Most Closely HLA-Matched Third Party Allogeneic CMV Specific T-Cells Generated Using the Cytokine Capture System for the Treatment of CMV Infections after Allogeneic Stem Cell Transplantation
Authors: Oran Betul , Muftuoglu Muharrem , Kaur Indreshpal , Li Li , Yvon Eric , Abueg Glorette , Bassett Roland , Thall Peter F , Marin David , Alousi Amin M. , Ahmed Sairah , Ciurea Stefan O. , Nina Shah , Hosing Chitra , Olson Amanda L. , Parmar Simrit , Jones Roy , Popat Uday R. , Andersson Borje S. , Champlin Richard E. , Shpall Elizabeth J. , Rezvani Katayoun 
Published: October 2019

Abstract
Adoptive immunotherapy with ex vivo expanded virus-specific T-cell lines from transplant donors has emerged as a potentially curative approach for the treatment of drug-refractory viral infections complicating allogeneic hematopoietic cell transplants (HSCT). In these studies, viral-specific T cells are generated in compliance with current good manufacturing practice (cGMP) following repeated rounds of antigen-driven stimulation over a number of weeks. Such protocols are logistically and technically demanding, requiring access to specialized GMP units and are difficult to apply in urgent clinical settings.
The cytokine-capture (gamma-catch) procedure is a fast and simple method that takes <30 hours to isolate polyclonal IFN-gamma-secreting CD8+ and CD4+ viral-specific T cells and can be performed outside a dedicated GMP facility. The cells are directly transfused to the patient without the requirement for ex vivo expansion. The use of allogeneic third party T-cell donors offers the option of selecting donors with high viral-specific T cell precursor frequencies and may serve as an alternative for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor.
Here, we report the interim results of a Phase 2 study in 11 patients with persistent CMV infection after HSCT, despite optimum anti-viral therapy for at least 14 days. We established a bank of cryopreserved peripheral blood mononuclear cells (PBMCs) from 36 healthy donors. Eligible patients were treated with small numbers of CMV-specific T cells selected by cytokine capture from the most closely HLA-matched third party donor. Manufacture of third party derived CMV specific CTLs was performed at the MDACC Core Facility. Cryopreserved PBMCs were thawed, washed and diluted at 1 × 109 cells in 100 mL MACS GMP TexMACS Reagent (Miltenyi Biotec) for approximately 16 hours at 37°C. The product was stimulated with MACS® GMP Peptivator® HCMV pp65 for approximately 4 hours at 37°C, CO2. Enrichment of cytokine-secreting cells was performed using the cytokine secretion system and the CliniMACS device for immunomagnetic separation. CMV-specific T cells were transfused directly after isolation without any further in vitro expansion.
To date, 11 HSCT recipients have received CMV-specific T-cells generated by gamma-catch. Of 11 patients, 2 had failed to respond to foscarnet, 12 to ganciclovir and 8 to both agents. One patient who had a complete response (CR) to his first CMV-specific T-cell infusion received a second infusion 166 days after the first dose for the treatment of CMV retinitis. The median number of infused CD3+IFN-gamma+ T-cells was 2598/kg (range, 61/kg-12717/kg). There were no immediate infusion-related adverse events. CMV-specific CTLs controlled infection in 9 of 11 evaluable infusions; 6 CR and 3 partial response (PR). The overall response and CR rates at 4 weeks were 77% (95%CI; 51%, 95%) and 54.5% (95%CI; 25.4%, 78.6%). Interestingly, the success of adoptive T-cell transfer was not related to the dose of infused T-cells. The median ratio of CD8+ IFN-gamma+ to CD4+ IFN-gamma+ T-cells was 2.7 (range, 0.17-13.4). Among 6 CR patients, 4 had a second episode of CMV infection at a median of 71 days (range, 61-355 days) after the first CTL infusion. Following infusion, there was expansion of both CD8+ and/or CD4+ CMV-specific T-cells, which persisted for a minimum of 1 month following infusion. Recurrence of CMV PCR after CTL infusion was associated with loss of CMV CTL response.
Despite the use of third-party donors with HLA disparity, only one patient developed de novo graft versus host disease (GVHD) of the gastrointestinal tract (grade 1) 3 months after CTL infusion. Three patients with a history of acute GVHD prior to CTL infusion had flares 5-6 months after CTL infusions and responded to systemic steroid use.
In summary, these interim results demonstrate the safety, feasibility and efficacy of immunotherapy with the most closely HLA-matched CMV specific CTLs generated by cytokine capture for the treatment of drug resistant CMV infection after HSCT patients. The clinical trial continues to enroll and an updated report of the study will be presented at the meeting.

Disclosures
Ciurea: Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership.