Martin Pule

Targeted immunotherapies have revolutionized outcomes for lymphoid malignancies, but success in myeloid neoplasms is limited by the lack of amenable targets and immunologically hostile tumor microenvironment (TME). Myeloproliferative neoplasms (MPNs) are chronic myeloid blood cancers, a third of which are driven by mutations in calreticulin. Calreticulin mutant protein (mutCALR) binds and activates thrombopoietin receptor (TpoR) and results in the display of the mutCALR-TpoR complex on the extracellular membrane of disease-driving cells, thus exposing a therapeutic vulnerability. Here, we present a chimeric antigen receptor T cell (CAR T cell) therapy that specifically targets mutCALR+ cells, both in vitro and in vivo. The CAR T cell therapy selectively depleted mutCALR+ stem cells in samples from patients with myelofibrosis without affecting healthy stem cells and improved survival in mutCALR leukemia xenograft models. To mimic myelofibrotic marrow, we developed a human “chimeroid” model and showed no decrease in the potency of CAR T cell–mediated target cell killing even in a fibrotic TME. We also devised a method to boost the cell surface expression of mutCALR in CD34+ cells isolated from patients with accelerated/blast phase MPNs (defined as >10% blasts in the peripheral blood or bone marrow), enhancing CAR T cell targeting. This study presents a therapeutic strategy with potential to eradicate mutCALR-driven malignancies and highlights an innovative strategy to evaluate blood cancer–targeting immunotherapies in a relevant TME.

Targeted immunotherapies have revolutionized outcomes for lymphoid malignancies, but success in myeloid neoplasms is limited by the lack of amenable targets and immunologically hostile tumor microenvironment (TME). Myeloproliferative neoplasms are chronic myeloid blood cancers, a third of which are driven by mutations in calreticulin (mutCALR). This yields a common neoepitope that binds to, and activates, the thrombopoietin receptor and results in display of the oncoprotein on the extracellular membrane of disease-driving cells, exposing a therapeutic vulnerability. Here, we present a first-in-class chimeric antigen receptor (CAR) T-cell therapy that specifically targets mutCALR+ cells, both in vitro and in vivo. The CAR T-cell therapy selectively depleted mutCALR+ stem cells from patients with myelofibrosis while sparing healthy stem cells, and improved survival in mutCALR leukemia xenografts. To mimic myelofibrotic marrow, we developed a bespoke human ‘chimeroid’ model and showed no decrease in the potency of CAR T cell-mediated target cell killing even in a fibrotic tumor microenvironment. We also devised a method to boost cell surface expression of mutCALR in CD34+ cells isolated from patients with accelerated/blast phase MPN (defined as >10 % blasts in peripheral blood or bone marrow), enhancing CAR T targeting. This study presents a therapeutic with potential to eradicate mutCALR-driven malignancies and highlights an innovative strategy to evaluate blood cancer-targeting immunotherapies in a relevant TME. One Sentence Summary: A first-in-class CAR T-cell therapy targeting mutant calreticulin selectively depletes malignant stem cells in vivo and in fibrotic human organoids.

Supplementary Table 1: Key Resources

Supplementary Table S2. Top 50 up-regulated GSEA pathways.

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor–specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

Patients with relapsed/refractory (r/r) T cell acute lymphoblastic leukemia (T-ALL) have a dismal prognosis, highlighting the urgent need for effective therapies. Chimeric antigen receptor (CAR)–T cell approaches targeting pan–T cell antigens may be limited by T cell aplasia and fratricide, necessitating “rescue” allogeneic hematopoietic stem cell transplantation. In this study, we identify CD21, a pan–B cell marker, as a promising target for T-ALL immunotherapy. CD21 is expressed in 50% of T-ALL cases at diagnosis but in fewer than 10% of mature T cells. We observed that CAR-T cells targeting membrane-distal CD21 epitopes were ineffective, likely because of the bulky, glycosylated nature of the antigen. However, when we engineered CAR-T cells to target membrane-proximal CD21 epitopes using an antigen-binding fragment (Fab)–CAR design, we demonstrated robust activity against T-ALL cell lines, primary tumors, and patient-derived xenografts in both in vitro and in vivo models. The enhanced efficacy of this Fab-CAR design was driven by its high stability and reduced surface expression, addressing limitations of traditional CAR constructs. In addition, pharmacological inhibition of the phosphatidylinositol 3-kinase axis up-regulated CD21 expression in T-ALL, further enhancing the potency of anti-CD21 CAR-T cells in vitro and in a patient-derived xenograft in vivo model. This study establishes CD21 as a viable CAR-T target and highlights advances in CAR design for bulky antigens, as well as the potential for pharmacological strategies to augment target expression. Anti-CD21 CAR-T cells represent a promising therapeutic option for improving outcomes for patients with T-ALL.

Relapsed/refractory peripheral T cell lymphomas (PTCLs) are aggressive tumors with a poor prognosis. Unlike B cell lymphomas, treatment of PTCL has not benefited from advances in immunotherapy. This is largely due to a lack of suitable target antigens that discriminate malignant from normal T cells, thus avoiding severe immunosuppression consequent to depletion of the entire T cell compartment. We recently described a targeting strategy based on the mutually exclusive expression of T cell antigen receptor beta-chain constant domain (TRBC) 1 and 2. Selective targeting of the T cell antigen receptor beta-chain expressed by the (clonal) malignancy spares normal T cells expressing the other chain. The LibraT1 study is an ongoing, multicenter, international, single-arm phase 1/2 study of TRBC1-directed autologous chimeric antigen receptor (CAR) T cells (AUTO4) in relapsed/refractory TRBC1-positive PTCL. Primary objectives were assessment of safety and tolerability of AUTO4 infusion. Key secondary endpoints included efficacy, CAR T cell expansion and persistence. Here we describe the findings from dose escalation in LibraT1 in the first ten patients, in a non-prespecified interim analysis. AUTO4 resulted in low frequency of severe immunotoxicity, with one of ten patients developing grade 3 cytokine release syndrome. Complete metabolic response was observed in four of ten evaluable patients, with remissions being durable beyond 1 year in two patients. While an absence of circulating CAR T cells was observed, CAR T cells were readily detected in lymph node biopsy samples from sites of original disease suggesting homing to tumor sites. These results support the continuing exploration of TRBC1 targeting in PTCL. ClinicalTrials.gov registration: NCT03590574.