Nirali N. Shah

T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.

BACKGROUND Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. METHODS This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. FINDINGS Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients). INTERPRETATION CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. FUNDING National Institutes of Health Intramural funds and St Baldricks Foundation.

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Treatment of Intraocular Retinoblastoma With Vincristine and Carboplatin

PURPOSE To evaluate the efficacy of chemoreduction using vincristine and carboplatin in preventing or delaying external-beam radiotherapy (EBRT) or enucleation in patients with intraocular retinoblastoma. PATIENTS AND METHODS Twenty-five patients (43 eyes) with newly diagnosed intraocular retinoblastoma received primary treatment with eight courses of vincristine and carboplatin. Focal treatments were delayed until documentation of disease progression. Outcome measures for each eye were length of time to disease progression, avoidance or delay of EBRT, and globe survival. Event-free survival was defined as the length of time to EBRT or enucleation. RESULTS Disease in all eyes responded to chemotherapy and progressed in only two patients before completion of the eight courses of therapy. Disease in all but four eyes progressed and required focal treatments. Event-free survival estimates at 2 years were 59.2% +/- 12.0% for Reese-Ellsworth group I, II, and III eyes and 26.3% +/- 9.2% for group IV and V eyes. Nineteen eyes (44.2%) required EBRT and 13 eyes (30.2%) were enucleated. The ocular salvage rate was 83.3% for Reese-Ellsworth group I to III eyes and 52.6% for group IV and V eyes. For those patients receiving EBRT, the median time from enrollment to EBRT was 9.5 months (median age at EBRT, 21 months). CONCLUSION In combination with appropriate early intensive focal treatments, chemoreduction with vincristine and carboplatin, without etoposide, may be an alternative treatment for patients with early-stage intraocular retinoblastoma, although additional studies are needed. Patients with advanced intraocular disease require more aggressive treatments.

Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T cell depleted stem cell transplantation

Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre–B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19− B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.Chimeric antigen receptor (CAR) T-cells targeting CD19 mediate potent effects in relapsed/refractory pre-B cell acute lymphoblastic leukemia (B-ALL) but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed on most B-ALL and usually retained following CD19 loss. We report results from a phase I trial testing a novel CD22-CAR in twenty-one children and adults, including 17 previously treated with CD19-directed immunotherapy. Dose dependent anti-leukemic activity was observed with complete remission in 73% (11/15) of patients receiving ≥ 1 × 106 CD22-CART cells/kg, including 5/5 patients with CD19dim/neg B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted escape from killing by CD22-CART cells. These results are the first to eastablish the clinical activity of a CD22-CAR in pre-B cell ALL, including in leukemia resistant to anti-CD19 immunotherapy, demonstrating comparable potency to CD19-CART at biologically active doses in B-ALL. They also highlight the critical role played by antigen density in regulating CAR function. (Funded by NCI Intramural Research Program)

Secondary solid cancer screening following hematopoietic cell transplantation.

Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients.

Characterization of CD22 expression in acute lymphoblastic leukemia

CD22 is a B‐lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL).

Transplantation for children with acute myeloid leukemia: a comparison of outcomes with reduced intensity and myeloablative regimens

The safety and efficacy of reduced-intensity conditioning (RIC) regimens for the treatment of pediatric acute myeloid leukemia is unknown. We compared the outcome of allogeneic hematopoietic cell transplantation in children with acute myeloid leukemia using RIC regimens with those receiving myeloablative-conditioning (MAC) regimens. A total of 180 patients were evaluated (39 with RIC and 141 with MAC regimens). Results of univariate and multivariate analysis showed no significant differences in the rates of acute and chronic graft-versus-host disease, leukemia-free, and overall survival between treatment groups. The 5-year probabilities of overall survival with RIC and MAC regimens were 45% and 48%, respectively (P = .99). Moreover, relapse rates were not higher with RIC compared with MAC regimens (39% vs 39%; P = .95), and recipients of MAC regimens were not at higher risk for transplant-related mortality compared with recipients of RIC regimens (16% vs 16%; P = .73). After carefully controlled analyses, we found that in this relatively modest study population, the data supported a role for RIC regimens for acute myeloid leukemia in children undergoing allogeneic hematopoietic cell transplantation. The data also provided justification for designing a carefully controlled randomized clinical trial that examines the efficacy of regimen intensity in this population.

Matched Related and Unrelated Donor Hematopoietic Stem Cell Transplantation for DOCK8 Deficiency

We performed allogeneic hematopoietic stem cell transplantation in 6 patients with mutations in the dedicator-of-cytokinesis-8 (DOCK8) gene using a myeloablative conditioning regimen consisting of busulfan 3.2 mg/kg/day i.v. for 4 days and fludarabine 40 mg/m(2)/day for 4 days. Three patients received allografts from matched related donors and 3 patients from matched unrelated donors. Two patients received peripheral blood stem cells and 4 patients bone marrow hematopoietic stem cells. Tacrolimus and short-course methotrexate on days 1, 3, 6, and 11 were used for graft-versus-host-disease (GVHD) prophylaxis. All 6 patients are alive at a median follow-up of 22.5 months (range, 14 to 35). All patients achieved rapid and high levels of donor engraftment and complete reversal of the clinical and immunologic phenotype. Adverse events consisted of acute skin GVHD in 2 patients and post-transplant pulmonary infiltrates in a patient with extensive bronchiectasis pretransplant. Thus, a uniform myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation in DOCK8 deficiency results in reconstitution of immunologic function and reversal of the clinical phenotype with a low incidence of regimen-related toxicity.

Vincristine Sulfate Liposomes Injection (VSLI, Marqibo®): Results From a Phase I Study in Children, Adolescents, and Young Adults With Refractory Solid Tumors or Leukemias.

Vincristine sulfate liposome injection (VSLI; Marqibo®) is an encapsulated preparation of standard vincristine in sphingomyelin/cholesterol liposomes. Clinical trials in adults have demonstrated safety, tolerability, and activity, leading to Food and Drug Administration (FDA) approval for adults with relapsed acute lymphoblastic leukemia (ALL). Pediatric experience with VSLI is limited.