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. 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. These fusion genes were cloned into the pDUAL lentivector expression system, which coexpress 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 and their phenotypes were characterized by flow cytometry analysis.
Results
Both constructs showed differential phenotypes and expression levels within different surface and intracellular molecules compared with their WT and CD3 activator controls, such as CD3, CD4, CD27, CD28, CD69, CD62L, CD45RA, PD1, LAG3, TIM-3, CTLA-4, KIR2DL1/S1/S3/S5, IFNg, IFNa/b, IL-12/IL-13, IL-4, IL-2 and IL-17A, among others.
Conclusions
These results showed that these molecules had functional PD-1 and LAG-3 constitutive inhibitory signalling in T cells leading T cell dysfunctionality. 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
Navarrabiomed, Instituto de Investigaciones Sanitarias de Navarra (IdiSNA), Universidad Pública de Navarra.
Funding
This work was supported and funded by the Spanish Association against Cancer (AECC, PROYE16001ESCO), Instituto de Salud Carlos III (ISCIII, FIS. PI17/02119), Biomedicine Project grant from the Department of Health of the Government of Navarre (BMED 050-2019).
Disclosure
All authors have declared no conflicts of interest.