Abstract 17P
Background
The lack of PD-1 or LAG-3 mutants with constitutive inhibitory activities prevents a systematic approach to dissect all the intracellular events regulated by PD-1 and LAG-3 that establish a strong T cell dysfunctionality in patients with intrinsic resistance to PD-1 therapies (Zuazo et al., 2019). It is thought that PD-1 and LAG-3 form a supramolecular complex together with TCR components in order to exert their inhibitory activity over TCR signal transduction. So far, no PD-1 and LAG-3 mutants with constitutive signalling activities have been described. Here we have constructed molecules that provide an initial TCR-dependent signal in T cells that lead to a sustained inhibitory activity of PD-1 and LAG-3.
Methods
To construct molecules with constitutive PD-1 and LAG-3 signaling activities in T cells, we replaced their immunoglobulin domains with a sequence encoding a single chain antibody that binds CD3. In addition, the coding sequence for GFP and mCherry were introduced at their c-terminus to track their expression in T cells. These fusion genes were cloned into the pDUAL lentivector expression system, which co-express antibiotic resistance for selection of cells lines. When expressed in T cells, the engagement with CD3 will trigger a TCR signal in neighbouring T cells, and these molecules will then transmit either PD-1 or LAG-3 signals. To test their expression, human T cells were transduced with lentivectors expressing these constructs singly or in combination, selected with antibiotics and visualized in living cells by fluorescence microscopy.
Results
Both constructs showed strong expression levels, with high accumulation especially at focal points of cell-to-cell contacts as would be expected for CD3-binding molecules. Similar expression patterns were observed in primary human T cells after lentivector modification. The growth of Jurkat cell lines expressing these molecules was evidently delayed compared to control untransduced T cells and to T cells constitutively expressing just the CD3-activating construct.
Conclusions
These results showed that these molecules had functional PD-1 and LAG-3 signaling in T cells leading to reduced cell growth. This will allow to study the reasons behind the intrinsic resistance to PD-1 blockade.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
OncoImmunology Research Unit, Navarrabiomed, Instituto de Investigaciones Sanitarias de Navarra (IdISNA), UPNA.
Funding
This research was supported by Asociación Española Contra el Cáncer (AECC, PROYE16001ESCO); Instituto de Salud Carlos III, Spain (FIS project grant PI17/02119); Gobierno de Navarra Biomedicine Project grant (BMED 050-2019); TRANSPOCART (Instituto de Salud Carlos III); “Precipita” Crowdfunding grant (FECYT); Crowdfunding grant from Sociedad Española de Inmunología (SEI); DESCARTHES project grant (Industry department, Government of Navarre).
Disclosure
All authors have declared no conflicts of interest.