Sandhya Sharma

CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies

Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7+) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies.

Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies

CAR T cells targeting malignant T cells can form unwanted cytotoxic immunologic synapses between themselves, impairing their survival. CAR-derived 4-1BB costimulation stabilized these synapses, and reversing CAR expression overcame unwanted fratricide while retaining antitumor activity. T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell–T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB–mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo. These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. Cancer Immunol Res; 6(1); 47–58. ©2017 AACR.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody–Activated Chimeric Antigen Receptor–Modified T Cells

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO+CCR7− effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28–activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell–derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro–expanded T cells.

Disparities in Receipt of Radiotherapy and Survival by Age, Sex, and Ethnicity among Patient with Stage I Follicular Lymphoma.

Background Radiotherapy (RT) is a first-line treatment option for stage I follicular lymphoma (FL). We studied disparities in receipt of RT and survival among patients with stage I FL. Methods Adult patients (age ≥18 years) with stage I FL, as the first primary cancer, diagnosed between 1992 and 2007 were identified using Surveillance, Epidemiology, and End Results (SEER) 18 database. Study population was divided into various subgroups based on age, sex, race, and marital status. Factors associated with receipt of RT and survival, among patients receiving RT, was evaluated using regression analysis and Cox PH modeling, respectively. SEER*Stat was used to compute 1- and 5-year RS for various subgroups and compared using Z score. Results Of the total 7315 patients (median age: 64 years), 2671 (36.5%) received RT. African-Americans, older age group, and single and separated/divorced/widow marital status predicted omission of RT. The 1- and 5-year RS were significantly better in patients receiving RT. In multivariate analysis, male sex, age <60 years, Caucasian race, and married marital status were found to be independent predictor of better RS among patients receiving RT (P < 0.0001). Conclusion This study showed that 36.5% patients with stage I FL received RT. Survival rates were significantly better for patients who received RT.

Increasing the purity, potency and specificity of ebv-specific T cells to improve the treatment of EBV-positive lymphoma

Meeting abstracts Up to ~40 % of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express type 2 viral latency proteins (T2LPs) EBNA-1, LMP-1, LMP-2 and BARF-1. EBV specific T cells (EBVSTs) can be generated from the blood of EBV+ individuals, but are usually