Background Tumor Infiltrating Lymphocytes (TIL) are a promising technology for the treatment of solid tumors. A key challenge for TIL efficacy, however, is overcoming the suppressive tumor microenvironment. TGFβ has been identified as a key pro-cancer cytokine that suppresses immune response. Here, TIL engineered with a dominant negative TGFβ receptor II (TGFβDNRII) were manufactured to treat melanoma patients for Cohort B of a Phase I Clinical Trial (NCT01955460). Characterization of the TGFβDNRII TIL Final Drug Product is important to: further understand product attributes, identify parameters that define successful product manufacturing, and inform future product development.

Methods TIL were manufactured from the surgical resections or core needle biopsies of patients enrolled in the study and treated at MD Anderson Cancer Center under an IRB approved protocol. Briefly, TIL were expanded from tumor fragments in Pre-REP culture, then transduced with a retroviral vector and expanded in REP culture. These cells were characterized for T cell phenotype, transduction efficiency, and TIL functionality using a matrix approach: T Cell Function by interferon-gamma secretion after anti-CD3/CD28 stimulation and function of the TGFβDNRII transgene by pSMAD3/4 signaling in response to TGFβ.

Results All lots were successfully manufactured with release specifications meeting acceptance criteria. Cells at the end of Pre-REP were mostly CD3⁺ T cells; the frequency of CD3⁺ cells continued to increase during REP. A majority of the Pre-REP cells were CD8⁺, as expected based on expansion conditions, although both CD4⁺ and gamma delta T cells were present. Transduction was monitored by%TGFβRII⁺, MFI of TGFβRII⁺, and Vector Copy Number of integrated transgene (VCN).%TGFβRII⁺ and VCN demonstrated a positive correlation. pSMAD signaling tended to decrease as transduction increased; pSMAD signaling correlated with VCN (R² = 0.7) and MFI (R² = 0.6). All T cells were functional and exhibited interferon-gamma secretion at varying levels.

Conclusions Characterizing the final drug product is an important aspect of Phase I Clinical trials. Here, lots of TGFβDNRII TIL were characterized. In addition to standard release testing (viability,% expression) T cell function and transgene function were also monitored. The correlations were strong between transduction and transgene function. Further data around product characteristics, limits and success of manufacture will be collected.