Background: Regimens using post-transplant cyclophosphamide (CY) have been developed to provide potent in vivo T cell depletion for patients undergoing human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT). Luznik, O'Donnell and colleagues (BBMT 2008) reported that when this immune suppression strategy is coupled with non-myeloablative conditioning (fludarabine 150 mg/m2, CY 29 mg/kg, 2 Gy total body irradiation) followed by marrow transplantation, it was well-tolerated with low rates of non-relapse mortality (NRM; 15% at 1 year). However, the 2 year overall survival (OS) and event-free survival were low at 36% and 26%, respectively, due to high relapse rates (51% at 1 year). One explanation could be that while post-HCT CY promoted low rates of acute graft-versus-host disease (GVHD), it also eliminated early T and natural killer (NK) cell clones important for disease surveillance. Based on this hypothesis, we developed a next-generation Phase I/II clinical trial incorporating a boost of donor NK cells on day +7 as an attempt to prevent relapse after transplant. In this study, CY 50 mg/kg as a single dose on day +3 was used for T cell depletion. Methods: Forty patients (pediatric, n=14; adult, n=26) with median age of 45 (range 8-75) years having ALL (n=11), AML (n=9), MDS (n=6), HL (n=6), MM (n=4), NHL (n=3), and CLL (n=1) underwent non-myeloablative transplantation using related, HLA-haploidentical marrow donors on this prospective clinical trial. Patients were high risk due to underlying disease, potential for relapse, and/or risk for transplant-related mortality (TRM). Most patients were heavily pre-treated, with median time from cancer diagnosis to transplant being 2 (0.3 - 12.1) years, including 18 patients having 26 prior HCTs (auto, n=14; allo, n=12). Twenty-five patients (63%) had HCT-CI scores ≥ 3 indicating high risk for TRM. In order to obtain NK cells, non-mobilized peripheral blood mononuclear cells were collected from donors on day +6 using apheresis and stored overnight. NK cells were isolated on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing or expansion. The Phase I dose-finding study (n=11) enrolled at 2 NK doses [2.5 or 5 x 106/kg +/- 20%, respectively], with extended enrollment at the 2nd dose level for Phase II (n=29) with 83% of patients meeting NK dose parameters. NK cell products had a median log T cell depletion of 5.4 (4.1-7.1), median NK recovery of 54% (33-68%), and median NK purity of 92% (74-99%). Excellent viability (>95%) was seen in all NK products. Results: One patient developed chest pain associated with NK cell infusion; otherwise all other patients tolerated their NK cell infusions well without fevers or other adverse reactions. Full donor chimerism (>95% CD3) was seen in 83% of patients at last follow-up, while 18% and 10% experienced graft rejection or graft failure, respectively. Cumulative incidence of grades 2-3 and grade 3 (no grade 4 seen) acute GVHD occurred in 36% and 8% of patients, respectively, at day +100. Of the 39 evaluable patients, 16% developed chronic extensive GVHD at 1 year. Relapse or progression occurred in 31% of patients by 1 year after HCT. With a median follow-up of 1.5 years (range, 0.1 - 4.9 years), 14 patients have died from relapse/progression (n=11) or infection/VOD (n=3), giving a probability of OS, relapse/progression-free survival (PFS), and NRM at 1 year of 73%, 62%, and 8%, respectively, and 2 year OS and relapse/PFS of 63% and 46%, respectively (Fig 1). Summary: We have demonstrated the safety of infusing donor NK cells early after HCT in a group of heavily-treated patients with high-risk hematological malignancies. In many patients, disease-free survival was possible with the aid of this prophylactic infusion of donor NK cells in combination with allogeneic HCT. These results provide a promising platform to further augment NK cell alloreactivity in the post-HCT setting to prevent relapse and disease progression. Figure 1 Incidence of Overall Survival and Relapse/Progression-Free Survival Figure 1. Incidence of Overall Survival and Relapse/Progression-Free Survival Disclosures Hari: Merck: Research Funding; BMS: Honoraria.

T-cell depletion (TCD) of the donor marrow graft has been shown to reduce the severity of graft-versus-host disease (GVHD) in patients with chronic-phase (CP) chronic myelogenous leukemia (CML) undergoing HLA-identical sibling allogeneic marrow transplantation. However, there has been a corresponding reduction in the graft-versus-leukemia effect so that any decrease in GVHD-related mortality has been offset by an increased rate of disease relapse. Therapy of recurrent disease with donor leukocyte infusions (DLI) has been proven to be effective salvage therapy for the majority of patients who relapse after allogeneic BMT with CP CML. However, the overall impact of salvage DLI therapy on the survival of CP CML patients initially transplanted with TCD marrow grafts is not defined. To address this question, we have evaluated a clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI in 25 CP CML patients undergoing allogeneic BMT from HLA-identical siblings. All patients received a standardized preparative regimen along with ex vivo TCD and posttransplant cyclosporine as GVHD prophylaxis. Durable engraftment was observed in all 25 patients. The incidence of grade II to IV acute GVHD was 8%. The cumulative incidence of transplant-related mortality (TRM) was 4%, and the 1-year probability of overall survival was 96%. The 3-year cumulative relapse incidence was 49%. All relapsed patients received DLI to reinduce remission. The total T-cell dose administered to these patients varied from 0.1 to 5.0 × 108 T cells/kg. Complete responses were observed in 12 of 14 patients, with 1 additional patient still too early to evaluate. Three patients died of GVHD after DLI, and 1 relapsed into blast crisis after a transient cytogenetic remission. Of the remaining 10 patients, 8 are in molecular remission, 1 is alive in relapse, and 1 is receiving DLI treatment. The median follow-up after infusion of surviving DLI patients in remission is 5.3 years. The probability of overall 5-year survival for the entire population is 80%, with a median follow-up of 6.4 years. We conclude that the clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI for those patients with evidence of recurrent disease is a viable transplant strategy for CP CML, resulting in 80% survival and a low risk of acute GVHD and transplant-related mortality.

Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients, including hematopoietic progenitor cell transplant patients. Studies in patients with allergic bronchopulmonary aspergillosis as well as murine models have demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. A. fumigatus allergens have been identified based on reactivity to IgG and IgE antibodies in serum from patients with allergic bronchopulmonary aspergillosis. One allergen, Asp f16 was shown to induce a protective Th1 T cell response to infection with aspergillus conidia in murine studies. We prepared a series of 104 overlapping pentadecapeptides spanning the entire 427 aa coding region of Asp f16. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Autologous monocytes from healthy donors were treated with GM-CSF and IL-4 for 2–3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-α) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5 of 5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. As early as 3 weeks the T cell line from donor #1 had a high frequency of IFNγ-producing CD4+ T cells (24.5±5.4%) in response to the peptide pool, was cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract pulsed DC, and peptide pool pulsed autologous BLCL. Supernatant from this culture killed fresh aspergillus conidia. Screening of 21 smaller pools containing 4–11 peptides showed reactivity to three smaller pools and could be narrowed by screening individual peptides shared by the pools to a single candidate sequence of TWSIDGAVVRT that elicited IFNγ production and lytic activity equal to the entire pool. Peptide pulsed BLCL matched with the donor for only 1 or 2 HLA alleles were used to demonstrate that CTL activity and IFNγ production was restricted by HLA-DRB1- 0301. 23% of the CD4+ cells in the culture expressed CD107a in response to peptide pool indicating degranulation. PBMC from DRB1-0301+ individuals proliferated strongly in response to peptide TWSIDGAVVRT. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD4+, HLA-DR-restricted aspergillus-specific Th1 type T cell response directed to a single peptide contained within the pool. Further characterization of this system is in progress to identify other immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.

Introduction Chimeric Antigen Receptor T (CAR-T) cells redirected against tumor antigens are an effective therapy for B cell malignancies refractory to standard treatments. The production of patient-derived CAR-T cells is complicated and thus far is limited to institutions with experienced researchers and expensive GMP facilities, or to those invited to participate in industry sponsored clinical trials. The outsourcing of CAR T-cell production to third party vendors where cells are collected locally, shipped to the manufacturing site, and then sent back to the institution for infusion can be both costly and timely. As a result, CAR-T cell therapies are not widely available and only patients with means to travel to participating sites and with disease that is stable enough to wait the 2-3 months needed to collect and produce CAR-T cells are eligible for these treatments. At our instution we have explored the use of the CliniMACS® Prodigy (Miltenyi Biotec, Inc) for the production of CAR-T cells. The CliniMACS® Prodigy is an automated device that can be used for cell processing within a closed GMP-compliant system. Using the CAR-T system that includes software, specialized tubing sets, and optimized reagents we demonstrate the processing of CAR-T cells, with similar characteristics to those produced in a more traditional manner, in a closed system that is suitable for clinical use without the need for a clean room manufacturing facility. Methods In collaboration with Miltenyi Biotec, we obtained pre-release and final versions of the CliniMACS® Prodigy TCT process software and the TS520 tubing set that allows for cell enrichment, transduction, wash steps, and expansion all within a single set. Starting material was MNC cells recovered from a leukoreduction system chamber (LRSC) used during platelet collections by apheresis. Materials and reagents included MACS CD4 & CD8 reagents for cell enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 zeta-cfrag vectors with either CD19 or CD20/CD19 Ab chains (Lentigen Technology Inc., A Miltenyi Biotec Company), TexMACS culture medium-3% HS-IL2, and PBS/EDTA buffer for wash steps. For two experiments, cells after CD4/CD8 enrichment were activated and transduced in 6 well plates and expanded after day 5 in G-Rex gas permerable devices. Total time for line preparation was 14±1 days. Transduction was measured by Protein L expression using flow cytometry. Line function was measured in 51Cr Release assays and by intracellular cytokine production. Results Starting cells were washed free of platelets and enriched for CD4+ and CD8+ cells using the Prodigy device. We achieved consistent high levels purity (99±3%) and good recovery (51.0±6%) of CD4+ and CD8+ cells (N=5). The enriched cells were 90±12% CD3+. The approximately 10% non-T cells were CD8+ NK cells, that were largely eliminated after cell activation through CD3/CD28 and expansion. A controlled number of 1 x 10E8 cells enriched for CD4+ plus CD8+ cells were retained in the Prodigy and in 2 experiments a smaller fraction of cells was cultured in 6 well plates for activation and initial transduction. Three preparations were conducted in the Prodigy, one using the CD19 vector and two with the CD19+CD20 vector. Transduction efficiency ranged from 21%-46% of total T cells with a modest preference for CD4+ cells. Expansion ranged from 26-40 fold and all of the lines recognized CD19 and/or CD20 targets based on 51Cr release assays or IFN-gamma production. The paired lines generated on the Prodigy versus manual methods showed similar overall transduction, phenotype, and function as shown in the figure for one representative preparation. Conclusions CAR-T cells generated in the Prodigy were similar to those prepared using manual methods in both phenotype and function. This process is timely, requiring 14 days for generation of the target CAR-T cell dose, and does not require outsourcing to third party vendors. All of the Prodigy CAR-T cell preparations met criteria for clinical use in our upcoming Phase I clinical trial. The ability to produce CAR-T cells suitable for clinical use in an entirely closed system without the need for a clean room should allow more centers and patients access to this novel form of immunotherapy. Disclosures Shah: Oncosec: Equity Ownership; Exelixis: Equity Ownership; Geron: Equity Ownership. Orentas:Lentigen Technology, Inc.: Employment. Dropulic:Lentigen Technology Inc. A Miltenyi Biotec Company: Employment. Hari:Merck: Research Funding; BMS: Honoraria.

e23551 Background: Patients with relapsed solid tumors have dismal predicted OS < 10-20% at 5 years with conventional salvage therapies. We hypothesized that a multi-faceted immunotherapy approach to optimize graft-versus-tumor (GVT) effects, in combination with a mammalian target of rapamycin (mTOR) inhibitor maintenance strategy, would enhance early disease-control rates (DCR), leading to improved progression-free survival (PFS) and OS for this high-risk population. Methods: Treatment consisted of HLA-haploidentical marrow (n = 12) or peripheral blood stem cell (n = 3) transplantation to optimize GVT effects, preceded by reduced-intensity conditioning (fludarabine, cyclophosphamide, and 3 Gy total body irradiation) to promote engraftment of these mismatched stem cells. Haplo-NK cells were given to boost this GVT effect. They were purified from non-mobilized donor mononuclear cells by CD3 depletion followed by CD56 selection using the Miltenyi CliniMACS system and were infused fresh on day +7 after HCT. Postgrafting immunosuppression included sirolimus maintenance, which continued until 6 months post-HCT. Results: Fifteen patients with relapsed Ewing sarcoma (EWS) (n = 9), rhabdomyosarcoma (n = 4), osteosarcoma (n = 1), and medulloblastoma (n = 1) having stable or undetectable gross disease were enrolled on this Phase II trial. Median age at HCT was 19 (4.5-37) years old and median performance status was 80%. Four patients underwent prior autologous transplants. Patients received a median NK dose of 6.5 (3.7-11.4) x 106/kg. NK cell products had a median log T cell depletion of 5.94 (5.18-6.75), median NK recovery of 62% (48-71%), and median NK purity of 92% (74%-97%). All donor NK infusions were well-tolerated without cytokine release syndrome. All patients engrafted, and all had sustained full donor chimerism ( > 95% CD3). Two patients developed grade II acute graft-versus-host disease (GVHD), and 2 patients developed chronic GVHD. No patients died from transplant-related causes. With a median follow-up of 1.3 years (range, 70 days – 5 years), 6-month DCR is 72%. 1- and 2-year OS for the entire cohort is estimated at 64% and 40%, respectively, while PFS is 29% and 22%, respectively. For patients with EWS,1- and 2- year -OS is estimated at 75% and 45%, and PFS is 38% and 25%, respectively. Conclusions: This dual immunotherapy approach followed by mTOR inhibition maintenance was well-tolerated, with better than expected OS for this high-risk set of diseases. Clinical trial information: NCT02100891 .