Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Infusion Product TNFα, Th2, and STAT3 Activities Are Associated with Clinical Responses to Transgenic T-cell Receptor Cell Therapy.

Transgenic T-cell receptor (TCR) T cell-based adoptive cell therapies for solid tumors are associated with dramatic initial response rates, but there remain many instances of treatment failure and disease relapse. The association of infusion product cytokine profiles with clinical response has not been explored in the context of TCR T-cell therapy products. Single-cell antigen-dependent secretomic and proteomic analysis of preinfusion clinical TCR T-cell therapy products revealed that TNFα cytokine functionality of CD8+ T cells and phospho-STAT3 signaling in these cells were both associated with superior clinical responsiveness to therapy. By contrast, CD4+ T-helper 2 cell cytokine profiles were associated with inferior clinical responses. In parallel, preinfusion levels of IL15, Flt3-L, and CX3CL1 were all found to be associated with clinical response to therapy. These results have implications for the development of therapeutic biomarkers and identify potential targets for enrichment in the design of transgenic TCR T-cell therapies for solid tumors.

Remodeling of the tumor microenvironment through PAK4 inhibition sensitizes tumors to immune checkpoint blockade.

PAK4 inhibition can sensitize tumors to immune checkpoint blockade (ICB) therapy, however, the underlying mechanisms remain unclear. We report that PAK4 inhibition reverses immune cell exclusion by increasing the infiltration of CD8 T cells and CD103+ dendritic cells (DCs), a specific type of DCs that excel at cross-presenting tumor antigens and constitute a source of CXCL10. Interestingly, in melanoma clinical datasets, PAK4 expression levels negatively correlate with the presence of CCL21, the ligand for CCR7 expressed in CD103+ DCs. Furthermore, we extensively characterized the transcriptome of PAK4 knock out (KO) tumors, in vitro and in vivo, and established the importance of PAK4 expression in the regulation of the extracellular matrix, which can facilitate immune cell infiltration. Comparison between PAK4 wild type (WT) and KO anti-PD-1 treated tumors revealed how PAK4 deletion sensitizes tumors to ICB from a transcriptomic perspective. In addition, we validated genetically and pharmacologically that inhibition of PAK4 kinase activity is sufficient to improve anti-tumor efficacy of anti-PD-1 blockade in multiple melanoma mouse models. Therefore, this study provides novel insights into the mechanism of action of PAK4 inhibition and provides the foundation for a new treatment strategy that aims to overcome resistance to PD-1 blockade by combining anti-PD-1 with a small molecule PAK4 kinase inhibitor.

Autoantibody Landscape in Patients with Advanced Prostate Cancer.

PURPOSE: Autoantibody responses in cancer are of great interest, as they may be concordant with T-cell responses to cancer antigens or predictive of response to cancer immunotherapies. Thus, we sought to characterize the antibody landscape of metastatic castration-resistant prostate cancer (mCRPC). EXPERIMENTAL DESIGN: Serum antibody epitope repertoire analysis (SERA) was performed on patient serum to identify tumor-specific neoepitopes. Somatic mutation-specific neoepitopes were investigated by associating serum epitope enrichment scores with whole-genome sequencing results from paired solid tumor metastasis biopsies and germline blood samples. A protein-based immunome-wide association study (PIWAS) was performed to identify significantly enriched epitopes, and candidate serum antibodies enriched in select patients were validated by ELISA profiling. A distinct cohort of patients with melanoma was evaluated to validate the top cancer-specific epitopes. RESULTS: SERA was performed on 1,229 serum samples obtained from 72 men with mCRPC and 1,157 healthy control patients. Twenty-nine of 6,636 somatic mutations (0.44%) were associated with an antibody response specific to the mutated peptide. PIWAS analyses identified motifs in 11 proteins, including NY-ESO-1 and HERVK-113, as immunogenic in mCRPC, and ELISA confirmed serum antibody enrichment in candidate patients. Confirmatory PIWAS, Identifying Motifs Using Next-generation sequencing Experiments (IMUNE), and ELISA analyses performed on serum samples from 106 patients with melanoma similarly revealed enriched cancer-specific antibody responses to NY-ESO-1. CONCLUSIONS: We present the first large-scale profiling of autoantibodies in advanced prostate cancer, utilizing a new antibody profiling approach to reveal novel cancer-specific antigens and epitopes. Our study recovers antigens of known importance and identifies novel tumor-specific epitopes of translational interest.