Key Points: T cells transduced with a CD5 CAR demonstrate limited and transient fratricide and expand ex vivo. CD5 CAR T cells eliminate T-ALL blasts in vitro and control disease progression in xenograft T-ALL mouse models.

The successful immunotherapy of acute myeloid leukemia (AML) has been hampered because most potential antigenic targets are shared with normal hematopoietic stem cells (HSCs), increasing the risk of sustained and severe hematopoietic toxicity following treatment. C-type lectin-like molecule 1 (CLL-1) is a membrane glycoprotein expressed by >80% of AML but is absent on normal HSCs. Here we describe the development and evaluation of CLL-1-specific chimeric antigen receptor T cells (CLL-1.CAR-Ts) and we demonstrate their specific activity against CLL-1+ AML cell lines as well as primary AML patient samples in vitro. CLL-1.CAR-Ts selectively reduced leukemic colony formation in primary AML patient peripheral blood mononuclear cells compared to control T cells. In a human xenograft mouse model, CLL-1.CAR-Ts mediated anti-leukemic activity against disseminated AML and significantly extended survival. By contrast, the colony formation of normal progenitor cells remained intact following CLL-1.CAR-T treatment. Although CLL-1.CAR-Ts are cytotoxic to mature normal myeloid cells, the selective sparing of normal hematopoietic progenitor cells should allow full myeloid recovery once CLL-1.CAR-T activity terminates. To enable elective ablation of the CAR-T, we therefore introduced the inducible caspase-9 suicide gene system and we show that exposure to the activating drug rapidly induced a controlled decrease of unwanted CLL-1.CAR-T activity against mature normal myeloid 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.

Objectives: As the prognosis of relapsed/refractory (R/R) acute myeloid leukaemia (AML) remains poor, novel treatment strategies are urgently needed. Clinical trials have shown that chimeric antigen receptor (CAR)‐T cells for AML are more challenging than those targeting CD19 in B‐cell malignancies. We recently developed piggyBac‐modified ligand‐based CAR‐T cells that target CD116/CD131 complexes, also known as the GM‐CSF receptor (GMR), for the treatment of juvenile myelomonocytic leukaemia. This study therefore aimed to develop a novel therapeutic method for R/R AML using GMR CAR‐T cells.Methods: To further improve the efficacy of the original GMR CAR‐T cells, we have developed novel GMR CAR vectors incorporating a mutated GM‐CSF for the antigen‐binding domain and G4S spacer. All GMR CAR‐T cells were generated using a piggyBac‐based gene transfer system. The anti‐tumor effect of GMR CAR‐T cells was tested in mouse AML xenograft models.Results: Nearly 80% of the AML cells predominant in myelomonocytic leukaemia were found to express CD116. GMR CAR‐T cells exhibited potent cytotoxic activities against CD116⁺ AML cells in vitro. Furthermore, GMR CAR‐T cells incorporating a G4S spacer significantly improved long‐term in vitro and in vivo anti‐tumor effects. By employing a mutated GM‐CSF at residue 21 (E21K), the anti‐tumor effects of GMR CAR‐T cells were also improved especially in long‐term in vitro settings. Although GMR CAR‐T cells exerted cytotoxic effects on normal monocytes, their lethality on normal neutrophils, T cells, B cells and NK cells was minimal.Conclusions: GMR CAR‐T cell therapy represents a promising strategy for CD116⁺ R/R AML.

We investigated the potential of nutritional and inflammatory parameters as prognostic factors for follicular lymphoma (FL), and also examined the predictive value of the early progression of disease within 24 months of first-line chemo-immunotherapy (POD24). We retrospectively analyzed 46 patients with FL admitted to Teikyo University Hospital and treated with chemo-immunotherapy between May 2009 and July 2019. Physical characteristics, blood parameters, and markers or scores for consumptive/inflammatory and nutritional conditions were used as variables. Nine parameters correlated with poor overall survival (OS) in univariate analysis: An Eastern Cooperative Oncology Group (ECOG) scale performance status (PS) ≥2, five or more involved nodal sites, positive bone marrow (BM) involvement, a serum albumin level 0.5 mg/dL, lactate dehydrogenase (LD) higher than the upper normal limit (UNL), high-density lipoprotein cholesterol (HDL-C) <40 mg/dL, modified Glasgow prognostic score of 1–2, and the geriatric nutritional risk index <82. In multivariate analysis, ECOG PS ≥2, positive BM involvement, and a serum HDL-C level UNL, and HDL-C <40 mg/dL by Fisher’s exact test. These results indicate that low serum HDL-C levels appear to be important for predicting the risk of POD24 and the worse prognosis of FL.

Although autologous hematopoietic cell transplantation (HCT) is an established therapy for patients with relapsed acute promyelocytic leukemia (APL) after returning to complete remission (CR), the role of allogeneic HCT remains unclear for treating relapsed APL. This study aimed to investigate allogeneic HCT outcomes in patients with relapsed APL, focusing particularly on those who underwent transplantation in non-CR and those who had relapsed after prior autologous HCT. We retrospectively analyzed Japanese nationwide transplantation registry data of patients with relapsed APL age ≥16 years who underwent allogeneic HCT between 2006 and 2020. A total of 195 patients were eligible for this analysis, including 69 who underwent transplantation in non-CR and 55 who relapsed after prior autologous HCT. The median duration of follow-up for survivors was 5.4 years. Multivariate analysis revealed that both non-CR at transplantation (hazard ratio [HR], 1.74; 95% confidence interval [CI], 1.12 to 2.71; P = .014) and prior autologous HCT (HR, 2.10; 95% CI, 1.28 to 3.44; P = .013) were associated with higher risks of overall mortality. The 5-year overall survival (OS) rates for patients who underwent transplantation in CR and non-CR were 58% and 39%, respectively (P = .085), if they did not have a history of prior autologous HCT. In the patients who had relapsed after prior autologous HCT, the 5-year OS rate was 47% for those who underwent allogeneic HCT in CR and 6% for those who did so in non-CR (P = .001). Allogeneic HCT still provides an opportunity for long-term survival for certain patients with relapsed APL for whom autologous HCT is unlikely to be effective. The dismal outcome of those with prior autologous HCT who underwent allogeneic HCT in non-CR poses a significant therapeutic challenge.