In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specific CAR-T Cells in B Cell Non-Hodgkin's Lymphomas.

Second-generation (2G) chimeric antigen receptors (CARs) targeting CD19 are highly active against B cell malignancies, but it is unknown whether any of the costimulatory domains incorporated in the CAR have superior activity to others. Because CD28 and 4-1BB signaling activate different pathways, combining them in a single third-generation (3G) CAR may overcome the limitations of each individual costimulatory domain. We designed a clinical trial in which two autologous CD19-specific CAR-transduced T cell products (CD19.CARTs), 2G (with CD28 only) and 3G (CD28 and 4-1BB), were infused simultaneously in 16 patients with relapsed or refractory non-Hodgkin's lymphoma. 3G CD19.CARTs had superior expansion and longer persistence than 2G CD19.CARTs. This difference was most striking in the five patients with low disease burden and few circulating normal B cells, in whom 2G CD19.CARTs had limited expansion and persistence and correspondingly reduced area under the curve. Of the 11 patients with measurable disease, three achieved complete responses and three had partial responses. Cytokine release syndrome occurred in six patients but was mild, and no patient required anti-IL-6 therapy. Hence, 3G CD19.CARTs combining 4-1BB with CD28 produce superior CART expansion and may be of particular value when treating low disease burden in patients whose normal B cells are depleted by prior therapy.

Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes for adoptive immunotherapy of HPV-associated malignancies.

Vaccines prevent human papillomavirus (HPV)-associated cancer but, although these tumors express foreign, viral antigens (E6 and E7 proteins), they have little benefit in established malignancies, likely due to negative environmental cues that block tumor recognition and induce T-cell anergy in vivo. We postulated that we could identify mechanisms by which ex vivo stimulation of T cells could reactivate and expand tumor-directed T-cell lines from HPV cancer patients for subsequent adoptive immunotherapy. A total of 68 patients with HPV-associated cancers were studied. Peripheral blood T cells were stimulated with monocyte-derived dendritic cells loaded with pepmixes [peptide libraries of 15-mers overlapping by 11 amino acids (aa)] spanning E6/E7, in the presence or absence of specific accessory cytokines. The resulting T-cell lines were further expanded with pepmix-loaded activated B-cell blasts. Interferon-γ release and cytotoxic responses to E6/E7 were assessed. We successfully reactivated and expanded (>1200-fold) E6-specific/E7-specific T cells from 8/16 cervical and 33/52 oropharyngeal cancer patients. The presence of the cytokines interleukin (IL)-6, IL-7, IL-12, and IL-15 is critical for this process. These T-cell lines possess the desirable characteristics of polyclonality, multiple T-cell subset representation (including the memory compartment) and a TH1 bias, and may eliminate E6/E7 targets. In conclusion, we have shown it is possible to robustly generate HPV16 E6/E7-directed T-cell lines from patients with HPV16-associated cancers. Because our technique is scalable and good-manufacturing procedures-compliant, these lines could be used for adoptive cellular immunotherapy of patients with HPV16 cancers.