Current limitations in using chimeric antigen receptor T(CART) cells to treat patients with hematological cancers include limited expansion and persistence in vivo that contribute to cancer relapse. Patients with chronic lymphocytic leukemia (CLL) have terminally differentiated T cells with an exhausted phenotype and experience low complete response rates after autologous CART therapy. Because PI3K inhibitor therapy is associated with the development of T-cell–mediated autoimmunity, we studied the effects of inhibiting the PI3Kδ and PI3Kγ isoforms during the manufacture of CART cells prepared from patients with CLL. Dual PI3Kδ/γ inhibition normalized CD4/CD8 ratios and maximized the number of CD8+ T-stem cell memory, naive, and central memory T-cells with dose-dependent decreases in expression of the TIM-3 exhaustion marker. CART cells manufactured with duvelisib (Duv-CART cells) showed significantly increased in vitro cytotoxicity against CD19+ CLL targets caused by increased frequencies of CD8+ CART cells. Duv-CART cells had increased expression of the mitochondrial fusion protein MFN2, with an associated increase in the relative content of mitochondria. Duv-CART cells exhibited increased SIRT1 and TCF1/7 expression, which correlated with epigenetic reprograming of Duv-CART cells toward stem-like properties. After transfer to NOG mice engrafted with a human CLL cell line, Duv-CART cells expressing either a CD28 or 41BB costimulatory domain demonstrated significantly increased in vivo expansion of CD8+ CART cells, faster elimination of CLL, and longer persistence. Duv-CART cells significantly enhanced survival of CLL-bearing mice compared with conventionally manufactured CART cells. In summary, exposure of CART to a PI3Kδ/γ inhibitor during manufacturing enriched the CART product for CD8+ CART cells with stem-like qualities and enhanced efficacy in eliminating CLL in vivo.

Chimeric antigen receptor (CAR) T cell therapy has been successful in treating B cell malignancies in clinical trials; however, fewer studies have evaluated CAR T cell therapy for the treatment of T cell malignancies. There are many challenges in translating this therapy for T cell disease, including fratricide, T cell aplasia, and product contamination. To the best of our knowledge, no tumor-specific antigen has been identified with universal expression on cancerous T cells, hindering CAR T cell therapy for these malignancies. Numerous approaches have been assessed to address each of these challenges, such as (i) disrupting target antigen expression on CAR-modified T cells, (ii) targeting antigens with limited expression on T cells, and (iii) using third party donor cells that are either non-alloreactive or have been genome edited at the T cell receptor α constant (TRAC) locus. In this review, we discuss CAR approaches that have been explored both in preclinical and clinical studies targeting T cell antigens, as well as examine other potential strategies that can be used to successfully translate this therapy for T cell disease.

Key Points: Number of prior chemotherapy cycles in cancer patients correlates with T-cell senescent phenotype and loss of CD27 and CD28 expression. Addition of PI3Kδ inhibitors and VIP antagonists increased ex vivo expansion, in vivo persistence, and anticancer cytotoxicity of T cells.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Lineage switch (LS), defined as the immunophenotypic transformation of acute leukemia, has emerged as a mechanism of relapse after antigen-targeted immunotherapy, which is associated with dismal outcomes. Through an international collaborative effort, we identified cases of LS after a host of antigen-targeted therapies (eg, CD19, CD22, CD38, and CD7), described how LS was diagnosed, reviewed treatment approaches, and analyzed overall outcomes for this form of postimmunotherapy relapse. Collectively, 75 cases of LS were evaluated, including 53 (70.7%) cases of B-cell acute lymphoblastic leukemia (B-ALL) transforming to acute myeloid leukemia (AML), 17 (22.7%) cases of B-ALL transforming to mixed phenotypic acute leukemia (MPAL)/acute leukemias of ambiguous lineage (ALAL), and 5 (6.7%) cases of rare LS presentation (ie, T-cell ALL to AML). An additional 10 cases with incomplete changes in immunophenotype, referred to as “lineage drift” were also described. With a primary focus on the 70 cases of LS from B-ALL to AML or MPAL/ALAL, LS emerged at a median of 1.5 months (range, 0-36.5) after immunotherapy, with 81.4% presenting with LS within the first 6 months from the most proximal immunotherapy. Although most involved KMT2A rearrangements (n = 45, 64.3%), other rare cytogenetic and/or molecular alterations were uniquely observed. Treatment outcomes were generally poor, with remission rates of <40%. The median overall survival after LS diagnosis was 4.8 months. Outcomes were similarly poor for those with rare immunophenotypes of LS or lineage drift. This global initiative robustly categorizes lineage changes after immunotherapy and, through enhanced understanding, establishes a foundation for improving outcomes of LS.