Translating combined PD1 and LAG3 inhibition from preclinical models to patients with refractory, DNA replication repair deficient (RRD) glioblastoma: An IRRDC study

Background and Aims: Glioblastoma driven by DNA Replication Repair Deficiency (RRD) account for 10% of all high-grade glioma in children and young adults, harbour high tumor mutation burden (TMB) and can respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority ultimately progress, highlighting the need for combinatorial therapies for sustained immune-surveillance. Methods: We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combined ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. Results: Human RRD-glioblastoma (n=80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2 LoxP/LoxP -POLE S459F/+ ) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1 -/- /NestinCre+/Trp53 LoxP/LoxP and therapy-induced hypermutant ENU/Trp53 -/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming, in-vivo, that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Seven children with refractory RRD-glioblastoma who had progressed after anti-PD1 treatment were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition for similar patients. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. Conclusions: LAG3 is an effective target in refractory RRD-glioblastoma. Combined inhibition with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses. The combination will now be tested in biomarker-driven clinical trials in RRD-glioblastoma and other immune-inflamed solid tumors.