A rapid method to assess the in vivo multi-functionality of adoptively transferred engineered TCR T cells

Abstract

Introduction: The clinical efficacy of chimeric antigen and T cell receptor (TCR) T cell immunotherapies is attributed to their ability to proliferate and persist in vivo. Since the interaction of the engineered T cells with the targeted tumour or its environment might suppress their function, their functionality should be characterized not only before but also after adoptive transfer.

Materials and methods: We sought to achieve this by adapting a recently developed Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid whole blood T cell assay to stimulate engineered TCR T cells in small volumes of whole blood (<1 ml) without in vitro cellular purification. As a proof-of-concept, we used this method to longitudinally study two patients with primary Hepatitis B Virus (HBV)-related hepatocellular carcinoma who received multiple dose-escalating infusions of transiently functional mRNA-engineered HBV-TCR T cells.

Results: We demonstrated that a simple pulsing of whole blood with a peptide corresponding to the epitope recognized by the specific HBV-TCR elicited Th1 cytokine secretion in both patients only after HBV-TCR T cell treatment and not before. The amount of cytokines secreted also showed an infusion-dose-dependent association.

Discussions: These findings support the utility of the whole blood cytokine release assay in monitoring the in vivo function and quantity of engineered T cell products following adoptive transfer.

Keywords: T cell therapy; adoptive cell transfer; hepatitis B; hepatocellular carcinoma.

Graphical Abstract

Figure 1.

Infusion and whole blood cytokine release assay analysis schedule. Autologous PBMCs from the primary HBV-HCC patients was isolated, expanded, and engineered to transiently express a specific HBV-TCR through mRNA electroporation. HBV-TCR T cells were infused every 14 days and whole blood was collected after each infusion at the indicated time points and analysed with the whole blood cytokine release assay. Created with BioRender.com.

Figure 2.

Assessment of the in vivo functionality of adoptively transferred HBV-TCR T cells. Concentration of cytokines in the plasma after overnight peptide stimulation of whole blood collected from patient 1 (A) and patient 2 (B) at different time points after HBV-TCR T cell transfer (second, third, and fourth infusion). Longitudinal monitoring of the peptide-induced IFN-γ (C) and IL-2 (D) secretion in the two primary HBV-HCC patients. The concentration of cytokines in the plasma after peptide stimulation (data from 3 h post-infusion are shown on the right for clarity) as well as the background from the unstimulated DMSO containing negative control wells (grey) are shown.

Figure 3.

In vitro cytokine secretion profile of mRNA-engineered HBV-TCR T cells. Indicated quantities of HBV-TCR T cells engineered from PBMCs obtained from two healthy donors were added into 320 μl of autologous whole blood. The concentration of cytokines released in the plasma after overnight peptide stimulation of the whole blood were analysed using the Protein Simple ELLA platform.