BACKGROUND. Adoptive transfer of donor-derived EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) can eradicate EBVassociated lymphomas (EBV-PTLD) after transplantation of hematopoietic cell (HCT) or solid organ (SOT) but is unavailable for most patients. METHODS.We developed a third-party, allogeneic, off-the-shelf bank of 330 GMP-grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (n = 33) or SOT (n = 13) with established EBV-PTLD, who had failed rituximab therapy, with third-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation. RESULTS.EBV-CTLs did not induce significant toxicities. One patient developed grade I skin graft-versus-host disease. Complete remission (CR) or sustained partial remission (PR) was achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, one year overall survival was 88.9% and 81.8%, respectively. In addition, 3 of 5 recipients with POD after a first cycle who received EBV-CTLs from a different donor achieved CR or durable PR (60%) and survived longer than 1 year. Maximal responses were achieved after a median of 2 cycles. CONCLUSION.Third-party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV-PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab-refractory EBVassociated lymphoma after transplantation.Authorship note: SP and ED contributed equally to this work. Conflict of interest: ED and ROR had consultancy agreements with Atara Biotherapeutics. ED and ROR are inventors on technology referenced in this work (SK2013-71 [patient ID], Proprietary Cell Banks for Use in 3rd-Party EBV-Specific T Cell Therapy; and SK2018-122 [patient ID], Methods of Selecting T Cell Lines for Adoptive Cellular Therapy). SP is an inventor on technology referenced in this work (SK2013-71 and SK2018-122) and has waived rights to revenue generated from these inventions. Memorial Sloan Kettering Cancer Center (MSK), which owns the technology, has licensed this technology to Atara, and MSK has interests in Atara through this licensing arrangement.

Adoptive immunotherapy with transplant donor-derived virus-specific T-cells has emerged as a potentially curative approach for the treatment of drug refractory EBV+lymphomas as well as CMV and adenovirus infections complicating allogeneic hematopoietic cell transplants. Adoptive transfer of HLA partially matched virus-specific T-cells from healthy third party donors has also shown promise in the treatment of these conditions, with disease response rates of 50–76% and strikingly low incidences of toxicity or GVHD recorded in initial trials. In this review, we examine the reported experience with transplant donor and third party donor-derived virus specific T-cells, identifying characteristics of the viral pathogen, the T-cells administered and the diseased host that contribute to treatment response or failure. We also describe the characteristics of virus-specific T-cell lines in our center’s bank and the frequency with which in vitro culture promotes expansion of immunodominant T-cells specific for epitopes that are presented by a limited array of HLA alleles that are highly prevalent which facilitates their broad applicability for treatment.

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We conducted a Phase I trial of allogeneic T-cells sensitized in vitro against a pool of 15-mer peptides spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with CMV viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but three of the patients had received T-cell depleted transplants without GVHD prophylaxis with immunosuppressive drugs post transplant. The CMVpp65-specific T-cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1–3 epitopes, presented by a limited set of HLA class I or II alleles. T-cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (N=16) or third party (N=1) CMVpp65CTLs, 15 cleared viremia including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T-cell receptor (TCR) Vβ usage in CMVpp65/HLA tetramer+ populations for period of 120 days to up to 2 years post infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post transplant period.

In models of HIV fusion, the glycoprotein gp41 is thought to form a six-helix bundle during viral fusion with the target cell. This bundle is comprised of three helical regions (from the heptad repeat 2, or HR2, region of gp41) bound to an inner, trimeric, coiled-coil core (from the HR1 region). Although much has been learned about the structure and thermodynamics of this complex, the energetics of the isolated HR1 self-associated oligomer remain largely unknown. By systematically studying self-association through a series of truncations based on a 51-mer HR1 peptide (T865), we have identified amino acid segments which contribute significantly to the stability of the oligomeric HR1 complex. Biophysical characterization of C-terminal truncations of T865 identifies a 10-15-amino acid region that is essential for HR1 oligomerization. This region coincides with a hydrophobic pocket that provides important contacts for the interaction of HR2 helices. Complete removal of this pocket abolishes HR1 oligomerization. Despite the dramatic reduction in stability, the monomeric HR1 peptides are still able to form stable six-helix bundles in the presence of HR2 peptides. Truncations on the N-terminal side of T865 have little effect on oligomerization but significantly reduce the stability of the HR1-HR2 six-helix bundle. Unlike the HR2 binding site, which extends along a hydrophobic groove on the HR1 oligomer, the residues that are critical for HR1 oligomerization are concentrated in a 10-15-amino acid region. These results demonstrate that there are localizations of binding energy, or "hot spots", in the self-association of peptides derived from the HR1 region of gp41.