Human Papillomavirus (HPV)--Specific T-Cells Can be Generated from Unimmunized Donors for Third Party Cell Therapy of HPV-Associated Neoplasms

HPV is a known cause of oropharyngeal cancer, cervical cancer, and other genital neoplasms. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T cell immunity is essential for disease control. As "off the shelf" third party EBV-specific T-cells have been successful in treating patients with EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third party setting to treat patients with HPV-associated cancers. Most protocols for generating virus-specific T-cells require prior exposure of the donor to the targeted virus. Since the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells is challenging. Hence, we implemented systematic changes to GMP-compliant protocols to improve antigen presentation, priming, and expansion for the manufacture of HPV-specific T cells.

By optimizing dendritic cell maturation and function using lipopolysaccharide (LPS) and IFNg, and adding IL-21 for priming, we achieved reliable expansion of naïve T-cells specific for oncoproteins E6 and E7 of the HPV16 serotype to clinically relevant numbers (mean 578-fold expansion, n=10). Ex vivo expanded, HPV-specific T cells secreted IFNγ (137±148 IFNγ spot forming cells, SFC/1x10⁵ cells and 68±145 IFNγ SFC/1x10⁵cells against HPV E6 and E7 respectively, compared to 7±8 IFNγ SFC/1x10⁵ cells against an irrelevant antigen, n=10) and other cytokines (in five evaluated products,T cells secreted high levels of IL6 (3792±966 pg/mL), IL7 (18000±0 pg/mL), IL8 (4766 ±2271 pg/mL), IP10 (234 ±220pg/mL), MIP1b (4915.848±2050.065 pg/mL) and TNFa (1945±823pg/mL). In contrast to T cells directed against other viral antigens, most T cells expanded against HPV antigens were CD4+ with a mean of 68.6±23.3% vs 18.2±20.9% CD8+ T cells (n=10). Expanded populations primarily recognized antigens through MHC class II confirmed by a decrease in responses to HPV antigen in the presence of HLA class II blocking antibodies

In summary, we successfully generated HPV16 specific T cells from healthy donors, and established the conditions for a robust and reproducible. GMP-compliant protocol using this product as an off the shelf T cell immunotherapeutic for patients with HPV-positive malignancies. Other groups have previously developed T cell therapies for HPV targets, but most efforts have focused on generating autologous products for patients with HPV malignancies. Other studies using TCR gene modified T cells have been developed, but the strategy limits the HLA types that can be targeted. Since immune dysfunction frequently accompanies patients with HPV malignancies, the development of an "off the shelf" third-party HPV-specific T-cell product potentially represents a readily available effective cell therapeutic for patients with HPV-associated cancers.