are considered unlikely to recur are eligible. All patients with previously treated in situ carcinoma (i.e., noninvasive) are eligible, as are patients with history of nonmelanoma skin cancer.

[0463] (10) Psychological, familial, sociological, or geographical conditions that do not permit compliance with the protocol.

[0464] (8) Pregnant or nursing (lactating) women.

[0465] (9) Require prohibited medications or treatments. The following medications are excluded:

[0466] Steroids: Therapeutic systemic doses of steroids must be stopped >72 hours prior to UCART7 infusion. However, the following physiological replacement doses of steroids are allowed: <12 mg/m²/day hydrocortisone or equivalent.

[0467] Allogeneic cellular therapy: Any donor lymphocyte infusions must be completed >6 weeks prior to UCART7 infusion.

[0468] GvHD therapies: Any systemic drug used for GvHD must be stopped >4 weeks prior to UCART7 infusion to confirm that GvHD recurrence is not observed [e.g., calcineurin inhibitors, methotrexate or other chemotherapy drugs, mycophenolate, rapamycin, thalidomide, or immunosuppressive antibodies such as anti-CD20 (rituximab), antitumor necrosis factor (anti-TNF), anti-interleukin 6 (anti-IL6) or anti-interleukin 6 receptor (anti- IL6R), systemic steroids]. GvHD therapy many be employed in the event it occurs after administration of UCART7.

[0469] Chemotherapy: Hydroxyurea must be stopped >24 hours prior to lymphodepletion. The following drugs must be stopped >1 week prior to UCART7 infusion and should not be administered concomitantly or following lymphodepleting chemotherapy: vincristine, 6-mercaptopurine, 6-thioguanine, methotrexate <25 mg/m², cytosine arabinoside <100 mg/m²/day, asparaginase (non-pegylated), nelarabine. The following drugs must be stopped >2 weeks prior to UCART7 infusion: salvage chemotherapy (e.g., clofarabine, cytosine arabinoside >100 mg/m², anthracyclines, cyclophosphamide, methotrexate ≥25 mg/m²), excluding the required lymphodepleting chemotherapy drugs. Pegylated- asparaginase must be stopped >4 weeks prior to UCART7 infusion. CNS prophylaxis treatment must be stopped >1 week prior to UCART7 infusion (e.g., intrathecal methotrexate).

[0470] Radiotherapy: Non-CNS site of radiation must be completed >2 weeks prior to UCART7 infusion. CNS directed radiation must be completed >8 weeks prior to UCART7 infusion.

[0471] Anti-T-cell antibodies: Administration of any T-cell lytic or toxic antibody (e.g., alemtuzumab) within 8 weeks prior to UCART7 is prohibited since residual lytic levels may destroy the infused UCART7 cells and/or prevent their in vivo expansion. If such an agent has been administered within 8 weeks prior to UCART7, the Medical Monitor will be contacted prior to enrollment in the study.

Example 5: Enrollment and Registration Procedures

[0472] Enrollment of Patients

[0473] The patient (or legal guardian for a patient aged less than 18 years) must willingly consent after being informed of the procedures to be followed, the experimental nature of the treatment, potential benefits, alternatives, side effects, risks, and discomforts. Eligible patients who wish to participate in the study will be enrolled into the study.

[0474] Replacement of Patients

[0475] Patients who discontinue study participation for any reason following the administration of UCART7 and after the dose-limiting toxicity (DLT) observation period will be asked to have all end-of-treatment safety evaluations performed as described in the protocol (see FIG. 1). If a patient withdraws or is withdrawn from the study either prior to treatment with UCART7, or prior to the completion of the DLT observation period for any reason other than DLT and does not meet the minimum requirements for inclusion in the maximum tolerated dose (MTD) -determining population as described herein, that patient will be replaced.

Example 6: Study Design

[0476] This first- in-human Phase 1 study will be a multicenter, dose-escalating, single- agent study conducted in patients with relapsed/refractory CD7-associated hematological cancers for which the Investigator determines there to be no other higher priority therapies available. All patients will have failed multiples lines of conventional systemic therapy applicable to their disease. The common diagnostic feature will be the presence of CD7 expression, as determined by: (1) T-cell ALL/LBL patients, including Relapsed/Refractory patients: >95% of patients have a high and homogeneous CD7 expression on >90% of blasts. CD7 expression on the malignant cells will be confirmed by a flow cytometry assay performed by a CLIA certified laboratory as part of standard clinical diagnostic testing. However, confirmation of CD7 expression using a validated Clinical Trial Assay, performed in a central laboratory, will not be required to enroll patients in the trial, but confirmation of CD7 expression will be performed after enrollment in the trial using a validate Clinical Trial Assay to assess correlation between CD7 expression and clinical responses; or (2) T-NHL: CD7 expression on the malignant cells will be confirmed by a flow cytometry or IHC assay performed by a CLIA certified laboratory as part of standard clinical diagnostic testing. In addition, because CD7 expression in T-NHL is heterogeneous, CD7 expression will be confirmed in a central laboratory using a validated Clinical Trial Assay.

[0477] The study will consist of 2 parts, Part A (dose escalation) and Part B (cohort expansion). In Part A of the study, dose escalation will proceed independently in three disease groups: (1) Adult T-ALL/LBL, (2) adult T-NHL, (3) Pediatric T-ALL. Enrollment in the pediatric T-ALL group will begin after acceptable safety of the first dose level has been demonstrated in the adult T-ALL group. Dose escalation will proceed as described herein, following a rule-based design methodology. This methodology will utilize prespecified dose increments, but intermediate dose levels may be explored, if warranted, based on real-time cellular pharmacokinetic (cPK) data. Part A of the study will assess the incidence of adverse effects, determine the RP2D, and assess WU-CART-007 expansion kinetics. Enrollment in the pediatric T-ALL arm will start after demonstration of safety of in the lowest dose level cohort of the adult T-ALL group. Further, once a limited number of adult patients with ALL have demonstrated acceptable safety at the first dose level, a cohort of pediatric patients with ALL will be explored. Upon attaining a Recommended Phase 2 Dose (RP2D), Part B of the study (cohort expansion) will commence. The RP2D may differ for T-ALL/LBL, adult T- NHL and pediatric T-ALL cohorts.

[0478] In Part B of the study, each disease cohort (T-ALL/LBL, adult T-NHL, and pediatric T-ALL) will be expanded to explore a total of 15 patients per cohort. Part B of the study will seek to confirm the applicable dose for each patient subgroup. Part B of the study will also seek early signals of efficacy. The study design overview is presented in FIG. 2. [0479] Prior to any study-specific activities, all patients or legal representatives will sign an informed consent form (ICF). Patients meeting the eligibility criteria will be enrolled and treated at the dose level specified by the dose-escalation scheme. Patients will receive a single dose of the CAR-T therapy. Logistically, the study is divided into observational windows of 28 days for data collection purposes.

[0480] All patients will be hospitalized for the first week following UCART7 administration, and in addition will be required to remain in the vicinity of the treating center for the following 3 weeks. Patients will be closely monitored for safety, being seen at the clinic on a weekly basis for the first 4 weeks, every 2 weeks for 4 weeks, then on months 3, 4, 5, 6, 9, 12, 15, 18, 21, and 24. Patients will then transition to another long-term follow up protocol where they are evaluated every 6 months for three years, then yearly for 10 years for a total of 15 years post WU-CART-007 infusion.

[0481 ] Initial Dose

[0482] The relevance of animal models to predict an appropriate starting dose of UCART7 is limited, the dose levels selected have been based in part on existing clinical precedent from historic data derived from commercially available autologous CAR-T therapies (which are not necessarily equivalent):

[0483] Axicabtagene ciloleucel: The target dose is 2xl0⁶ CAR-positive viable T cells per kg body weight.

[0484] Tisagenlecleucel: A single dose of 0.2 to 5.0xl0⁶ CAR-positive viable T cells per kg of body weight for patients 50 kg or less, or 0.1 to 2.5xl0⁸ CAR-positive viable T cells for patients more than 50 kg.

[0485] The proposed dose levels are shown below. The first dose level of 2 x 10⁶ viable CAR-positive cells/kg is within the range of several allogenic CAR-T programs and 3-fold lower than the lowest dose of TruUCAR™ GC027 in T-ALL. Dose level 2 is 3-fold higher than dose level , dose level 3 is 2-fold higher than dose level 2, and dose level 4 is only 1.5- fold higher that dose level 3..

Example 7: Treatment Plan

[0486] All patients entering this study will receive a single dose of UCART7 by intravenous injection (IV) at a dose level based on data evaluation from the prior cohort(s). Patients will be admitted to the hospital and will receive treatment according to the following schedule.

[0487] Treatment Plan:

[0488] Lymphodepleting chemotherapy

[0489] UCART7 infusion

[0490] Hospitalization

[0491] Response assessment

[0492] (A) Lymphodepleting chemotherapy

[0493] Upon successful screening, each patient will receive lymphodepleting chemotherapy, described below.

[0494] Criteria for Treatment

[0495] Patients should commence treatment within 48 hours of enrollment. Failure to commence treatment within this time period will require re-confirmation of inclusion criteria. Any changes from the inclusion criteria must be discussed with the Medical Monitor prior to commencing the preconditioning lymphodepletion. Patients will not be treated if any of the following criteria are noted to be outside the inclusion criteria: (a) performance status, (b) hypoxia; or (c) active infection. In addition, the patients should not be hypotensive or display neurological symptoms. Upon confirmation that the patient remains suitable for UCART7 treatment, the patient will receive cyclophosphamide 500 mg/m² IV daily for 3 days (days -5, -4, -3), and fludarabine 30 mg/m² IV daily for 3 days.

[0496] (B) Antiviral and Bacterial Prophylaxis

[0497] Patients will be screened for Epstein-Barr virus (EBV), cytomegalovirus (CMV), Herpes Simplex (HSV), (Varicella-zoster virus) (VZV), human herpesvirus 6 (HHV6), adenovirus, aspergillus, and respiratory infections prior to starting therapy. Post UCART7 infusion, patients should receive anti-viral prophylaxis with acyclovir or valacyclovir for one year, PJP prophylaxis for at least 6 months or until when CD4 is more than 200/μL whichever is longer, and antibacterial prophylaxis (e.g., Ciprofloxacin 500 BID) and antifungal prophylaxis (e.g., Fluconazole 400 mg PO daily) when ANC is less than 1000/μL. EBV PCR and CMV PCR should be done at baseline, weekly for the first month, every 2 weeks in the second month, and monthly thereafter for another 4 months. Alternatives should be discussed with the medical monitor.

[0498] (C) Bridging Therapy Guidelines

[0499] The first 5 patients enrolled may receive bridging therapy while awaiting collection of bone marrow by the donor. This should be discussed with the sponsor prior to initiation. Subsequent patients will not incur a delay in study participation and will not require bridging therapy.

[0500] (D) UCART7 Administration

[0501] Criteria for Treatment

[0502] Patients should then receive UCART7 over 30 minutes at the designated IV dose 72 hours following Day 3 of the lymphodepletion protocol (i.e., on Day 0), providing they meet the following criteria: (a) no evidence of infection; (b) blood oxygenation within range of inclusion criteria; (c) no major complications of preconditioning lymphodepletion regimen. Patients will remain as inpatients for 7 days following the administration of UCART7.

[0503] (E) Selection of Starting Dose

[0504] As the relevance of animal models to predict an appropriate start dose is limited, the dose levels selected have been primarily based on existing clinical precedent from historic data derived from commercially available autologous CAR-T therapies:

[0505] Axicabtagene ciloleucel: The target dose is 2xl0⁶ CAR-positive viable T cells per kg body weight.

[0506] Tisagenlecleucel: A single dose of 0.2 to 5.0x10⁶ CAR-positive viable T cells per kg of body weight for patients 50 kg or less, or 0.1 to 2.5x10⁸ CAR-positive viable T cells for patients more than 50 kg.

[0507] To facilitate the treatment of young patients, UCART7 will be dosed on a cells- per-body-weight basis. To accommodate the logistic of drug supply, patients will be dosed according to the body weight range applicable to the patient, as described in Table 4. The first dose level of 1.4 x 10⁵ cells/kg is more than an order of magnitude lower than the dose currently used for axicabtagene ciloleucel and is approximately half of the lowest dose for tisagenlecleucel.

[0508] Toxicology

[0509] The starting dose was further confirmed from the toxicology study in immunocompromised NSG mice harboring CCRF-CEM human T-ALL tumor cells. These cells were engineered to express luciferase to facilitate weekly measurements of tumor burden by whole body imaging. Mice were inoculated with 0.5 x 10⁶ cells on day 0 and were randomized to receive vehicle (Group 1), low (2xl0⁶ cells, Group 2) or high (6.6xl0⁶ cells, Group 3) dose of UCART7 4 days later. These cell numbers translate to approximately 66xl0⁶ and 220xl0⁶ cells/kg, respectively. Vehicle treated mice were taken down on day 21 when the tumor burden was maximum and UCART7 treated mice were euthanized on 43 to assess toxicity endpoints (hematology, clinical chemistry and tissue histology). UCART7 at both doses reduced tumor burden >99% with no significant adverse effects on body weight, blood chemistry or the emergence of clinical signs of GvHD. Tissue histology revealed infiltration of neoplastic mononuclear cells in many organs including the brain, femur/bone marrow, kidney, liver, lung, spleen, heart, ovary, etc., in all three groups. Group 1 mice also had necrosis of the bone marrow, osteoarthrosis of the stifle joint, and sometimes pulmonary hemorrhage, likely due to homing of tumor cells to these tissues. There were no histological signs of GvHD. In summary, UCART7 was well tolerated at both dose levels.

[0510] The proposed clinical start dose of 2xl0⁶ is 220-fold lower than the well tolerated high dose of WU-CART-007 in the toxicology study.

[0511] (F) Study Part A: Dose-Escalation Procedure

[0512] This study will follow the illustrative dose-escalation design in Table 4 below. It is anticipated that approximately 4 dose cohorts will be evaluated during the trial. The actual number of dose cohorts and dose levels explored will depend upon the maximum tolerated dose (MTD) and the safety profile observed during the conduct of the trial. Typically, the dose-escalation scheme will continue until greater than 1 patient out of 6 experiences DLT as defined herein. Intermediate dose levels may be explored, if deemed appropriate.

[0513] The study will follow a standard 3-by-3 dose-escalation design. The dose of UCART7 will be escalated in sequential patient cohorts. For each cohort, the decision whether to dose-escalate will be made once all patients have been enrolled into the cohort and the last patient enrolled has been followed for 28 days (DLT observation period). Enrollment into each cohort will be staggered, with the first patient of each cohort being observed for 28 days prior to the enrollment of the remaining patients in that cohort. FIG. 3 presents the overall dose-escalation methodology.

[0514] The following dose-escalation rules will be used:

[0515] Initially, 1 patient will be entered into the first dose level and observed for 28 days. If the patient either experiences or dies from life-threatening toxicity, the study will be interrupted, and alternate dose levels will be considered.

[0516] In the absence of life-threatening or lethal toxicity, the next 2 patients will be enrolled at the first dose level: (a) If 0/3 patients demonstrate a DLT, dose escalation to the next dose level will occur, (b) If 1/3 patients experience a DLT, enroll a further 3 patients at the current dose level. If 1/6 patients demonstrate a DLT, dose escalation to the next dose level will occur. If ≥2/6 patients experience a DLT, the MTD has been exceeded, and lower dose levels will be explored, (c) If >1/3 patients experience DLT, the MTD has been exceeded, and lower dose levels will be explored.

[0517] Dose escalation may continue according to these rules until the top maximum protocol stipulated dose has been administered or until DLT is documented.

[0518] Intermediate Dose Levels

[0519] Intermediate dose levels, or dose levels lower than the target dose stipulated in Table 4, may be explored following the demonstration of a DLT to ascertain a safe and effective dose.

Table 4. Dose-Escalation Schedule

[0520] Dose-Limiting Toxicity [0521] In general, toxicity will be assessed utilizing the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 5 (available online at ctep.cancer.gov/protocoldevelopment/electronic_applications/ctc.htm#ctc_50), unless otherwise specified. Cytokine release syndrome (CRS), neurotoxicity, and tumor lysis syndrome (TLS) will be evaluated according to the ASTCT Consensus Grading For Cytokine Release Syndrome And Neurologic Toxicity Associated With Immune Effector Cells Criteria (Lee et al., Biol Blood Marrow Transplant 25:625-638, 2019Error! Reference source not found.).

[0522] A toxicity will be considered dose-limiting if it occurs within 28 days of dosing. Dose- limiting toxicides will be defined as follows:

[0523] Any Grade 3 or higher neurotoxicity (ASBMT/ASTCT 2018 Consensus Criteria) [0524] Any Grade 4 CRS of any duration

[0525] Grade 3 CRS that does not improve to ≤Grade 2 within 72 hours

[0526] Grade ≥3 infusion reactions

[0527] Grade ≥3 vital organs toxicity

[0528] Grade ≥2 GVHD

[0529] Renal failure requiring dialysis >3 days

[0530] Any intubation

[0531] Any other Grade 3/4 nonhematological toxicity lasting for more than 7 days [0532] Determination of Dose-Limiting Toxicides

[0533] The patient population used for determination of DLTs will consist of patients who have received the intended dose of UCART7 and have met the minimum 28-day DLT observation period.

[0534] Maximum Tolerated Dose

[0535] The MTD is defined as the highest dose level of UCART7 at which no more than 1 out of 6 patients experiences DLT during the first cycle of therapy.

[0536] Recommended Phase 2 Dose (RP2D)

[0537] The RP2D will not be greater than the MTD. However, the RP2D may be a lower dose level in certain circumstances as follows:

[0538] If emerging toxicity at the MTD is unpredictable or undesirable for other reasons.

[0539] If clear evidence of efficacy is noted at lower doses with a cleaner safety profile.

[0540] If the cPK profile that emerges results is similar numbers of clonal cells over time that is independent of administered dose level. [0541] If longer follow-up on earlier patient cohorts suggests the emergence of delayed toxicity.

[0542] (G) Study Part B: Dose-Expansion Group at the RP2D

[0543] Once the MTD, if relevant, and the applicable RP2D have been determined for each patient cohort, up to 15 patients will be enrolled, at the selected RP2D level, into each of the 4 different disease groups. No more than 45 patients total will be enrolled in Part B of the study. Each patient group will be characterized by the presence of the CD7 antigen, and the 4 phenotypic disease groups will separately include adult T-ALL, pediatric T-ALL, AML, and NHL. This will allow further characterization of the safety and cPK profile, confirmation or modification of the ultimate UCART7 dose to be used in future Phase 2 studies, and early exploration of efficacy.

[0544] (H) Duration of Therapy

[0545] Patients will receive a single dose of UCART7. There will be no repeat cycles.

[0546] (I) Concomitant Anticancer Therapies

[0547] Patients will be instructed not to take any additional cancer medications during the course of the study. At each visit, the patient will be asked about any new medications he is taking or has taken after the start of the study drug.

[0548] (J) Special Precautions

[0549] Management of Cytokine Release Syndrome (CRS)

[0550] CRS is an expected toxicity of therapies such as UCART7, and the primary treatment for CRS is currently tocilizumab. Although it is expected that low doses of UCART7 will show reduced CRS compared to other CAR-T therapies, prior to patient dosing, each clinical site is required to have at least 2 doses of tocilizumab per patient immediately available at the site pharmacy prior to administration of UCART7. FIG. 4 provides the recommended algorithm for the evaluation of CRS.

[0551] For confirmed cases of CRS requiring treatment, tocilizumab should be administered. The recommended dosage is:

[0552] Patients weighing less than 30 kg: 12 mg/kg

[0553] Patients weighing 30 kg or greater: 8 mg/kg

[0554] This may be administered as a single agent or in combination with corticosteroids.

[0555] (K) Concomitant Medications

[0556] Premedication with anti-emetics is not required but is allowed according to standard practice guidelines. Medications may be administered for maintenance of existing conditions prior to study enrollment or for a new condition that develops while on study. [0557] Other medications considered necessary for the patient’s safety and well-being may be given at the discretion of the Investigator with the exception of those prohibited by the study design as described herein.

[0558] Prohibited Concomitant Medications

[0559] The following treatments are prohibited while in this study: No other investigational therapy should be given to patients. No anticancer agents other than the study medications should be given to patients. If such agents are required for a patient, then the patient must first be withdrawn from the study.

[0560] (L) Study Termination and Patient Discontinuation

[0561] Patient Discontinuation from Study Treatment

[0562] This is a single-dose study. While patients receiving UCART7 may request to withdraw from the study at any time, any patient receiving UCART7 should be followed as clinically appropriate for the collection of safety data and to protect their own ongoing safety. [0563] Site and Study Discontinuation

[0564] This study may be terminated prior to completion if circumstances warrant. Any clinical center may elect to discontinue patient enrollment or withdraw their participation from the study for any reason. In these circumstances, every effort will be made by the clinical trial site to provide all outstanding data for patients previously enrolled. Conditions that may warrant termination of the study include, but are not limited to, emergence of an unexpected, serious, or unacceptable risk to the patients in the study.

Example 8: Study Assessments and Evaluations

[0565] All patients should visit the study center on the days specified within this protocol. The complete Schedule of Assessments for this study is shown in FIG. 1.

[0566] Informed consent must be obtained, e.g., ≤21 days prior to initiation of treatment and before any protocol- specific procedures are performed. The screening physical examination, medical history, performance status, complete blood counts (CBC), differential and platelets, CMP, urinalysis, PT/PTT, and other clinically directed physiological status evaluations should be done ≤7 days prior to initiation of treatment.

[0567] Any initial examinations obtained within 72 hours of commencement of the lymphodepletion protocol do not have to be repeated. A pregnancy test for women of childbearing potential must be performed within 72 hours of lymphodepletion computerized tomography (CT) scans and other relevant imaging studies should be performed ≤21 days prior to initiation of treatment. [0568] The screening assessments described in FIG. 1 will be collected, reviewed, and determined to be acceptable by the site Principal Investigator (PI) or designee after obtaining informed consent prior to the initiation of treatment.

[0569] Scheduled tests (safety and efficacy evaluations) should be conducted as close as possible to the nominal schedule date. Where clinic or patient factors render this impractical, unless otherwise stated, these assessments must be completed within either a 3-day window (for laboratory tests) or a 5-day window (for imaging studies) of the scheduled date.

[0570] (A) Central Laboratory

[0571] All laboratory, imaging, and other safety studies performed are expected to be conducted at local laboratories. Should early efficacy data suggest an expedited regulatory path, a central laboratory may be implemented in a subsequent protocol amendment.

Similarly, independent central review of key efficacy data may be implemented if warranted by regulatory considerations.

[0572] (B) Baseline Study Assessments (Protocol Day -28 to Day 1)

[0573] The following information will be collected and procedures will be performed for each patient at screening ≤3 days prior to initiation of lymphodepletion treatment unless otherwise noted:

[0574] Written informed consent prior to any other study -related procedures (≤28 days prior to initiation of treatment) [0575] Medical history

[0576] Physical examination, measurements of height (first visit), weight, and vital signs (resting heart rate, blood pressure [BP], respiratory rate, and oral temperature)

[0577] Relevant performance status (see Tables 5 and 6)

[0578] 12-lead electrocardiogram in ECG (triplicate)

[0579] Bone marrow aspirate (within 7 days) for acute leukemias

[0580] Concomitant medication review

[0581] CBC including hemoglobin, hematocrit, WBC with 5 -part differential, platelets plus reticulocytes

[0582] Comprehensive metabolic profile (CMP) to include: glucose, blood urea nitrogen, creatinine, sodium, potassium, chloride, calcium, carbon dioxide (CO2), aspartate aminotransferase (AST), ALT, total bilirubin, total protein, and albumin, LDH, uric acid, Mg, Phos

[0583] PT/PTT fibrinogen and C-reactive protein (CRP)

[0584] Urine testing dipstick [0585] Serum or urine pregnancy test for women of childbearing potential

[0586] Appropriate tumor imaging studies (CT or magnetic resonance imaging [MRI] scans), if relevant, for nodal or extramedullary sites of disease within 14 days of treatment.

Table 5. ECOG and Karnofsky Performance Status Criteria

Table 6. Lansky Performance Scale

[0587] (C) Study Treatment Assessments

[0588] Protocol Days -5 to -3 (Lymphodepletion Period}

[0589] Unless otherwise stated, the following assessments do not need to be repeated if previously conducted within 72 hours of treatment:

[0590] Physical examination, measurements of weight and vital signs (resting heart rate,

BP and oral temperature)

[0591] Performance status

[0592] AE assessment

[0593] Concomitant medication review [0594] PT/PTT fibrinogen and CRP

[0595] Urine testing dipstick

[0596] The following tests to be completed daily during lymphodepletion:

[0597] CBC including hemoglobin, hematocrit, WBC with 5 -part differential, platelets plus reticulocytes

[0598] CMP, LDH, uric acid, Mg, Phos

[0599] Protocol Day 0

[0600] The following assessments to be done prior to UCART7 administration:

[0601] Physical examination, measurements of weight and vital signs (resting heart rate, blood pressure [BP] and oral temperature)

[0602] Performance status

[0603] AE assessment

[0604] Concomitant medication review

[0605] CBC including hemoglobin, hematocrit, WBC with 5-part differential, platelets plus reticulocytes

[0606] CMP, LDH, uric acid, Mg, Phos

[0607] PT/PTT fibrinogen and CRP

[0608] UCART7 administration at the relevant dose

[0609] cPK Samples commence with UCART7 treatment, designated Day 1

[0610] Twice Weekly from Protocol Day 7 to Day 21 (Post-UCART7 Dose Weeks 1 and

21

[0611] CBC including hemoglobin, hematocrit, WBC with 5-part differential, platelets plus reticulocytes

[0612] CMP, LDH, uric acid, Mg, Phos

[0613] PT/PTT Fibrinogen and CRP

[0614] cPK Samples

[0615] Weekly from Protocol Day 22 to Day 50 (Post-UCART7 Dose Weeks 3 to 6)

[0616] CBC including hemoglobin, hematocrit, WBC with 5-part differential, platelets plus reticulocytes

[0617] CMP

[0618] PT/PTT Fibrinogen and CRP

[0619] cPK Samples

[0620] Day 35 (DLT Observation Period, 28 Days After UCART7) [0621] The following assessments to be done at the end of the initial 28-day post- treatment observation period:

[0622] Physical examination measurements of weight and vital signs (resting heart rate, BP, and oral temperature)

[0623] Performance status

[0624] Urine dipstick

[0625] Serum or urine pregnancy test for women of childbearing potential

[0626] AE assessment

[0627] Concomitant medication review

[0628] CBC including hemoglobin, hematocrit, WBC with 5-part differential, platelets plus reticulocytes

[0629] CMP

[0630] PT/PTT Fibrinogen and CRP

[0631] cPK S amples

[0632] Monthly from Protocol Week 7 to Month 6

[0633] Patients will be seen in the clinic no less frequently than monthly for routine safety follow-up, to include:

[0634] Physical examination measurements of weight and vital signs (resting heart rate,

BP, and oral temperature)

[0635] Performance status

[0636] AE assessment

[0637] Concomitant medication review

[0638] CBC including hemoglobin, hematocrit, WBC with 5-part differential, platelets plus reticulocytes

[0639] CMP

[0640] cPK Samples

[0641] At Month 3 the patients will be assessed for efficacy, to include bone marrow evaluation and appropriate radiological imaging studies, if relevant.

[0642] Long-Term Follow-Up

[0643] All patients will be followed up for a minimum of 15 years or until patient death, whichever occurs first, to evaluate the persistence of the UCART7 cell clones and to explore any unanticipated genetic consequences secondary to the introduction of genetically modified cells. The detailed follow-up schedule will be determined by the Investigator but will generally fall within the following guidelines: [0644] Monthly until such time as there is no UCART7 clone detected; thereafter:

[0645] Quarterly for 2 years

[0646] Annually through Year 15

[0647] Cellular PK samples will be taken on each scheduled protocol visit until no UCART7 cells are detected. Sampling will cease once upon the previous sample failing to detect any cells.

[0648] Logistically, the long term follow up may be achieved through a separate long term follow up clinical protocol.

[0649] (D) Pharmacokinetic Assessments

[0650] The cPK parameters (including AUC(0-∞ ), AUC(0-Ƭ ), and maximum drug concentration in body after dosing [Cmax].) of UCART7 following IV administration will be assessed by analysis of peripheral blood samples.

[0651] cPK blood samples will be taken at the following timepoints counting from the day of UCART7 administration:

[0652] Day of UCART7 administration: (Day 0)

[0653] Predose

[0654] 30 minutes

[0655] Days 3, 7, 10, 14, 21, and 28

[0656] One sample on each of the days specified as early in the morning as feasible.

Record the precise time of sample collection.

[0657] Monthly and long-term sampling

[0658] A cPK sample will be taken at 1 sample on each of the days specified as early in the morning as feasible. Record the precise time of sample collection.

Example 9: Response Evaluation Criteria and Measurements

[0659] Response and progression will be evaluated in this study using relevant efficacy criteria, such as:

[0660] Non-Hodgkin’s Lymphoma (NHL): Lugano criteria (Cheson, Chin Clin Oncol 4(1):5, 2015)

[0661] Acute Leukemias: Center for International Blood & Marrow Transplant Research (CIBMTR) for ALL and European Leukemia Net (ELN) for AML (Cheson et al., J Clinical Oncol 21(24):4642-4649, 2003)

[0662] The overall remission rate (ORR) during the 3 months following UCART7 administration will be assessed. This includes complete remission (CR) and CR with incomplete blood count recovery (CRi) as determined by an Independent Review Committee (IRC) assessment.

[0663] Hematologic Complete Remission (CR or CRh}

[0664] Hematologic CR (CRh) is defined as meeting all of the following response criteria for at least 4 weeks: (1) <5% blasts in the bone marrow; (2) normal maturation of all cellular components in the bone marrow; (3) no extramedullary disease (e.g., CNS, soft tissue disease); (4) absolute neutrophil count ≥1000/μL (>1 x 10⁹/L); (5) platelets ≥100,000/μL (>100 x 10⁹/L); (6) <1% circulating blasts; and (7) transfusion independent, i.e., no platelet or neutrophil transfusion for ≤7 days.

[0665] Alternative post-transplant CR criteria are accepted in the setting of pediatric ALL when the center does not routinely perform bone marrow biopsies post-transplant and the patient was in CR pretransplant. These criteria are not used for pretransplant ALL disease status. The criteria are as follows:

[0666] Complete donor chimerism (≥95% donor chimerism without recipient cells detected)

• No extramedullary disease (e.g., CNS, soft tissue disease)

• Neutrophils ≥1000/μL

• Platelets ≥100,000/μL

• Transfusion independent

[0667] In some cases, there may not be a 4-week interval between completion of therapy and the pretransplant disease assessment; in this case, CR should still be reported as the status at transplant, since it represents the “best assessment” prior to HCT. This is an exception to the criteria that CR be durable beyond 4 weeks. The pretransplant disease status should not be changed based on early relapse or disease assessment post-transplant.

[0668] Include recipients with persistent cytogenetic or molecular abnormalities who meet the above CR criteria for hematologic CR.

[0669] Include recipients meeting the above CR criteria regardless of how many courses of therapy were required to achieve CR.

[0670] The number of this CR can be determined by using the following guidelines:

[0671] First CR: no prior relapse

[0672] Second CR: 1 prior relapse

[0673] Third or higher: 2 or more prior relapses [0674] CRh meets all CR criteria above except for neutrophils >0.5x10⁹/L and platelets >50xl0⁹/L.

[0675] CR with Incomplete Hematologic Recovery ( CRi)

[0676] Hematologic CR with incomplete hematologic recovery is defined as meeting all of the following response criteria for at least 4 weeks: (1) <5% blasts in the bone marrow; (2) normal maturation of all cellular components in the bone marrow; (3) no extramedullary disease (e.g., CNS, soft tissue disease); (4) transfusion independent (Please note, if the physician documents transfusion dependence related to treatment and not the patient’s underlying ALL, CRi can be reported). CRi is a CR except for neutrophils ≤ 1 x 10⁹/L and/or platelets ≤ 1 x 10¹¹/L.

[0677] Primary Induction Failure (PIF)

[0678] The patient received treatment for ALL but never achieved CR or CRi at any time. PIF is not limited by the number of unsuccessful treatments; this disease status only applies to recipients who have never been in CR or CRi.

[0679] Relapse (REL)

[0680] Relapse is defined as the recurrence of disease after CR, meeting at least 1 of the following criteria: (1) ≥5% blasts in the marrow or peripheral blood; (2) extramedullary disease; (3) disease presence determined by a physician upon clinical assessment.

[0681] The number of this relapse can be determined by using the following guidelines: [0682] First relapse: 1 prior CR

[0683] Second relapse: 2 prior CRs

[0684] Third or higher: 3 or more CRs

[0685] Do not include a partial response (PR) when determining number of relapse. Recipients who achieve a PR to treatment should be classified as either PIF or relapse; PR in ALL is generally of short duration and is unlikely to predict clinical benefit.

[0686] No Treatment

[0687] The recipient was diagnosed with acute leukemia and never received therapeutic agents. Include patients who have received only supportive therapy, including growth factors and/or blood transfusions (Center for International Blood & Marrow Transplant Research, ALL Response Criteria, 2018, available at cibmtr.org/manuals/fim/ 1/en/topic/alLresponse- criteria).

Example 10: Statistical Methodology

[0688] This is a Phase 1, multicenter, open-label (i.e., no blinding procedures), dose- escalation, 2-part study of UCART7 in patients with relapsed or refractory CD7+ve T- ALL/LBL, or T-NHL. Part A (dose escalation) will enroll patients with CD7+ve adult T- ALL/LBL, adult T-NHL, and pediatric T-ALL into cohorts of 3 patients at each dose level. Upon reaching the maximum tolerated dose or RP2D, Part B (cohort expansion) of the study will enroll up to 45 patients to explore preliminary efficacy.

[0689] (A) Sample Size Considerations

[0690] Phase 1 Dose Escalation (Part A)

[0691] Dose escalation will proceed according to the 3-by-3 escalation scheme specified. The methodology will utilize prespecified dose increments, but intermediate dose levels may be explored if warranted by emerging safety and cPK data. Each patient will participate in only 1 dose cohort. The total number of patients to be enrolled in the dose-escalation portion of the study is dependent upon the observed safety profile, which will determine the number of patients per dose cohort, as well as the number of dose escalations required to achieve the MTD and subsequent RP2D. The number of patients in Part A of the study might range from as few as 3 patients (assuming unacceptable toxicity at the first dose level) to as many as 36 patients (assuming a total of 4 dose levels per CD7+ve disease with 3 patients per group).

[0692] RP2D Expansion (Part B)

[0693] Up to 45 patients with CD7+ve disease will be treated in the final dose expansion groups. Fifteen patients will be enrolled into each of the 4 CD7+ve disease groups (adult T- ALL/LBL, T-NHL, pediatric T-ALL) to assess safety. The enrollment of these 45 patients at the RP2D will provide sufficient clinical experience to identify any significant toxicity associated with the administration of UCART7. In addition, efficacy will be assessed in each of these groups.

[0694] (B) Analysis Population

[0695] The following analysis populations will be used:

[0696] (1) A safety analysis population will include all patients receiving a dose of study drug. Patients will be included in the treatment dose group for which they were actually treated.

[0697] (2) An efficacy analysis population will be informal and exploratory. All patients with CD7+ve disease receiving a full dose of study drug will be included in the efficacy population.

[0698] (C) Data Analysis

[0699] Descriptive statistics, including mean, median, standard deviations and ranges for all continuous measures will be tabulated and reported. Percentages and frequencies for all categorical measures will also be presented. If relevant, time to events endpoints will be reported using Kaplan- Meier estimates, with 95% confidence intervals for median time to event.

[0700] Demographics and Baseline Characteristics

[0701] Demographic and baseline disease characteristics will be summarized in order to assess the comparability of the treatment groups descriptively. Data to be tabulated will include demographic features such as age, sex and race, as well as disease-specific characteristics.

[0702] The number and percentages of patients screened, enrolled, treated, completed the treatment/study, and withdrawn from treatment/study for any reasons will be presented overall and also by dose level.

[0703] Efficacy Analysis

[0704] No formal efficacy analysis will be conducted during the dose-escalation phase of the study (Part A). Any evidence of efficacy will be reported descriptively.

[0705] An efficacy assessment will be conducted on all patients treated at the RP2D level in Part B of the study who received the full prescribed cell dose.

[0706] The overall remission rate (ORR) during the 3 months following UCART7 administration will be assessed. This includes complete remission (CR) and CR with incomplete blood count recovery (CRi) as determined by an Independent Review Committee (IRC) assessment. Efficacy criteria are discussed in detail above.

[0707] Progression-free survival (PFS), defined as the time from the first day of study drug administration (Day 0) to disease progression as defined by >5% of blast cells in the bone marrow.

[0708] Overall survival (OS), defined as the time from the first day of study drug administration (Day 0) or death on study. Patients who are alive will be censored at the date of last known date alive.

[0709] For ORR, the estimates and the associated 95% CI (based on the Clopper-Pearson method) in each treatment group will be calculated.

[0710] For PFS and OS, Kaplan-Meier curves will be generated and the median time to event and the associated 95% CI will be provided. The hazard ratio and the 95% CI for these endpoints between the 2 treatment groups will be calculated.

[0711] Safety Analysis

[0712] Safety will be assessed through the analysis of the reported incidence of treatment-emergent adverse events (AEs). Treatment-emergent AEs are those with an onset on or after the initiation of therapy, and with the exception of cytokine release syndrome (CRS) and neurotoxicity, will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) Version 5 (available at ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_Refer ence_8.5xl l.pdf). CRS, neurotoxicity and TLS will be evaluated according to the ASTCT Consensus Grading For Cytokine Release Syndrome And Neurologic Toxicity Associated With Immune Effector Cells Criteria (Center for International Blood & Marrow Transplant Research [Internet]. ALL Response Criteria. 2018 [cited 2020 Feb 2013]. Available on the internet at cibmtr.org/manuals/fim/ 1 /en/topic/all-response-criteriaError! Reference source not found.)-

[0713] The AEs will be coded using Medical Dictionary for Regulatory Activities (MedDRA), and summarized using system organ class and preferred term by dose level for all patients in the Safety Population. In addition, summaries of serious adverse events, AEs leading to treatment discontinuation, AEs by maximum NCI CTCAE grade, and AEs related to study treatment will also be presented by dose level.

[0714] Other safety endpoints including laboratory results, vital signs, and ECG findings will be summarized for all patients in the Safety Population.

[0715] Concomitant medications will be coded using the World Health Organization- Drug Dictionary and they will be listed and summarized by dose level.

[0716] Cellular Pharmacokinetics/Pharmacodynamics

[0717] Plasma concentrations of UCART7 will be used to calculate the cPK parameters. Parameters evaluated will include plasma concentration-time profiles and C_max, Tmax, AUCiast, and AUCinf. These parameters will be listed by individual patient and summarized by descriptive statistics (means, medians, ranges, standard deviations, and coefficient of variation as appropriate, by treatment group/cohort).

[0718] (D) Analysis Timepoints

[0719] Final Analysis

[0720] The final analysis of the study will occur when the first of the either of the following circumstances occurs: (1) The last patient completes treatment with a minimum of 3-month follow up; (2) Three years following the enrollment of the first patient.

[0721] No formal interim analysis is planned. This is an open-label study. Patient safety and efficacy will be monitored and reviewed on an ongoing basis.

Example 12: Safety Reporting and Analysis

[0722] Safety assessments will consist of monitoring and recording protocol-defined AEs and SAEs, measurement of protocol- specified hematology, clinical chemistry, and urinalysis variables, measurement of protocol- specified vital signs, and other protocol-specified tests that are deemed critical to the safety evaluation of the study drug.

[0723] All AEs regardless of seriousness or relationship to UCART7 treatment (called study treatment), spanning from the start of study treatment, until 30 calendar days after discontinuation or completion of study treatment as defined by the study for that patient, will be recorded.

[0724] All AEs resulting in discontinuation from the study should be followed until resolution or stabilization.

[0725] (C) Assessment of Adverse Events

[0726] All AEs and SAEs whether volunteered by the patient, discovered by study personnel during questioning, or detected through physical examination, laboratory test, or other means will be reported appropriately. Each reported AE or SAE will be described by its duration (i.e., start and end dates), regulatory seriousness criteria if applicable, suspected relationship to the study drug (see following guidance), and actions taken.

[0727] To ensure consistency of AE and SAE causality assessments, Investigators should apply the following general guideline:

[0728] YES: There is a plausible temporal relationship between the onset of the AE and administration of the study medication, and the AE cannot be readily explained by the patient’s clinical state, intercurrent illness, or concomitant therapies; and/or the AE follows a known pattern of response to the study drug; and/or the AE abates or resolves upon discontinuation of the study drug or dose reduction and, if applicable, reappears upon re-challenge.

[0729] NO: Evidence exists that the AE has an etiology other than the study drug (e.g., pre-existing medical condition, underlying disease, intercurrent illness, or concomitant medication); and/or the AE has no plausible temporal relationship to study drug administration (e.g., cancer diagnosed 2 days after first dose of study drug).

[0730] (D) Serious Adverse Event Reporting by Investigators

[0731] AEs classified by the treating Investigator as serious require expeditious handling and reporting in order to comply with regulatory requirements.

[0732] (E) Recording of Adverse Events and Serious Adverse Events

[0733] Diagnosis Versus Signs and Symptoms

[0734] All AEs should be recorded individually in the patient’s own words (verbatim) unless, in the opinion of the Principal Investigator or designated physician, the AEs constitute components of a recognized condition, disease, or syndrome. In the latter case, the condition, disease, or syndrome should be named rather than each individual sign or symptom. If a constellation of signs and/or symptoms cannot be medically characterized as a single diagnosis or syndrome at the time of reporting, each individual event should be recorded as an AE or SAE. If a diagnosis is subsequently established, it should be reported as follow-up information is available. If a diagnosis is determined subsequent to the reporting of the constellation of symptoms, the signs/symptoms should be updated to reflect the diagnosis. [0735] Progression of malignancy (including fatal outcomes), if documented by use of appropriate method (for example, as per RECIST criteria for solid tumors), should not be reported as an SAE.

[0736] Persistent or Recurrent Adverse Events

[0737] A persistent AE is one that extends continuously, without resolution, between patient evaluation timepoints. Such events should only be recorded once. If a persistent AE becomes more severe or lessens in severity, it should be recorded separately.

[0738] A recurrent AE is one that occurs and resolves between patient evaluation timepoints, and subsequently recurs.

[0739] Abnormal Laboratory Values

[0740] Laboratory abnormalities, whether clinically significant or not, will not be recorded as AEs. All laboratory data will be separately graded, coded, and analyzed according to the NCI-CTCAE system. In this manner the consistent grading of laboratory data provides a greater level of transparency to abnormalities and renders reporting of laboratory anomalies as AEs duplicative and redundant.

[0741] Deaths

[0742] Deaths that occur during the protocol- specified AE reporting period that are attributed solely to progression of disease will be recorded. All other on study deaths, regardless of attribution, will be recorded separately.

[0743] Hospitalization, Prolonged Hospitalization, or Surgery

[0744] Any AE that results in hospitalization of >24 hours or prolongation of pre-existing hospitalization should be documented and reported. There are some hospitalizations that do not require reporting as an SAE.

[0745] Treatment within or admission to the following facilities is not considered to meet the criteria of “inpatient hospitalization” (although if any other SAE criteria are met, the event must still be treated as an SAE and immediately reported):

[0746] Emergency Department or Emergency Room [0747] Outpatient or same-day surgery units [0748] Observation or short-stay unit

[0749] Rehabilitation facility

[0750] Hospice or skilled nursing facility

[0751] Nursing homes, Custodial care or Respite care facility

[0752] Hospitalization during the study for a preplanned surgical or medical procedure (one that was planned prior to entry in the study), does not require reporting as a SAE.

[0753] Pre-Existing Medical Conditions

[0754] A pre-existing medical condition is one that is present at the start of the study. Such conditions should be recorded in the General Medical History. A pre-existing medical condition should be recorded as an AE or SAE only if the frequency, severity, or character of the condition worsens during the study.

[0755] New Cancers

[0756] The development of a new primary cancer should be regarded as an AE and will generally meet at least 1 of the seriousness criteria as described herein. New primary cancers are those that are not the primary reason for the administration of the study treatment and have developed after the inclusion of the patient into the study. They do not include metastases of the original cancer. Symptoms of metastasis or the metastasis itself should not be reported as an AE/SAE, as they are considered to be disease progression.

[0757] Pregnancy, Abortion, Birth Defects/Congenital Anomalies

[0758] Pregnancies of female patients, and of female partner of male patients, that occur during the course of the study, should be reported. Every effort should be made to follow the pregnancy for the final pregnancy outcome. Abortions (spontaneous, accidental, or therapeutic) should also be reported.

[0759] Congenital anomalies/birth defects always meet SAE criteria, and should therefore be expeditiously reported as an SAE.

[0760] UCART7 Overdose

[0761] Symptomatic and non-symptomatic overdose must be reported. Any accidental or intentional overdose with the study treatment that is symptomatic, even if not fulfilling a seriousness criterion, is to be reported.

[0762] (F) Protocol-Defined Events of Special Interest

[0763] The following are events of special interest, and will need to be reported expeditiously:

[0764] (G) Serious Adverse Event Reporting Requirements [0765] Relevant SAEs shall be reported to the Competent Authority, other applicable regulatory authorities, and participating Investigators, in accordance with ICH guidelines, FDA regulations.

Example 13: Quality Assurance and duality control

[0766] (A) Monitoring

[0767] Site monitoring shall be conducted to ensure that patient protection, study procedures, laboratory, study intervention administration, and data collection processes are of high quality and meet Sponsor, GCP/ICH and, when appropriate, regulatory guidelines.

[0768] (B) Audits and Inspections

[0769] Study-related quality audits and inspections of all study-related documents (e.g., source documents, regulatory documents, data collection instruments, case report forms) will be permitted.

Example 14: Guidelines for Women of Childbearing Potential and Fertile Males

[0770] (A) Female Patients

[0771 ] Acceptable Contraception Methods:

[0772] Women of childbearing potential, defined as all women physiologically capable of becoming pregnant, must use highly effective contraception during the study and for 12 months after stopping treatment.

[0773] All women of childbearing potential must use highly effective methods of contraception for a period of 1 year after the UCART7 infusion. Highly effective contraception methods include the following:

[0774] Total abstinence (when this is in line with the preferred and usual lifestyle of the patient). Periodic abstinence (e.g., calendar, ovulation, symptothermal, postovulation methods) and withdrawal are NOT acceptable methods of contraception.

[0775] Female sterilization (has had surgical bilateral oophorectomy with or without hysterectomy), total hysterectomy, or tubal ligation at least 6 weeks before taking study treatment. In case of oophorectomy alone, only when the reproductive status of the woman has been confirmed by follow up hormone level assessment.

[0776] Male partner sterilization (at least 6 months prior to screening). For female patients on the study the vasectomized male partner should be the sole partner for that patient. [0777] Use of oral, injected, or implanted hormonal methods of contraception or other forms of hormonal contraception that have comparable efficacy (failure rate < 1%), for example hormone vaginal ring or transdermal hormone contraception [0778] Use of IUDs are not acceptable due to increased risks of infection and bleeding in this population. However, IUD inserted prior to consent may remain in place, and a second method of contraception is mandated.

[0779] In case of use of oral contraception, women must be stable on the same pill for a minimum of 3 months before taking study treatment.

[0780] Women who are not of reproductive potential (defined as postmenopausal for at least 24 consecutive months [i.e., have had no menses] or have undergone hysterectomy, bilateral salpingectomy, and/or bilateral oophorectomy) are eligible without requiring the use of contraception. If applicable, women who are not yet of reproductive potential are to agree to use acceptable forms of contraception when they reach reproductive potential if within 1 year of UCART7 or if CART cells are present in the blood by PCR. Acceptable documentation supporting the lack of reproductive potential includes 1 of the following: [0781 ] Primary care physician report/letter

[0782] Operative report or other source documentation in the patient record or patient discharge summary

[0783] Follicle stimulating hormone measurement elevated into the menopausal range [0784] Unacceptable Contraception Methods for women of childbearing potential include:

[0785] IUD progesterone T

[0786] Female condom

[0787] Natural family planning (rhythm method) or breastfeeding

[0788] Fertility awareness

[0789] Withdrawal

[0790] Cervical shield

[0791] (B) Male Patients

[0792] Fertile male patients, defined as all males physiologically capable of conceiving offspring, with female partners of childbearing potential must use condoms plus spermicidal agent during the study treatment period and for 12 months after the last dose of study drug, and should not father a child during this period.

[0793] Male patients must also refrain from donating sperm during for the same time period

[0794] Reporting of Unintended Pregnancies

[0795] To ensure patient safety, each pregnancy in a patient on study treatment should be reported within 24 hours of learning of its occurrence. The pregnancy should be followed up for 3 months after the termination of the pregnancy to determine outcome, including spontaneous or voluntary termination, details of the birth, and the presence or absence of any birth defects, congenital abnormalities, or maternal and/or newborn complications.

[0796] Pregnancy should be recorded on a Clinical Study Pregnancy Form and reported. Pregnancy follow-up should be recorded and should include an assessment of the possible relationship to the study drug of any pregnancy outcome. Any SAE experienced during pregnancy must be reported.

[0797] Pregnancy outcomes must be collected for the female partners of any males who took study treatment in this study. Consent to report information regarding these pregnancy outcomes should be obtained from the mother.

Other Embodiments

[0798] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present disclosure. However, the disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description, which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

Claims

CLAIMS What is claimed is:

1. A single dose of a pharmaceutical composition comprising from about 1 x 10⁵ to about 6.3 x 10⁸ chimeric antigen receptor (CAR)-bearing immune effector cells per kg of body weight, wherein the immune effector cells each comprise one or more chimeric antigen receptors (CARs) targeting one or more T-cell antigens, wherein the immune effector cells are deficient in cell surface expression of one or more subunits of the T cell receptor complex (TCR), wherein the immune effector cells are deficient in cell surface expression of the at least one or more T-cell antigens to which the one or more CAR(s) specifically bind(s), and wherein the composition does not elicit persistent grade 3 or grade 4 cytokine release syndrome (CRS).

2. The single dose of a pharmaceutical composition of claim 1 , wherein the composition comprises from about 2.0 x 10⁶ to about 18.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

3. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 2.0 x 10⁶ to about 12 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

4. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 2.0 x 10⁶ to about 6 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

5. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 6.0 x 10⁶ to about 18 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

6. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 2.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

7. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 6.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

8. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 12.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

9. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 18.0 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

10. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 100 x 10⁶ to about 900 x 10⁶ CAR-bearing immune effector cells.

11. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 100 x 10⁶ to about 600 x 10⁶ CAR-bearing immune effector cells.

12. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 100 x 10⁶ to about 600 x 10⁶ CAR-bearing immune effector cells.

13. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 300 x 10⁶ to about 900 x 10⁶ CAR-bearing immune effector cells.

14. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 100 x 10⁶ CAR-bearing immune effector cells.

15. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 300 x 10⁶ CAR-bearing immune effector cells.

16. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 600 x 10⁶ CAR-bearing immune effector cells.

17. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises about 900 x 10⁶ CAR-bearing immune effector cells.

18. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 1.0 x 10⁵ to about 5 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

19. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 1.1 x 10⁵ to about 5.4 x 10⁶ CAR-bearing immune effector cells per kg of body weight.

20. The single dose of a pharmaceutical composition of claim 1, wherein the composition comprises from about 1.4 x 10⁵ to about 8.6 x 10⁵ CAR-bearing immune effector cells per kg of body weight.

21. The single dose of a pharmaceutical composition of any of claims 1-20, wherein the T-cell antigen targeted by the CAR, and in which the cells are deficient in cell surface expression, is CD7.

22. The single dose of a pharmaceutical composition of claim 21, wherein the CAR- bearing immune effector cells are T-cells.

23. The single dose of a pharmaceutical composition of claim 22, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is one or more of the T cell receptor complex alpha chain (TCRα/TRAC), the T cell receptor complex beta chain (TCRβ ), the T cell receptor complex delta chain (TCR5), and the T cell receptor complex gamma chain (TCRγ).

24. The single dose of a pharmaceutical composition of claim 23, wherein the subunit of the TCR in which the cells are deficient (in cell surface expression) is the T cell receptor complex alpha chain (TCRα/TRAC).

25. The single dose of a pharmaceutical composition of claim 24, wherein the CAR targeting CD7 comprises: a CD28 co- stimulatory domain; or a 4- IBB co-stimulatory domain; or a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain.

26. The single dose of a pharmaceutical composition of claim 25, wherein the CAR targeting CD7 comprises a 4- IBB co- stimulatory domain and a CD3-zeta effector domain.

27. The single dose of a pharmaceutical composition of claim 25 or 26, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

28. The single dose of a pharmaceutical composition of claim 27, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

29. The single dose of a pharmaceutical composition of claim 28, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

30. The single dose of a pharmaceutical composition of claim 29, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

31. The single dose of a pharmaceutical composition of claim 30, wherein the CAR comprises the amino acid sequence SEQ ID NO:24.

32. The single dose of a pharmaceutical composition of claim 30, wherein the CAR comprises the amino acid sequence SEQ ID NO:30.

33. The single dose of a pharmaceutical composition of any of claims 1-26, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO:22.

34. The single dose of a pharmaceutical composition of claim 33, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NO: 11.

35. The single dose of a pharmaceutical composition of claim 34, wherein the CAR comprises a 4-1BB costimulatory domain comprising SEQ ID NO:9.

36. The single dose of a pharmaceutical composition of claim 35, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

37. The single dose of a pharmaceutical composition of claim 36, wherein the CAR comprises: a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

38. The single dose of a pharmaceutical composition of claim 25 or 26, wherein the CAR is encoded by a nucleotide sequence encoding the amino acid sequence(s) as recited in any of claims 27-37.

39. The single dose of a pharmaceutical composition claim 38, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

40. A pharmaceutical composition comprising a dose of genetically modified T-cells transduced with one or more chimeric antigen receptors (CARs), wherein the one or more CARs targets CD7, has a CD28 costimulatory domain or a 4- 1BB costimulatory domain or both a CD28 costimulatory domain and a 4-1BB costimulatory domain, and has a CD3-zeta effector domain; wherein the T-cells are genetically modified to be deficient in cell surface expression of TCRα/TRAC, and wherein the T-cells are genetically modified to be deficient in cell surface expression of CD7, wherein the dose is effective to treat a T-cell malignancy and prevent grade 3 or grade 4 cytokine release syndrome (CRS).

41. The pharmaceutical composition of claim 40, wherein the genetically modified T cells a) are deficient in CD7 surface expression due to deletion or disruption of the CD7 gene and/or b) express a functionally disabled T-cell receptor (TCR) due to deletion of the T-cell receptor alpha chain (TCRα/TRAC) gene.

42. The pharmaceutical composition of claim 41, wherein the CD7 and/or TCRα/TRAC gene(s) is/are deleted by CRISPR/Cas9 gene editing.

43. The pharmaceutical composition of any of claims 1-42, wherein the T-cell malignancy is a hematological malignancy.

44. The pharmaceutical composition of claim 43, wherein the hematological malignancy is a T-cell malignancy.

45. The pharmaceutical composition of claim 44, wherein the hematological malignancy is a CD7+ T-cell malignancy.

46. The pharmaceutical composition of either of claims 32 or 33, wherein the T-cell malignancy is AML, T-ALL, and non-Hodgkin’s lymphoma.

47. The pharmaceutical composition or single dose thereof of any of claims 1-46, wherein the CRS is grade 3 or grade 4 CRS.

48. The pharmaceutical composition of any of claims 40-47, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

49. The pharmaceutical composition of claim 48, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

50. The pharmaceutical composition of claim 49, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

51. The pharmaceutical composition of claim 50, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

52. The pharmaceutical composition of claim 51, wherein the CAR comprises the amino acid sequence SEQ ID NO: 24.

53. The pharmaceutical composition of claim 51, wherein the CAR comprises the amino acid sequence SEQ ID NO:30.

54. The pharmaceutical composition of any of claims 40-47, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO: 22.

55. The pharmaceutical composition of claim 54, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NO: 11.

56. The pharmaceutical composition of claim 55, wherein the CAR comprises a 4-1BB costimulatory domain comprising SEQ ID NO:9.

57. The pharmaceutical composition of claim 56, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

58. The pharmaceutical composition of claim 57, wherein the CAR comprises: a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

59. The pharmaceutical composition of any of claims 40-47, wherein the CAR is encoded by a nucleotide sequence encoding the amino acid sequence(s) as recited in any of claims 48- 58.

60. The pharmaceutical composition claim 59, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

61. A method of treatment of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 that does not elicit cytokine release syndrome (CRS).

62. A method of achieving complete remission of one or more T-cell malignancies in a patient comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

63. A method of reducing CD7+ malignant cells without causing cytokine release syndrome (CRS) in a patient having a T-cell malignancy, the method comprising administering a dose of UCART7 cells, wherein the CD7+ malignant cells are reduced to an undetectable number of malignant cells.

64. A method of treatment of T-cell malignancies in a patient comprising administration of a dose of UCART7 cells, wherein the levels of interleukin (IL)-l, IL-2, IL-5, IL-6, IL-8, IL-10, IL-13, TNF-α, granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN)γ, and/or fracktalkine are not elevated to a level that would correspond with or result in grade 3 or 4 CRS as a result of treatment.

65. A method of achieving an overall remission in the majority of a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

66. A method of achieving complete remission (CR) in a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

67. A method of achieving complete remission with incomplete hematologic recovery (CRi) in a population of patients having one or more T-cell malignancy, comprising administration of a dose of UCART7 cells, wherein the cells do not elicit cytokine release syndrome (CRS).

68. A method of preventing cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome in a patient being treated for a CD7+ T-cell malignancy, the method comprising administering a dose of UCART7 cells, wherein the dose of UCART7 cells prevents the occurrence of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome.

69. A method of preventing grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose of UCART7 cells to the patient; and evaluating CRS symptoms in the patient.

70. A method of treating grade 3 or higher cytokine release syndrome (CRS) during treatment of T-cell malignancies in a patient, comprising: administering a dose of UCART7 cells to the patient; evaluating CRS symptoms in the patient; wherein if the patient is classified as having grade 3 CRS, administering a therapy for treating CRS in the patient, and if the CRS in the patient reduces to grade 2 or below as a result of the therapy for treating CRS, continuing treatment with UCART7 cells.

71. The method of any of claims 62-70, wherein the T-cell malignancy is a hematological malignancy.

72. The method of claim 71, wherein the hematological malignancy is a T-cell malignancy.

73. The method of claim 72, wherein the hematological malignancy is a CD7+ T-cell malignancy.

74. The method of any of claims 72 or 73, wherein the T-cell malignancy is AML, T- ALL, or lymphoma.

75. The method of claim 74, wherein the lymphoma is non-Hodgkin’s lymphoma.

76. The pharmaceutical composition of any of claims 40-60, or the method of any of claims 61-75, wherein the composition comprises: from about 1 x 10⁵ to about 6.3 x 10⁸ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 18.0 x 10⁶ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 12 x 10⁶ CAR-T cells per kg of body weight; from about 2.0 x 10⁶ to about 6 x 10⁶ CAR-T cells per kg of body weight; from about 6.0 x 10⁶ to about 18 x 10⁶ CAR-T cells per kg of body weight; about 2.0 x 10⁶ CAR-T cells per kg of body weight; about 6.0 x 10⁶ CAR-T cells per kg of body weight; about 12.0 x 10⁶ CAR-T cells per kg of body weight; about 18.0 x 10⁶ CAR-T cells per kg of body weight; from about 100 x 10⁶ to about 900 x 10⁶ CAR-T cells; from about 100 x 10⁶ to about 600 x 10⁶ CAR-T cells; from about 100 x 10⁶ to about 300 x 10⁶ CAR-T cells; from about 300 x 10⁶ to about 900 x 10⁶ CAR-T cells; about 100 x 10⁶ CAR-T cells; about 300 x 10⁶ CAR-T cells; about 600 x 10⁶ CAR-T cells; about 900 x 10⁶ CAR-T cells; from about 1.0 x 10⁵ to about 5 x 10⁶ CAR-T cells per kg of body weight; from about 1.1 x 10⁵ to about 5.4 x 10⁶ CAR-T cells per kg of body weight; or from about 1.4 x 10⁵ to about 8.6 x 10⁵ CAR-T cells per kg of body weight.

77. The method of any of claims 61-86, wherein the dose of UCART7 is administered intravenously in a single dose.

78. The method of any of claims 61-76, wherein the dose of UCART7 is administered intravenously in more than one dose.

79. The method of any of claims 87-77, wherein the dose of UCART7 cells is administered in a clinical setting or a hospital.

80. The method of any of claims 61-79 wherein determining the efficacy of a dose of UCART7 cells comprises evaluation of progression-free survival (PFS) and/or duration of response (DoR) in the patient.

81. The method of any of claims 61-79, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of cytokine release syndrome (CRS), neurotoxicity, and/or tumor lysis syndrome (TLS) in the patient.

82. The method of any of claims 61-79, wherein determination of the safety of a dose of UCART7 cells comprises evaluation of treatment-related toxicity in the patient.

83. The method of any of claims 61-79, wherein toxicity of a dose of UCART7 cells is classified as: any grade 3 or higher neurotoxicity within 28 days of administration of UCART7 cells; any grade 4 CRS of any duration; grade 3 CRS that does not improve to grade 2 within 72 hours; grade 3 or greater infusion reaction; grade 3 or greater vital organ toxicity; grade 2 or greater GVHD; renal failure requiring dialysis for longer than 3 days; any intubation; and/or any grade 3 or 4 nonhematological toxicity lasting for more than 7 days.

84. The method of claim 70, wherein the therapy for treating CRS comprises a monoclonal antibody.

85. The method of claim 84, wherein the monoclonal antibody for treating CRS comprises an anti-interleukin-6 receptor antibody.

86. The method of claim 85, wherein the monoclonal antibody for treating CRS comprises tocilizumab.

87. The method of claim 86, wherein the therapy for treating CRS comprises a small molecule kinase inhibitor.

88. The method of claim 87, wherein the small molecule kinase inhibitor comprises a Janus kinase (JAK) inhibitor.

89. The method of either of claims 87 or 88, wherein the small molecule kinase inhibitor comprises Ruxolitinib (Jakofi®), Baricitinib, itacitinib, or Dasatinib.

90. The method of any of claims 61-89, wherein the patient has not had any anti-CD7 therapy prior to treatment.

91. The method of any of claims 61-90, wherein the patient does not have grade 3 or 4 graft- versus-host-disease (GVHD) .

92. The method of any of claims 61-91, wherein the patient has grade 2 GVHD treatable with topical therapy.

93. The method of any of claims 61-92, wherein the patient is pre-treated with cyclophosphamide at a dose of 500 mg/m² IV daily for 3 days, and fludarabine at a dose of 30 mg/m² IV daily for 3 days before treatment with UCART7.

94. The method of any of claims 61-93, wherein the patient does not exhibit profound and persistent T cell aplasia before administration of UCART7 cells.

95. The method of any of claims 61-94, wherein the patient exhibits T cell aplasia as a result of chemotherapeutic therapy before administration of UCART7 cells.

96. The method of any of claims 61-95, wherein lymphodepletion occurs on days 1-3, wherein UCART7 cells are administered 3 days after the lymphodepletion period.

97. The method of any of claims 61-96, wherein the patient is observed twice weekly for 3 weeks after administration of UCART7 cells, then weekly for 4 weeks, then monthly for a period of 5 months, then monthly until no more UCART7 cells are detectable in the patient, then quarterly for 2 years, then annually through year 15.

98. The method of any of claims 61-97, wherein cellular PK samples are obtained from the patient at each visit until no UCART7 cells are detectable in the patient.

99. The method of any of claims 61-98, wherein cellular PK samples comprise one or more of AUC(0-∞ ), AUC(_0-Ƭ), maximum drug concentration in the body after dosing [C_max], and Tmax-

100. The method of any of claims 61-99, wherein complete remission (CR) for a patient having ALL is classified as:

101. The method of any of claims 61-99, wherein complete remission (CR) for a pediatric patient having ALL is classified as: complete donor chimerism (≥ 95% donor chimerism without recipient cells detected); no extramedullary disease; neutrophil count ≥ 1000/μL; platelets ≥ 100,000/μL; and transfusion independent.

102. The method of any of claims 100 or 101, wherein the extramedullary disease comprises CNS or soft tissue disease.

103. The method of any of claims 61-99, wherein complete remission with incomplete hematologic recovery (CRi) for a patient having ALL is classified as:

104. The method of any of claims 61-103, wherein the CAR comprises an amino acid sequence having at least 95% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

105. The method of claim 104, wherein the CAR comprises an amino acid sequence having at least 98% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

106. The method of claim 105, wherein the CAR comprises an amino acid sequence having at least 99% identity to a sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

107. The method of claim 106, wherein the CAR comprises an amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:30.

108. The method of claim 107, wherein the CAR comprises the amino acid sequence SEQ ID NO:24.

109. The method of claim 107, wherein the CAR comprises the amino acid sequence SEQ ID NO:30.

110. The method of any of claims 61-103, wherein the CAR comprises a light chain variable region (V_L) comprising SEQ ID NO:23 and a heavy chain variable region (V_H) comprising SEQ ID NO:22.

111. The method of claim 110, wherein the CAR comprises a CD3 zeta signaling domain comprising SEQ ID NO: 11.

112. The method of claim 111, wherein the CAR comprises a 4- IBB costimulatory domain comprising SEQ ID NO:9.

113. The method of claim 112, wherein the CAR comprises: a CD8α hinge comprising SEQ ID NO:6; a peptide linker comprising SEQ ID NO: 13 between the (V_L) and (V_H) domains; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; and a CD8α signal peptide comprising SEQ ID NO:1.

114. The method of claim 113, wherein the CAR comprises : a CD8α signal peptide comprising SEQ ID NO:1; a CD28 Transmembrane (T_m) domain comprising SEQ ID NO. 7; a light chain variable region comprising SEQ ID NO: 23; a peptide linker comprising SEQ ID NO: 13; a heavy chain variable region comprising SEQ ID NO: 22; a CD8α hinge comprising SEQ ID NO:6; a 4- IBB costimulatory domain comprising SEQ ID NO:9; and a CD3 zeta signaling domain comprising SEQ ID NO: 11.

115. The method of any of claims 61-103, wherein the CAR is encoded by a nucleotide sequence chosen from SEQ ID NO:31 and SEQ ID NO:32.

116. The method of any of claims 1-39, or the pharmaceutical composition of any of claims 40-60, or the method of any of claims 61-115, wherein the CAR-bearing immune effector cell or CAR-T cell or UCART7 does not comprise a membrane-bound IL- 15 protein or IL-15/IL- 15R fusion protein or a constitutively active IL-7R protein.