A significant number of cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers with high T-cell infiltration. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic and epigenetic pathways. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. These results uncovered distinct degrees of T-cell dysfunctionality. Global changes were cross-evaluated among T-cell lines with multiomic biopsy data to identify genetic, metabolic, and proteomic programmes regulated by PD-1/LAG-3 dysfunctionality. One of these relied on differential regulation of E3 ubiquitin ligases CBL-B and C-CBL. PD-1/LAG-3 co-blockade with a bispecific drug under clinical development but not with a combination of anti-PD-1/anti-LAG-3 antibodies achieved both CBL-B and C-CBL inhibition, reverting T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade.

C.J.E, J.L. and D.E. are inventors of the Humabody CB213 (WO/2019/158942. Crescendo Biologics Ltd.). The rest of the authors declare no conflicts of interest.

This research was supported by The Spanish Association against Cancer, Instituto de Salud Carlos III (ISCIII)_FEDER, Department of Health of the Government of Navarre_FEDER, Strategic projects from the Department of Industry, Government of Navarre; European Union Horizon 2020 ISOLDA project, under grant agreement ID 848166.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The prospective observational study was approved by the Ethics Committee of Clinical Investigations at the University Hospital of Navarre (reference number: PI_2020/115). Informed consent was obtained from all subjects, and all experiments conformed to the principles in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report. Clinical details and composition of the cohort under study are described elsewhere (Arasanz et al., 2020; Bocanegra et al., 2022; Zuazo et al., 2019).

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Mass-spectrometry data and search results files were deposited in the Proteome Xchange Consortium via the JPOST partner repository (https://repository.jpostdb.org; Reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210561/) with the identifier PXD040408 for ProteomeXchange and JPST002062 for jPOST (for reviewers: https://repository.jpostdb.org/preview/118930299263fc6310a6249 ; Access key: 7060).

https://repository.jpostdb.org/preview/118930299263fc6310a6249