Introduction
Despite recent advances in treatment for a number of cancers with immune checkpoint blockade (ICB), immunotherapy has had limited efficacy in glioblastoma (GBM). The recent ...multi-centered CheckMate 143 trial in first time recurrent GBM and the Checkmate 498 trial in newly diagnosed unmethylated GBM showed that antibodies against programmed cell death protein 1 (PD-1) failed to improve overall survival in patients with GBM. Recent preclinical and clinical studies have explored combining ICB with several other therapies including additional ICB against alternative checkpoint molecules, activation of costimulatory checkpoint molecules such as 4-1BB, radiation-induced tumor cell lysis and immunogenic recruitment, local chemotherapy, neoadjuvant ICB therapy, and myeloid cell reactivation.
Methods
We have reviewed the literature on ICB seminal to the progression of several preclinical studies and clinical trials in order to provide a compendium of the current state of combination immunotherapy for GBM. For ongoing clinical trials without associated publications, we searched
clinicaltrials.gov
for ongoing studies using the keywords, “GBM” and “glioblastoma”, as well as names of checkpoint molecules.
Results
Recent trends from clinical trials demonstrate that despite a variety of different combination strategies involving ICB, GBM remains largely elusive to current immunotherapies. There is a discordance of survival outcomes between GBM pre-clinical models and clinical trials, likely due to the heterogeneity of GBM in patients as well as other adaptive immune mechanisms not otherwise represented in murine models. However, in clinical studies, neoadjuvant ICB in GBM was found to diversify the T cell receptor (TCR) repertoire and increase chemokine mRNA transcripts when comparing pre- and post- surgical time points. Moreover, an increase in peripheral and tumor-infiltrating lymphocyte (TIL) clonotypes were also observed when comparing adjuvant and neoadjuvant cohorts.
Discussion
Despite the lack of clinical survival benefit, immune modulation was observed in multiple different combination strategies for GBM in both preclinical and clinical studies, indicating that ICB combination therapy results in a significant immunological impact on the tumor microenvironment.
PD-1 blockade unleashes CD8 T cells
, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell ...transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens
, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay
in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIT
TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.
BackgroundWe conducted the first trial of neoadjuvant PD-1 blockade in resectable non-small cell lung cancer (NSCLC), finding nivolumab monotherapy to be safe and feasible with an encouraging rate of ...pathologic response. Building on these results, and promising data for nivolumab plus ipilimumab (anti-CTLA-4) in advanced NSCLC, we expanded our study to include an arm investigating neoadjuvant nivolumab plus ipilimumab.MethodsPatients with resectable stage IB (≥4 cm)–IIIA (American Joint Committee on Cancer Tumor Node Metastases seventh edition), histologically confirmed, treatment-naïve NSCLC received nivolumab 3 mg/kg intravenously plus ipilimumab 1 mg/kg intravenously 6 weeks prior to planned resection. Nivolumab 3 mg/kg was given again approximately 4 and 2 weeks preoperatively. Primary endpoints were safety and feasibility with a planned enrollment of 15 patients. Pathologic response was a key secondary endpoint.ResultsWhile the treatment regimen was feasible per protocol, due to toxicity, the study arm was terminated early by investigator consensus after 9 of 15 patients were enrolled. All patients received every scheduled dose of therapy and were fit for planned surgery; however, 6 of 9 (67%) experienced treatment-related adverse events (TRAEs) and 3 (33%) experienced grade ≥3 TRAEs. Three of 9 patients (33%) had biopsy-confirmed tumor progression precluding definitive surgery. Of the 6 patients who underwent resection, 3 are alive and disease-free, 2 experienced recurrence and are actively receiving systemic treatment, and one died postoperatively due to acute respiratory distress syndrome. Two patients who underwent resection had tumor pathologic complete responses (pCRs) and continue to remain disease-free over 24 months since surgery. Pathologic response correlated with pre-treatment tumor PD-L1 expression, but not tumor mutation burden. Tumor KRAS/STK11 co-mutations were identified in 5 of 9 patients (59%), of whom two with disease progression precluding surgery had tumor KRAS/STK11/KEAP1 co-mutations.ConclusionsThough treatment was feasible, due to toxicity the study arm was terminated early by investigator consensus. In light of this, and while the long-term disease-free status of patients who achieved pCR is encouraging, further investigation of neoadjuvant nivolumab plus ipilimumab in patients with resectable NSCLC requires the identification of predictive biomarkers that enrich for response.
T-cell receptor sequencing (TCRseq) enables tracking of T-cell clonotypes recognizing the same antigen over time and across biological compartments. TCRseq has been used to test if cross-reactive ...antitumor T cells are responsible for development of immune-related adverse events (irAEs) following immune checkpoint blockade. Prior studies have interpreted T-cell clones shared among the tumor and irAE as evidence supporting this, but interpretations of these findings are challenging, given the constraints of TCRseq. Here we capitalize on a rare opportunity to understand the impact of potential confounders, such as sample size, tissue compartment, and collection batch/timepoint, on the relative proportion of shared T-cell clones between an irAE and tumor specimens. TCRseq was performed on tumor-involved and -uninvolved tissues, including an irAE, that were obtained throughout disease progression and at the time of rapid autopsy from a patient with renal cell carcinoma treated with programmed death-1 (PD-1) blockade. Our analyses show significant effects of these confounders on our ability to understand T-cell receptor overlap, and we present mitigation strategies and study design recommendations to reduce these errors. Implementation of these strategies will enable more rigorous TCRseq-based studies of immune responses in human tissues, particularly as they relate to antitumor T-cell cross-reactivity in irAEs following checkpoint blockade.
HIV-1 positive elite controllers or suppressors control viral replication without antiretroviral therapy, likely via CTL-mediated elimination of infected cells, and therefore represent a model of an ...HIV-1 functional cure. Efforts to cure HIV-1 accordingly rely on the existence or generation of antigen-specific cytotoxic T lymphocytes (CTL) to eradicate infected cells upon reversal of latency. Detecting and quantifying these HIV-1-specific CTL responses will be crucial for developing vaccine and T cell-based immunotherapies. A recently developed assay, called MANAFEST, uses T cell receptor (TCR) Vβ sequencing of peptide-stimulated cultures followed by a bioinformatic pipeline to identify neoantigen-specific T cells in cancer patients. This assay is more sensitive than conventional immune assays and therefore has the possibility to identify HIV-1 antigenic targets that have not been previously explored for vaccine or T cell immunotherapeutic strategies. Here we show that a modified version of the MANAFEST assay, called ViraFEST, can identify memory CD8
T cell responses against autologous HIV-1 Gag and Nef epitope variants in an elite suppressor. Nine TCR Vβ clonotypes were identified and 6 of these were cross-reactive for autologous variants or known escape variants. Our findings are a proof of principle that the ViraFEST assay can be used to detect and monitor these responses for downstream use in immunotherapeutic treatment approaches.
Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade due to its highly immunosuppressive tumor milieu. Moreover, current ...anti-PD-1 treatment requires multiple infusions with adverse systemic effects. Therefore, we used a PCL:PEG:PCL polymer gel loaded with anti-PD-1 and implanted at the site of lymph nodes in an attempt to maximize targeting of inactivated T cells as well as mitigate unnecessary systemic exposure.
Mice orthotopically implanted with GL261 glioma cells were injected with hydrogels loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with ex vivo flow cytometric staining for L/D, CD45, CD3, CD4, CD8, TNF-α and IFN-y and co-culture ELISpots were done for immune cell activation assays.
Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with anti-PD-1 (P = .0185). Flow cytometric analysis of brain tissue and co-culture of lymph node T cells from mice implanted with gels demonstrated increased levels of IFN-y and TNF-α compared to mice treated with systemic anti-PD-1, indicating greater reversal of immunosuppression compared to systemic treatment.
Our data demonstrate proof of principle for using localized therapy that targets lymph nodes for GBM. We propose an alternative treatment paradigm for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.
Several predictive biomarkers are currently approved or are under investigation for the selection of patients for checkpoint blockade. Tumor PD-L1 expression is used for stratification of non-small ...cell lung (NSCLC) patients, with tumor mutational burden (TMB) also being explored with promising results, and mismatch-repair deficiency is approved for tumor site-agnostic disease. While tumors with high PD-L1 expression, high TMB, or mismatch repair deficiency respond well to checkpoint blockade, tumors with lower PD-L1 expression, lower mutational burdens, or mismatch repair proficiency respond much less frequently.
We studied two patients with unexpected responses to checkpoint blockade monotherapy: a patient with PD-L1-negative and low mutational burden NSCLC and one with mismatch repair proficient colorectal cancer (CRC), both of whom lack the biomarkers associated with response to checkpoint blockade, yet achieved durable clinical benefit. Both maintained T-cell responses in peripheral blood to oncogenic driver mutations - BRAF-N581I in the NSCLC and AKT1-E17K in the CRC - years after treatment initiation. Mutation-specific T cells were also found in the primary tumor and underwent dynamic perturbations in the periphery upon treatment.
These findings suggest that T cell responses to oncogenic driver mutations may be more prevalent than previously appreciated and could be harnessed in immunotherapeutic treatment, particularly for patients who lack the traditional biomarkers associated with response. Comprehensive studies are warranted to further delineate additional predictive biomarkers and populations of patients who may benefit from checkpoint blockade.
Antibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in ...which little progress has been made during the past decade.
In this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses.
Neoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1-positive and PD-L1-negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab.
Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute-Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621 .).
LAD is an inventor of licensed intellectual property related to technology for circulating tumor DNA analyses and mismatch repair deficiency for diagnosis and therapy (WO2016077553A1) from Johns ...Hopkins University. ...in the past 5 years, LAD has participated as a paid consultant for one-time engagements with Caris, Lyndra, Genocea Biosciences, Illumina and Cell Design Labs. J.R.B. is a consultant/advisory board member of BMS, Merck, Medimmune/AstraZeneca, Johnson and Johnson, Syndax, Celgene, Amgen, EliLilly. R.A.A. is a consultant/advisory board member for Bristol Myers Squibb, Merck, AstraZeneca, Adaptive Biotechnologies and has received research funding from Bristol Myers Squibb, Stand-up to Cancer and contractual work from Five Prime Therapeutics and FLX Bio.
INTRODUCTION: Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade against programmed cell death protein 1 (PD-1) on T ...cells. The highly immunosuppressive tumor milieu of GBM prevents rescue of inactivated T cells due to decreased numbers of infiltrating T cells at the tumor site as well as increased recruitment of myeloid cells. Moreover, the current strategy of using antibodies against PD-1 (anti-PD-1) requires multiple intravenous infusions every two or three weeks with debilitating systemic effects. METHODS: Mice orthotopically implanted with GL261 glioma cells were injected with PCL:PEG:PCL hydrogel polymers loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with subsequent ex vivo flow cytometric staining for CD4, CD8, IFN-y, TNF-a, Foxp3, and PD-1. RESULTS: Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with three doses of anti-PD-1 (p < 0.001). Flow cytometric analysis of lymph nodes and brain tissue in mice injected with this gel demonstrated increased levels of IFN-y, indicating greater reversal of immunosuppression compared to standard treatment. CONCLUSIONS: Our data demonstrates proof of principle of the advantages of using localized therapy that targets lymph nodes for GBM. We propose a paradigm shift for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.
PDF
