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

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

A Pilot Trial of the Combination of Transgenic NY-ESO-1–reactive Adoptive Cellular Therapy with Dendritic Cell Vaccination with or without Ipilimumab

PURPOSE:Transgenic adoptive cell therapy (ACT) targeting the tumor antigen NY-ESO-1 can be effective for the treatment of sarcoma and melanoma. Preclinical models have shown that this therapy can be improved with the addition of dendritic cell (DC) vaccination and immune checkpoint blockade. We studied the safety, feasibility, and antitumor efficacy of transgenic ACT with DC vaccination, with and without CTLA-4 blockade with ipilimumab. PATIENTS AND METHODS:Freshly prepared autologous NY-ESO-1-specific T-cell receptor (TCR) transgenic lymphocytes were adoptively transferred together with NY-ESO-1 peptide-pulsed DC vaccination in HLA-A2.1-positive subjects alone (ESO, NCT02070406) or with ipilimumab (INY, NCT01697527) in patients with advanced sarcoma or melanoma. RESULTS:Six patients were enrolled in the ESO cohort, and four were enrolled in the INY cohort. Four out of six patients treated per ESO (66%), and two out of four patients treated per INY (50%) displayed evidence of tumor regression. Peripheral blood reconstitution with NY-ESO-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Tracking of transgenic T cells to the tumor sites was demonstrated in on-treatment biopsies via TCR sequencing. Multiparametric mass cytometry of transgenic cells demonstrated shifting of transgenic cells from memory phenotypes to more terminally differentiated effector phenotypes over time. CONCLUSIONS:ACT of fresh NY-ESO-1 transgenic T cells prepared via a short ex vivo protocol and given with DC vaccination, with or without ipilimumab, is feasible and results in transient antitumor activity, with no apparent clinical benefit of the addition of ipilimumab. Improvements are needed to maintain tumor responses

Autoantibody Landscape in Patients with Advanced Prostate Cancer

PurposeAutoantibody 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 designSerum 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.ResultsSERA 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.ConclusionsWe 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

Ipilimumab with or without nivolumab in PD-1 or PD-L1 blockade refractory metastatic melanoma: a randomized phase 2 trial

In this randomized phase 2 trial, blockade of cytotoxic T-lymphocyte protein 4 (CTLA-4) with continuation of programmed death protein 1 (PD-1) blockade in patients with metastatic melanoma who had received front-line anti-PD-1 or therapy against programmed cell death 1 ligand 1 and whose tumors progressed was tested in comparison with CTLA-4 blockade alone. Ninety-two eligible patients were randomly assigned in a 3:1 ratio to receive the combination of ipilimumab and nivolumab, or ipilimumab alone. The primary endpoint was progression-free survival. Secondary endpoints included the difference in CD8 T cell infiltrate among responding and nonresponding tumors, objective response rate, overall survival and toxicity. The combination of nivolumab and ipilimumab resulted in a statistically significant improvement in progression-free survival over ipilimumab (hazard ratio = 0.63, 90% confidence interval (CI) = 0.41-0.97, one-sided P = 0.04). Objective response rates were 28% (90% CI = 19-38%) and 9% (90% CI = 2-25%), respectively (one-sided P = 0.05). Grade 3 or higher treatment-related adverse events occurred in 57% and 35% of patients, respectively, which is consistent with the known toxicity profile of these regimens. The change in intratumoral CD8 T cell density observed in the present analysis did not reach statistical significance to support the formal hypothesis tested as a secondary endpoint. In conclusion, primary resistance to PD-1 blockade therapy can be reversed in some patients with the combination of CTLA-4 and PD-1 blockade. Clinicaltrials.gov identifier: NCT03033576

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

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