Adoptive Transfer of CMV-Specific Donor T Cells Following Allogeneic Transplantation Leads to Rapid and Safe Systemic Reconstitution.

Abstract 44

Reactivation of CMV remains a significant problem following allogeneic hematopoietic stem cell transplantation. Antiviral drug therapy is effective but toxic, and resistant strains of CMV are increasingly being reported. Virus-specific T lymphocytes are necessary for the control of viral reactivation. Adoptive transfer of donor derived CMV-specific T cells has been reported previously but most methods to produce such cells have involved several weeks of in vitro culture or have produced a therapeutic product restricted to CD8 T cells. The current method involves a short incubation of donor peripheral blood mononuclear cells with either CMV-pp65 protein (20 hours) or a pool of peptides from pp65 (6 hours) with subsequent isolation of interferon-gamma secreting cells by CliniMACS using IFNψ capture microbeads (Miltenyi Biotec). This technique permits rapid isolation of an enriched IFNψ secreting T cell product, manufactured to clinical grade, which is then cryopreserved in dosed aliquots for subsequent infusion. Here we report the outcome of a single arm phase I/II in which CMV-T cells given pre-emptively at first detection (qPCR) of CMV DNA in peripheral blood, or at day +40-60 as prophylaxis. CMV replication was monitored by weekly PCR and reconstitution of CMV-specific T cells by pentamer labelling and/or IFNψ secretion assay. Conventional antiviral drug therapy was instituted if the viral load rose above institutional threshold. 30 recipients of T cell depleted low intensity transplants from HLA-matched CMV-seropositive related donors were enrolled between 2006 and 2008. Donors underwent a second, short apheresis procedure approximately 15 days after collection of the mobilised HPC-A for the collection of CMV-T cells. 26 clinical-grade products were produced to full cGMP standards; four donors were unsuitable or withdrew. The mean yield of cells following enrichment was 41.7% with a median purity of 43.9% (range 1.4-81.8). Adequate CMV-T cells were isolated from all donors. Both pp65 and peptide stimulated products contained both CD4 and CD8 reactive T cells. Median dose of CMV-specific CD4 T cells was 2840/kg and of CMV-specific CD8 was 630/kg. Eighteen patients received a single dose of 1×10^4 CD3+/kg; 13 were CMV seropositive; 11 were treated pre-emptively and 7 prophylactically. 83% had received T cell deplete regimens. Within 2 weeks of infusion in vivo expansion of CMV-T cells was observed in 17 of 18 patients. One patient required 4 weeks to generate detectable CMV-T cell in his peripheral blood. TCR-BV usage of the CMV-T cells post infusion matched that of the cells which had been infused. The 7 patients who had cells infused prophylactically all showed expansions of CMV-T cells in the absence of detectable viral DNA in peripheral blood. Subsequent low level CMV-reactivation was seen in one of these and was associated with rapid CMV-T cell expansion with clearance of virus without anti-viral drug therapy. One developed subsequent extensive chronic GvHD and required antiviral treatment for multiple reactivation episodes following introduction of steroids. Of the 11 patients treated pre-emptively, 9 received antiviral therapy for the initial reactivation, although in 7 patients this was required for only 7-15 days. (compared to a median of 21 days in historical controls). Three patients had a further CMV reactivation event. One followed prednisolone therapy for acute grade II GvHD. The second was the patient who had shown poor T cell expansion post infusion and had required prolonged anti-viral therapy (33 days) for the initial CMV reactivation. The third patient received no treatment and cleared virus following a further in vivo expansion of CMV-reactive T cells, suggesting the presence of a functional memory population. GVHD incidence and severity was no worse than seen in comparable historical controls. 3 patients suffered grade 2-3 acute GvHD. 3/17 evaluable patients developed extensive chronic GvHD (2 were recipients of T replete grafts). 16/18 patients were alive at the end of the 6 month monitoring period and CMV-reactive T cells were detectable in all 16. CMV-specific donor T cells can be readily produced to cGMP compliance which can be safely infused and lead to early immune reconstitution in at-risk patients. Cells expand in response to subsequent CMV-reactivation and patients appear to require fewer anti-viral treatment episodes which is being tested in an ongoing phase III trial.