Adoptive T Cell Therapy for Polyomaviral Infections and Related Diseases

Introduction: Polyomaviruses (PyV) are a family of DNA-viruses responsible for serious morbidity and mortality in immunocompromised individuals including those with HIV or receiving immunosuppressive treatments for transplant or other conditions. BK polyomavirus (BKV) causes hemorrhagic cystitis and nephropathy in stem cell and organ transplant recipients. JC polyomavirus (JCV) causes progressive multifocal encephalopathy (PML) - an often fatal complication in immunosuppressed transplant recipients and in patients treated with natalizumab for multiple sclerosis. Merkel Cell Carcinoma (MCC) is an aggressive skin cancer of elderly or immunocompromised patients caused by Merkel Cell polyomavirus (MCV). There are no established antiviral drugs targeting PyVs and therefore current strategy is limited to reversing immunosuppression where possible. There is strong evidence that PyV can be controlled through T-cell based immunity and adoptive T cell transfer can cure or prevent BKV and JCV viral diseases after stem cell transplants (SCT). Similarly,evidence exists for immune susceptibility of MCC to T cell-mediated immune attack. JCV, BKV, and MCV are closely related and display a high degree of homology in the large T (LT) and VP1 antigens at the protein sequence level. Therefore, generation of a single T cell product containing a polyclonal cross-reactive donor T cell repertoire recognizing antigens from multiple PyV is an attractive strategy with potential clinical application for adoptive immunotherapy to treat patients suffering from HC, nephropathy, PML, MCC, and possibly other PyV-related diseases.

Methods: T cells recognizing LT and VP1 antigens from BKV, MCV, and JCV were generated from healthy donor peripheral blood lymphocytes in two weekly stimulations against autologous, mature dendritic cells (DCs) pulsed with relevant peptide libraries or lentivirally transduced to express full length proteins. The cultures were supplemented with cytokines IL-7 and IL-15 to promote memory formation, and IL-2 was added after the second stimulation to promote expansion. These ex vivo generated T cells were tested against autologous DC targets pulsed with the relevant or irrelevant peptide libraries. In addition, to more accurately assess their potential activity in vivo, the T cells were tested against autologous DC targets lentivirally transduced to express naturally-processed epitopes of BKV and MCV antigens and analyzed for their ability to secrete polyfunctional cytokines by flow cytometry.

Results: Flow cytometry revealed polyfunctional activity of BKV-primed cells against cognate BK peptide libraries and antigen-expressing transduced DCs as well as JC peptide libraries and MCV-expressing DCs in the production of TNFa, IFNg, and IL2 in both CD4 and CD8 subsets upon stimulation. The lentivirally engineered antigen-expressing DCs proved to be effective stimulators in generating BKV and MCV-recognizing T cells. While a high degree of cross-reactivity was observed among LT antigens of each virus, cross-reactivity among VP1 antigens was lower and largely donor dependent. T cells separately generated against VP1 antigens from Ia and IV serotypes of BKV showed no significant difference in activity between targets, indicating high overlap in TCR recognition of both serotypes. The antigen-primed T cultures could be highly enriched for antigen-specific T cells by CD137 (4-1BB) sorting 24 hours following simulation. T cells generated against JCV peptide libraries and MCV antigen expressing DCs displayed similar polyfunctional cross-reactivity among antigens from the three polyomaviruses, thus establishing the feasibility of generating a single human polyomavirus-targeted T cell product capable of recognizing multiple viruses within the polyomaviradae family.

Conclusions: We have demonstrated successful generation of polyclonal cross reactive T cell repertoires targeted against the LT or VP1 antigens from BKV, JCV or MCV. The LT antigen-specific T cells were highly cross reactive among these PyV. There is a high degree of cross reactivity among the VP1-specific T cells from different serotypes of BKV. These universal PyV-specific T cells could have potential clinical application in the treatment and prevention of PyV infections and related diseases.