Cary F. Opel

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.

Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody-cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R-expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibody-antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.

Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture.