Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response
T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner.
Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response
Tumors with mismatch repair deficiency (MMR-d) are characterized by sequence alterations in microsatellites and can accumulate thousands of mutations. This high mutational burden renders tumors immunogenic and sensitive to programmed cell death-1 (PD-1) immune checkpoint inhibitors. Yet, despite their tumor immunogenicity, patients with MMR-deficient tumors experience highly variable responses, and roughly half are refractory to treatment. We present experimental and clinical evidence showing that the degree of microsatellite instability (MSI) and resultant mutational load, in part, underlies the variable response to PD-1 blockade immunotherapy in MMR-d human and mouse tumors. The extent of response is particularly associated with the accumulation of insertion-deletion (indel) mutational load. This study provides a rationale for the genome-wide characterization of MSI intensity and mutational load to better profile responses to anti-PD-1 immunotherapy across MMR-deficient human cancers.
CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy
Cancer immunotherapy restores or enhances the effector function of CD8(+) T cells in the tumour microenvironment(1,2). CD8(+) T cells activated by cancer immunotherapy clear tumours mainly by inducing cell death through perforin-granzyme and Fas-Fas ligand pathways(3,4). Ferroptosis is a form of cell death that differs from apoptosis and results from iron-dependent accumulation of lipid peroxide(5,6). Although it has been investigated in vitro(7,8), there is emerging evidence that ferroptosis might be implicated in a variety of pathological scenarios(9,10). It is unclear whether, and how, ferroptosis is involved in T cell immunity and cancer immunotherapy. Here we show that immunotherapy-activated CD8(+) T cells enhance ferroptosis-specific lipid peroxidation in tumour cells, and that increased ferroptosis contributes to the anti-tumour efficacy of immunotherapy. Mechanistically, interferon gamma (IFNgamma) released from CD8(+) T cells downregulates the expression of SLC3A2 and SLC7A11, two subunits of the glutamate-cystine antiporter system xc(-), impairs the uptake of cystine by tumour cells, and as a consequence, promotes tumour cell lipid peroxidation and ferroptosis. In mouse models, depletion of cystine or cysteine by cyst(e)inase (an engineered enzyme that degrades both cystine and cysteine) in combination with checkpoint blockade synergistically enhanced T cell-mediated anti-tumour immunity and induced ferroptosis in tumour cells. Expression of system xc(-) was negatively associated, in cancer patients, with CD8(+) T cell signature, IFNgamma expression, and patient outcome. Analyses of human transcriptomes before and during nivolumab therapy revealed that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNgamma and CD8. Thus, T cell-promoted tumour ferroptosis is an anti-tumour mechanism, and targeting this pathway in combination with checkpoint blockade is a potential therapeutic approach.
Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy
Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer(1-3). However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients(4). In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2(-/-)Il2rg(-/-) mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
Endogenous CD4+ T cells recognize neoantigens in lung cancer patients, including recurrent oncogenic KRAS and ERBB2 (Her2) driver mutations
T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Much of the focus has been on identifying epitopes presented to CD8+ T cells by class I MHC. However, CD4+ class II MHC-restricted T cells have been shown to have an important role in antitumor immunity. Unfortunately, the vast majority of neoantigens recognized by CD8+ or CD4+ T cells in cancer patients result from random mutations and are patient-specific. Here, we screened the blood of 5 NSCLC patients for T-cell responses to candidate mutation-encoded neoepitopes. T-cell responses were detected to 8.8% of screened antigens, with 1-7 antigens identified per patient. A majority of responses were to random, patient-specific mutations. However, CD4+ T cells that recognized the recurrent KRASG12V and the ERBB2 (Her2) internal tandem duplication (ITD) oncogenic driver mutations, but not the corresponding wildtype sequences, were identified in two patients. Two different T-cell receptors (TCRs) specific for KRASG12V and one T-cell receptor specific for Her2-ITD were isolated and conferred antigen specificity when transfected into T cells. Deep sequencing identified the Her2-ITD-specific TCR in the tumor but not non-adjacent lung. Our results showed that CD4+ T-cell responses to neoantigens, including recurrent driver mutations, can be derived from the blood of NSCLC patients. These data support the use of adoptive transfer or vaccination to augment CD4+ neoantigen-specific T cells and elucidate their role in human antitumor immunity.
Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma
BACKGROUND: Although mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs. METHODS: In-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data. RESULTS: The approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations. CONCLUSIONS: This study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.
Identification of Neoantigen-Reactive Tumor-Infiltrating Lymphocytes in Primary Bladder Cancer
Immune checkpoint inhibitors are effective in treating a variety of malignancies, including metastatic bladder cancer. A generally accepted hypothesis suggests that immune checkpoint inhibitors induce tumor regressions by reactivating a population of endogenous tumor-infiltrating lymphocytes (TILs) that recognize cancer neoantigens. Although previous studies have identified neoantigen-reactive TILs from several types of cancer, no study to date has shown whether neoantigen-reactive TILs can be found in bladder tumors. To address this, we generated TIL cultures from patients with primary bladder cancer and tested their ability to recognize tumor-specific mutations. We found that CD4(+) TILs from one patient recognized mutated C-terminal binding protein 1 in an MHC class II-restricted manner. This finding suggests that neoantigen-reactive TILs reside in bladder cancer, which may help explain the effectiveness of immune checkpoint blockade in this disease and also provides a rationale for the future use of adoptive T cell therapy targeting neoantigens in bladder cancer.
Efficacy and Safety Analysis of Nelipepimut-S Vaccine to Prevent Breast Cancer Recurrence: A Randomized, Multicenter, Phase III Clinical Trial
PURPOSE: In phase I/II studies, nelipepimut-S (NP-S) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) vaccine was well tolerated and effectively raised HER2-specific immunity in breast cancer patients. Results from a prespecified interim analysis of a phase III trial assessing NP-S+GM-CSF are reported. EXPERIMENTAL DESIGN: This multicenter, randomized, double-blind phase III study enrolled females >/=18 years with T1-T3, HER2 low-expressing (immunohistochemistry 1+/2+), node-positive breast cancer in the adjuvant setting. Patients received 1000 mug NP-S+250 microg GM-CSF or placebo+GM-CSF monthly for 6 months, then every 6 months through 36 months. The primary objective was disease-free survival (DFS). Protocol-specified imaging occurred annually. New abnormalities were categorized as recurrence events; biopsy confirmation was not mandated. The interim analysis was conducted as specified in the protocol after 73 DFS events. RESULTS: Seven hundred fifty-eight patients (mean age 51.8 years) were randomized. Adverse events were similar between groups; most common were injection-associated: erythema (84.3%), induration (55.8%), pruritus (54.9%). There was no significant between-arms difference in DFS events at interim analysis at median follow-up (16.8 months). In the NP-S arm, imaging detected 54.1% of recurrence events in asymptomatic patients versus 29.2% in the placebo arm (P=0.069). CONCLUSIONS: NP-S was well tolerated. There was no significant difference in DFS events between NP-S and placebo. Use of mandated annual scans and image-detected recurrence events hastened the interim analysis contributing to early trial termination. Trial registration ID: NCT01479244.
A library of Neo Open Reading Frame peptides (NOPs) as a sustainable resource of common neoantigens in up to 50% of cancer patients
Somatic mutations in cancer can result in neoantigens against which patients can be vaccinated. The quest for tumor specific neoantigens has yielded no targets that are common to all tumors, yet foreign to healthy cells. Single base pair substitutions (SNVs) at best can alter 1 amino acid which can result in a neoantigen; with the exception of rare site-specific oncogenic driver mutations (such as RAS) such mutations are private. Here, we describe a source of common neoantigens induced by frame shift mutations, based on analysis of 10,186 TCGA tumor samples. We find that these frame shift mutations can produce long neoantigens. These are completely new to the body, and indeed recent evidence suggests that frame shifts can be highly immunogenic. We report that many different frame shift mutations converge to the same small set of 3' neo open reading frame peptides (NOPs), all encoded by the Neo-ORFeome. We find that a fixed set of only 1,244 neo-peptides in as much as 30% of all TCGA cancer patients. For some tumor classes this is higher; e.g. for colon and cervical cancer, peptides derived from only ten genes (saturated at 90 peptides) can be applied to 39% of all patients. 50% of all TCGA patients can be achieved at saturation (using all those peptides in the library found more than once). A pre-fabricated library of vaccines (peptide, RNA or DNA) based on this set can provide off the shelf, quality certified, 'personalized' vaccines within hours, saving months of vaccine preparation. This is crucial for critically ill cancer patients with short average survival expectancy after diagnosis.
Adaptive Immune Resistance Emerges from Tumor-Initiating Stem Cells
Our bodies are equipped with powerful immune surveillance to clear cancerous cells as they emerge. How tumor-initiating stem cells (tSCs) that form and propagate cancers equip themselves to overcome this barrier remains poorly understood. To tackle this problem, we designed a skin cancer model for squamous cell carcinoma (SCC) that can be effectively challenged by adoptive cytotoxic T cell transfer (ACT)-based immunotherapy. Using single-cell RNA sequencing (RNA-seq) and lineage tracing, we found that transforming growth factor beta (TGF-beta)-responding tSCs are superior at resisting ACT and form the root of tumor relapse. Probing mechanism, we discovered that during malignancy, tSCs selectively acquire CD80, a surface ligand previously identified on immune cells. Moreover, upon engaging cytotoxic T lymphocyte antigen-4 (CTLA4), CD80-expressing tSCs directly dampen cytotoxic T cell activity. Conversely, upon CTLA4- or TGF-beta-blocking immunotherapies or Cd80 ablation, tSCs become vulnerable, diminishing tumor relapse after ACT treatment. Our findings place tSCs at the crux of how immune checkpoint pathways are activated.