Fast tumor-infiltrating lymphocytes (TILs): rapid manufacture of an adoptive cellular therapeutic from pleural infiltrating T cells for intrapleural administration.

Abstract

Background aims: Cancers of many different tissue origins can metastasize to the pleura, a space with a unique immune environment that predisposes to aggressive tumor behavior and the development of effusions, an exudative leakage of serous fluid accompanied by an immune infiltrate. Effusions are drained therapeutically to relieve dyspnea, often several times per week. Characteristically, they contain 50-1000 × 10⁶ viable pleural T cells (PITs), which can be reliably activated and expanded in culture, making them an ideal source for generation of a cellular therapeutic. We sought to determine the feasibility of a Good Manufacturing Practice-compliant, rapidly manufactured adoptive cellular therapeutic from pleural-infiltrating T cells and to determine the cytolytic activity against autologous tumor, cytokine secretion profile, and immune check point molecule (ICM) expression.

Methods: Six products were generated from consecutively collected malignant pleural effusions (MPEs) drained from patients with breast (4) or non-small cell lung (2) cancer metastatic to the pleura. CD4+ and CD8+ cells were immunomagnetically selected, stimulated with anti-CD3/anti-CD28 and expanded in the presence of interleukin (IL)-7 and IL-15 (12.5 ng/mL each) using the Miltenyi CliniMACS Prodigy device. Cells were cultured for 8- 12 days. Cytokines were assayed in the MPE and in the culture medium before harvest using a multiplexed bead assay. Cytolytic activity of the final cellular product formulation against an autologous tumor was measured in a 4-hour killing assay by lactate dehydrogenase release. T-cell content, ICM expression and intracellular interferon ɣ were assessed by flow cytometry.

Results: All MPEs successfully generated products containing 0.7 to 3.2 × 10⁹ viable T cells. All final products showed no growth in bacterial or fungal cultures. T-cell purity was 98.3 ± 1.7% (mean, standard deviation), viability was 97.6 ± 1.7% and T-cell fold expansion was 14.3 ± 10.6. Twenty cytokines (excluding IL-7 and IL-15) were present in the culture supernatants at ≥10 pmol/L. These include granzyme B, interferon-ɣ, IL-13, perforin, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α. In the final cellular product formulation, 69 ± 28% of CD4+ T cells, and 75 ± 27% of CD8+ T-cells produced interferon-ɣ without additional stimulation. ICM expression was well correlated between CD4+ and CD8+ T cells and was relatively low, with TIGIT (44.6 ± 10.9%) and programmed cell death protein 1(21.1 ± 6.7%) being the highest. All products evidenced cytolytic activity against an autologous tumor, with maximal lysis ranging from 19.4% to 100% and cytolytic indices ranging from 4.3 to 21.1.

Conclusions: We conclude that Fast tumor-infiltrating lymphocytes (Fast TIL), an adoptive cellular therapeutic generated from drained MPEs, can be rapidly and reliably manufactured using the Prodigy system. The products have demonstrable in vitro effector activity against an autologous tumor. Expression of interferon-ɣ in the majority of cells, without accompanying elevated expression of ICM, suggests that the cells have not been exhausted during expansion. Based in part on the results presented here, the US Food and Drug Administration has issued Investigational New Drug status (IND # 30892) for the rapid manufacture of PIT-based cellular therapeutic (Fast TIL), paving the way for a first-in-human clinical trial (supported by CDMRP-TTSA CA230972). We plan to infuse this product intrapleurally, accompanied by low-dose intrapleural IL-2 with the expectation that infused cells will immediately encounter tumor antigens, continue to expand in the pleural space, and migrate to the peripheral circulation.