Colette Chaney

CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

BACKGROUND T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION ClinicalTrials.gov NCT01865617. FUNDING R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8 + and CD4 + CD19-specific chimeric antigen receptor-modified T cells

A CD19 chimeric antigen receptor–modified T cell product with defined composition has potent antitumor activity. Standardizing the CAR assembly line Chimeric antigen receptor (CAR)–modified T cells are engineered to recognize specific tumor antigens. They have shown promising results in clinical trials, primarily in leukemia so far, but it has been difficult to predict therapeutic efficacy and toxicity for individual patients. To address this issue, Turtle et al. treated non-Hodgkin’s lymphoma patients with CAR-T cells prepared from strictly defined subsets. By carefully controlling the ratio of CD4 to CD8 T cells, the authors were able to identify some of the treatment characteristics that correlate with therapeutic response and toxicity, including the role of the drug regimen used for lymphodepletion before CAR-T cell treatment. CD19-specific chimeric antigen receptor (CAR)–modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4+/CD8+ ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin’s lymphoma after cyclophosphamide (Cy)–based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4+/CD8+ ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients

Donor-derived WT1-specific CD8+ T cells transferred after hematopoietic cell transplantation mediate antileukemic activity and can establish persistent responses without toxicity to normal tissues in high-risk leukemia patients. Targeting Leukemic Cells for Destruction After a patient is diagnosed with leukemia, the first line of treatment is generally chemotherapy. If it doesn’t work, the patient can get a bone marrow transplant, which can sometimes cure otherwise untreatable leukemia. However, for patients who have already relapsed after a transplant, or whose cancer has particularly unfavorable characteristics, few options remain. Such patients’ prognosis is generally very poor, with a high risk of relapse and death from leukemia. Immune cells derived from the donor bone marrow can help fight the cancer by attacking malignant cells inside the graft recipient. At the same time, however, they often attack the recipient’s healthy cells as well and cause graft-versus-host disease (GVHD), which can itself be lethal. Now, Chapuis and coauthors report a way to harness the power of the donors’ immune cells against some leukemias, without triggering GVHD in the bone marrow recipients. In this pilot trial, the authors enrolled 11 patients who had leukemia with poor prognostic characteristics and who had each undergone a bone marrow transplant. The patients then received T cells that were derived from their respective donors and selected for their ability to recognize Wilms tumor antigen 1 (WT1). Very small amounts of WT1 are present in some healthy cells, but its expression in malignant cells is much greater and corresponds to the aggressiveness of the cancer. In the first seven patients, the authors found that T cells that recognized WT1 could suppress the leukemia temporarily, but the new cells disappeared within 14 days, and the cancer rebounded. In the last four patients, the authors tried a modified protocol, pretreating the cells with interleukin-21 (IL-21) before infusion into the recipients. T cells pretreated with IL-21 developed characteristics of memory cells, including a greatly extended life span. At this time, all four patients who received IL-21–treated T cells are still alive, with no recurrence of their cancer up to 30 months after the T cell infusion. Notably, none of the patients who received WT1-specific T cells in this study have developed GVHD, supporting the idea that WT1 targeting is specific to the tumor cells and safe for patient use. The study by Chapuis et al. is a small pilot trial, and its results must be replicated with larger groups of patients before this protocol can become standard practice. Nevertheless, the combination of targeting WT1 in leukemia and pretreating leukemia-targeting T cells with IL-21 to extend their life span looks very promising and offers a potential safe and effective treatment for patients who have few other options. Relapse remains a leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk leukemias. The potentially beneficial donor T cell–mediated graft-versus-leukemia (GVL) effect is often mitigated by concurrent graft-versus-host disease (GVHD). Providing T cells that can selectively target Wilms tumor antigen 1 (WT1), a transcription factor overexpressed in leukemias that contributes to the malignant phenotype, represents an opportunity to promote antileukemic activity without inducing GVHD. HLA-A*0201–restricted WT1-specific donor-derived CD8+ cytotoxic T cell (CTL) clones were administered after HCT to 11 relapsed or high-risk leukemia patients without evidence of on-target toxicity. The last four treated patients received CTL clones generated with exposure to interleukin-21 (IL-21) to prolong in vivo CTL survival, because IL-21 can limit terminal differentiation of antigen-specific T cells generated in vitro. Transferred cells exhibited direct evidence of antileukemic activity in two patients: a transient response in one patient with advanced progressive disease and the induction of a prolonged remission in a patient with minimal residual disease (MRD). Additionally, three treated patients at high risk for relapse after HCT survive without leukemia relapse, GVHD, or additional antileukemic treatment. CTLs generated in the presence of IL-21, which were transferred in these latter three patients and the patient with MRD, all remained detectable long term and maintained or acquired in vivo phenotypic and functional characteristics associated with long-lived memory CD8+ T cells. This study supports expanding efforts to immunologically target WT1 and provides insights into the requirements necessary to establish potent persistent T cell responses.

Outcomes of acute leukemia patients transplanted with naive T cell–depleted stem cell grafts

BACKGROUND Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (TN) cause more severe GVHD than memory T cells (TM). We hypothesized that selective depletion of TN from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and TM to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively. METHODS In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with TN-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of TM purged of CD45RA+ TN. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution. RESULTS All recipients of TN-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell-replete grafts. TM in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years. CONCLUSION Depletion of TN from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory. TRIAL REGISTRATION ClinicalTrials.gov (NCT 00914940).

Leukemia-associated minor histocompatibility antigen discovery using T-cell clones isolated by in vitro stimulation of naïve CD8 + T cells

T-cell immunotherapy that targets minor histocompatibility (H) antigens presented selectively by recipient hematopoietic cells, including leukemia, could prevent and treat leukemic relapse after hematopoietic cell transplantation without causing graft-versus-host disease. To provide immunotherapy that can be applied to a majority of transplantation recipients, it is necessary to identify leukemia-associated minor H antigens that result from gene polymorphisms that are balanced in the population and presented by common human leukocyte antigen alleles. Current approaches for deriving minor H antigen-specific T cells, which provide essential reagents for the molecular identification and characterization of the polymorphic genes that encode the antigens, rely on in vivo priming and are often unsuccessful. We show that minor H antigen-specific cytotoxic T lymphocyte precursors are found predominantly in the naive CD8(+) T-cell subset and provide an efficient strategy for in vitro priming of native T cells to generate T cells to a broad diversity of minor H antigens presented with common human leukocyte antigen alleles. We used this approach to derive a panel of stable cytotoxic T lymphocyte clones for discovery of genes that encode minor H antigens and identify a novel antigen expressed on acute myeloid leukemia stem cells and minimally in graft-versus-host disease target tissues.