Shelly Heimfeld

Genome-wide association studies have identified many noncoding variants associated with common diseases and traits. We show that these variants are concentrated in regulatory DNA marked by deoxyribonuclease I (DNase I) hypersensitive sites (DHSs). Eighty-eight percent of such DHSs are active during fetal development and are enriched in variants associated with gestational exposure-related phenotypes. We identified distant gene targets for hundreds of variant-containing DHSs that may explain phenotype associations. Disease-associated variants systematically perturb transcription factor recognition sequences, frequently alter allelic chromatin states, and form regulatory networks. We also demonstrated tissue-selective enrichment of more weakly disease-associated variants within DHSs and the de novo identification of pathogenic cell types for Crohn's disease, multiple sclerosis, and an electrocardiogram trait, without prior knowledge of physiological mechanisms. Our results suggest pervasive involvement of regulatory DNA variation in common human disease and provide pathogenic insights into diverse disorders.

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.

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.

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.

BACKGROUND: In recipients of allogeneic hematopoietic-cell transplants, peripheral-blood cells mobilized with the use of filgrastim (recombinant granulocyte colony-stimulating factor) engraft more rapidly than bone marrow. However, the relative effects of these techniques on the rates of acute and chronic graft-versus-host disease, overall survival, and disease-free survival have not been determined in randomized studies. METHODS: Between March 1996 and July 1999, 172 patients (12 to 55 years of age) with hematologic cancer were randomly assigned to receive either bone marrow or filgrastim-mobilized peripheral-blood cells from HLA-identical relatives for hematopoietic rescue after the treatment of hematologic cancer with high doses of chemotherapy, with or without radiation. RESULTS: The recovery of both neutrophils and platelets was faster with peripheral-blood cells than with marrow (P<0.001 for both comparisons). The cumulative incidence of grade II, III, or IV acute graft-versus-host disease at 100 days was 64 percent with peripheral-blood cells and 57 percent with marrow (hazard ratio, 1.21; 95 percent confidence interval, 0.81 to 1.81; P=0.35). The cumulative incidence of chronic graft-versus-host disease was 46 percent with peripheral-blood cells and 35 percent with marrow (hazard ratio, 1.16; 95 percent confidence interval, 0.71 to 1.90; P=0.54). The estimated overall probability of survival at two years was 66 percent with peripheral-blood cells and 54 percent with marrow (hazard ratio for death, 0.62; 95 percent confidence interval, 0.38 to 1.02; P=0.06). The rate of disease-free survival at two years was 65 percent with peripheral-blood cells and 45 percent with marrow (hazard ratio for relapse or death, 0.60; 95 percent confidence interval, 0.38 to 0.95; P=0.03). CONCLUSIONS: In patients given high-dose chemotherapy, with or without radiation, for the treatment of hematologic cancer, allogeneic peripheral-blood cells used for hematopoietic rescue restore blood counts faster than allogeneic bone marrow, without increasing the risk of graft-versus-host disease.