Characterization and Risk Factor Analysis of Osteoporosis in a Large Cohort of Patients with Chronic Graft-versus-Host Disease.

The National Institutes of Health Chronic Graft-versus-Host Disease (cGVHD) Consensus Project Ancillary and Supportive Care Guidelines recommend annual assessment of bone mineral density (BMD) to monitor bone health. The study of osteoporosis in patients with cGVHD has been limited to small numbers of patients, and the guidelines are based on experience with other chronic diseases and expert opinion. We hypothesized that the prevalence of osteoporosis is high in a cohort of 258 patients with moderate to severe cGVHD because of prolonged exposure to risk factors for osteoporosis after allogeneic hematopoietic stem cell transplantation. We defined osteoporosis using BMD criteria (T-score ≤-2.5) at 3 anatomic sites-the femoral neck (FN), lumbar spine (LS), and total hip (TH)-and characterized risk factors through univariate and multivariate analyses. We found that low body weight (FN, P < .0001; LS, P = .0002; TH, P < .0001), malnutrition (FN, P = .0002; LS, P = .03; TH, P = .0076), higher platelet count (FN, P = .0065; TH, P = .0025), higher average National Institutes of Health organ score (FN, P = .038), higher prednisone dose (LS, P = .032), lower complement component 3 (LS, P = .0073), and physical inactivity (FN, P = .01) were associated with osteoporosis in at least 1 site. T-scores were significantly lower in the FN compared with the LS or TH (P < .0001 for both). The prevalence of osteoporosis and osteopenia was high (17% and 60%, respectively), supporting current recommendations for frequent monitoring of BMD. The association of higher platelet count in patients with cGVHD and osteoporosis has not been reported previously and represents a new area of interest in the study of osteoporosis after allogeneic hematopoietic stem cell transplantation.

Phase 2 clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell transplantation.

In experimental models, ex vivo induced T-cell rapamycin resistance occurred independent of T helper 1 (Th1)/T helper 2 (Th2) differentiation and yielded allogeneic CD4(+) T cells of increased in vivo efficacy that facilitated engraftment and permitted graft-versus-tumor effects while minimizing graft-versus-host disease (GVHD). To translate these findings, we performed a phase 2 multicenter clinical trial of rapamycin-resistant donor CD4(+) Th2/Th1 (T-Rapa) cells after allogeneic-matched sibling donor hematopoietic cell transplantation (HCT) for therapy of refractory hematologic malignancy. T-Rapa cell products, which expressed a balanced Th2/Th1 phenotype, were administered as a preemptive donor lymphocyte infusion at day 14 post-HCT. After T-Rapa cell infusion, mixed donor/host chimerism rapidly converted, and there was preferential immune reconstitution with donor CD4(+) Th2 and Th1 cells relative to regulatory T cells and CD8(+) T cells. The cumulative incidence probability of acute GVHD was 20% and 40% at days 100 and 180 post-HCT, respectively. There was no transplant-related mortality. Eighteen of 40 patients (45%) remain in sustained complete remission (range of follow-up: 42-84 months). These results demonstrate the safety of this low-intensity transplant approach and the feasibility of subsequent randomized studies to compare T-Rapa cell-based therapy with standard transplantation regimens.

Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation.

New treatments are needed for B-cell malignancies persisting after allogeneic hematopoietic stem cell transplantation (alloHSCT). We conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. T cells for genetic modification were obtained from each patient's alloHSCT donor. All patients had malignancy that persisted after alloHSCT and standard donor lymphocyte infusions (DLIs). Patients did not receive chemotherapy prior to the CAR T-cell infusions and were not lymphocyte depleted at the time of the infusions. The 10 treated patients received a single infusion of allogeneic anti-CD19-CAR T cells. Three patients had regressions of their malignancies. One patient with chronic lymphocytic leukemia (CLL) obtained an ongoing complete remission after treatment with allogeneic anti-CD19-CAR T cells, another CLL patient had tumor lysis syndrome as his leukemia dramatically regressed, and a patient with mantle cell lymphoma obtained an ongoing partial remission. None of the 10 patients developed graft-versus-host disease (GVHD). Toxicities included transient hypotension and fever. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. These results show for the first time that donor-derived allogeneic anti-CD19-CAR T cells can cause regression of B-cell malignancies resistant to standard DLIs without causing GVHD.

Differential gene expression profile of first-generation and second-generation rapamycin-resistant allogeneic T cells.

BACKGROUND AIMS: We completed a phase II clinical trial evaluating rapamycin-resistant allogeneic T cells (T-rapa) and now have evaluated a T-rapa product manufactured in 6 days (T-rapa(6)) rather than 12 days (T-Rapa(12)). METHODS: Using gene expression microarrays, we addressed our hypothesis that the two products would express a similar phenotype. The products had similar phenotypes using conventional comparison methods of cytokine secretion and surface markers. RESULTS: Unsupervised analysis of 34,340 genes revealed that T-rapa(6) and T-rapa(12) products clustered together, distinct from culture input CD4(+) T cells. Statistical analysis of T-rapa(6) products revealed differential expression of 19.3% of genes (n = 6641) compared with input CD4(+) cells; similarly, 17.8% of genes (n = 6147) were differentially expressed between T-rapa(12) products and input CD4(+) cells. Compared with input CD4(+) cells, T-rapa(6) and T-rapa(12) products were similar in terms of up-regulation of major gene families (cell cycle, stress response, glucose catabolism, DNA metabolism) and down-regulation (inflammatory response, immune response, apoptosis, transcriptional regulation). However, when directly compared, T-rapa(6) and T-rapa(12) products showed differential expression of 5.8% of genes (n = 1994; T-rapa(6) vs. T-rapa(12)). CONCLUSIONS: Second-generation T-rapa(6) cells possess a similar yet distinct gene expression profile relative to first-generation T-rapa(12) cells and may mediate differential effects after adoptive transfer.

Allogeneic T Cells That Express an Anti-CD19 Chimeric Antigen Receptor Induce Remissions of B-Cell Malignancies That Progress After Allogeneic Hematopoietic Stem-Cell Transplantation Without Causing Graft-Versus-Host Disease.

PURPOSE: Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. METHODS: We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipient's alloHSCT donor. RESULTS: Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. CONCLUSION: Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT.

High-Dose Sirolimus and Immune-Selective Pentostatin plus Cyclophosphamide Conditioning Yields Stable Mixed Chimerism and Insufficient Graft-versus-Tumor Responses.

PURPOSE: We hypothesized that lymphoid-selective host conditioning and subsequent adoptive transfer of sirolimus-resistant allogeneic T cells (T-Rapa), when combined with high-dose sirolimus drug therapy in vivo, would safely achieve antitumor effects while avoiding GVHD. EXPERIMENTAL DESIGN: Patients (n = 10) with metastatic renal cell carcinoma (RCC) were accrued because this disease is relatively refractory to high-dose conditioning yet may respond to high-dose sirolimus. A 21-day outpatient regimen of weekly pentostatin (P; 4 mg/m(2)/dose) combined with daily, dose-adjusted cyclophosphamide (C; ≤200 mg/d) was designed to deplete and suppress host T cells. After PC conditioning, patients received matched sibling, T-cell-replete peripheral blood stem cell allografts, and high-dose sirolimus (serum trough target, 20-30 ng/mL). To augment graft-versus-tumor (GVT) effects, multiple T-Rapa donor lymphocyte infusions (DLI) were administered (days 0, 14, and 45 posttransplant), and sirolimus was discontinued early (day 60 posttransplant). RESULTS: PC conditioning depleted host T cells without neutropenia or infection and facilitated donor engraftment (10 of 10 cases). High-dose sirolimus therapy inhibited multiple T-Rapa DLI, as evidenced by stable mixed donor/host chimerism. No antitumor responses were detected by RECIST criteria and no significant classical acute GVHD was observed. CONCLUSIONS: Immune-selective PC conditioning represents a new approach to safely achieve alloengraftment without neutropenia. However, allogeneic T cells generated ex vivo in sirolimus are not resistant to the tolerance-inducing effects of in vivo sirolimus drug therapy, thereby cautioning against use of this intervention in patients with refractory cancer.