Chimeric antigen receptor (CAR) T cell therapy can achieve outstanding response rates in heavily pretreated patients with hematological malignancies. However, relapses occur and they limit the efficacy of this promising treatment approach. The cellular composition and immunophenotype of the administered CART cells play a crucial role for therapeutic success. Less differentiated CART cells are associated with improved expansion, long-term in vivo persistence, and prolonged anti-tumor control. Furthermore, the ratio between CD4+ and CD8+ T cells has an effect on the anti-tumor activity of CART cells. The composition of the final cell product is not only influenced by the CART cell construct, but also by the culturing conditions during ex vivo T cell expansion. This includes different T cell activation strategies, cytokine supplementation, and specific pathway inhibition for the differentiation blockade. The optimal production process is not yet defined. In this review, we will discuss the use of different CART cell production strategies and the molecular background for the generation of improved CART cells in detail.

Despite high response rates after initial chemotherapy in patients with acute myeloid leukemia (AML), relapses occur frequently, resulting in a five-year-survival by <30% of the patients. Hitherto, allogeneic hemotopoietic stem cell transplantation (allo-HSCT) is the best curative treatment option in intermediate and high risk AML. It is the proof-of-concept for T cell-based immunotherapies in AML based on the graft-versus-leukemia (GvL)-effect, but it also bears the risk of graft-versus-host disease. CD19-targeting therapies employing chimeric antigen receptor (CAR) T cells are a breakthrough in cancer therapy. A similar approach for myeloid malignancies is highly desirable. This article gives an overview on the state-of-the art of preclinical and clinical studies on suitable target antigens for CAR T cell therapy in AML patients.

Introduction: Therapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (T_N) vs. effector (T_E) T cells, T_N cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the T_N/T_E ratio of CART cells. Materials and methods: CART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays. Results: IL-7/IL-15 preferentially induced differentiation into T_N, stem cell memory (T_SCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (T_EM), CD56+ and CD4+ T regulatory (T_Reg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CART_N cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CART_N cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CART_N cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CART_N cells in untreated CLL patients. Final T_N/T_E ratio stayed 2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CART_N expansion. Conclusion: Untreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of T_N among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the T_N/T_E ratio within the CART product.

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B‐cell lymphoma or acute lymphoblastic leukemia. Impaired T cell fitness of CLL patients may be involved in treatment failure. Less‐differentiated naïve‐like T cells play an important role in CART expansion and long‐term persistence in vivo . These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B‐cell receptor inhibitor ibrutinib targeting bruton tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin‐2‐inducible T cell kinase (ITK) which is involved in T cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells (PBMCs) from 9 healthy donors (HD) and 8 CLL patients were used to generate CART cells. T cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient‐derived CART cells. Furthermore, ibrutinib enriched CART cells with less‐differentiated naïve‐like phenotype and decreased expression of exhaustion markers including PD‐1, TIM‐3, and LAG‐3. In addition, ibrutinib increased the cytokine release capacity of CLL patient‐derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient‐derived CART cell products.