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Although immune check-point inhibitors (CPIs) revolutionized treatment of Merkel cell carcinoma (MCC), patients with CPI-refractory MCC lack effective therapy. More than 80% of MCC express T-antigens encoded by Merkel cell polyomavirus, which is an ideal target for T-cell receptor (TCR)-based immunotherapy. However, MCC often repress HLA expression, requiring additional strategies to reverse the downregulation for allowing T cells to recognize their targets. We identified TCRMCC1 that recognizes a T-antigen epitope restricted to human leukocyte antigen (HLA)-A*02:01. Seven CPI-refractory metastatic MCC patients received CD4 and CD8 T cells transduced with TCRMCC1 (TTCR-MCC1) preceded either by lymphodepleting chemotherapy or an HLA-upregulating regimen (single-fraction radiation therapy (SFRT) or systemic interferon gamma (IFNγ)) with concurrent avelumab. Two patients who received preceding SFRT and IFNγ respectively experienced tumor regression. One experienced regression of 13/14 subcutaneous lesions with 1 'escape' lesion and the other had delayed tumor regression in all lesions after initial progression. Although TTCR-MCC1 cells with an activated phenotype infiltrated tumors including the 'escape' lesion, all progressing lesions transcriptionally lacked HLA expression. While SFRT/IFNγ did not immediately upregulate tumor HLA expression, a secondary endogenous antigen-specific T cell infiltrate was detected in one of the regressing tumors and associated with HLA upregulation, indicating in situ immune responses have the potential to reverse HLA downregulation. Indeed, supplying a strong co-stimulatory signal via a CD200R-CD28 switch receptor allows TTCR-MCC1 cells to control HLA-downregulated MCC cells in a xenograft mouse model, upregulating HLA expression. Our results demonstrate the potential of TCR gene therapy for metastatic MCC and propose a next strategy for overcoming epigenetic downregulation of HLA in MCC.
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Dysregulation of the bone marrow (BM) niche in multiple myeloma (MM) alters the composition and state of resident immune cells, potentially impeding anti-tumor immunity. One common mechanism of immune inhibition in solid tumors is the induction of exhaustion in tumor-specific T cells. However, the extent of T cell tumor recognition and exhaustion is not well-characterized in MM. As the specific mechanisms of immune evasion are critical for devising effective therapeutic strategies, we deeply profiled the CD8+ T cell compartment of newly-diagnosed MM (NDMM) patients for evidence of tumor reactivity and T cell exhaustion. We applied single-cell multi-omic sequencing and antigen-specific mass cytometry to longitudinal BM and peripheral blood (PB) samples taken from timepoints spanning from diagnosis through induction therapy, autologous stem cell transplant (ASCT), and maintenance therapy. We identified an exhausted-like population that lacked several canonical exhaustion markers, was not significantly enriched in NDMM patients, and consisted of small, nonpersistent clones. We also observed an activated population with increased frequency in the PB of NDMM patients exhibiting phenotypic and clonal features consistent with homeostatic, antigen-nonspecific activation. However, there was no evidence of "tumor-experienced" T cells displaying hallmarks of terminal exhaustion and/or tumor-specific activation/expansion in NDMM patients at any timepoint.
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Relapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (HCT) for leukemia. T cells engineered by gene transfer to express T cell receptors (TCR; TCR-T) specific for hematopoietic-restricted minor histocompatibility (H) antigens may provide a potent selective anti-leukemic effect post-HCT. We conducted a phase I clinical trial employing a novel TCR-T product targeting the minor H antigen HA-1 to treat or consolidate treatment of persistent or recurrent leukemia and myeloid neoplasms. The primary objective was to evaluate the feasibility and safety of administration of HA-1 TCR-T post-HCT. CD8+ and CD4+ T cells expressing the HA-1 TCR and a CD8-co-receptor were successfully manufactured from HA-1 disparate HCT donors. One or more infusions of HA-1 TCR-T following lymphodepleting chemotherapy were administered to nine HCT recipients who had developed disease recurrence post-HCT. TCR-T cells expanded and persisted in vivo after adoptive transfer. No dose-limiting toxicities occurred. Although the study was not designed to assess efficacy, four patients achieved or maintained complete remissions following lymphodepletion and HA-1 TCR-T, with one ongoing at >2 years. Single-cell RNA sequencing of relapsing/progressive leukemia after TCR-T therapy identified upregulated molecules associated with T cell dysfunction or cancer cell survival. HA-1 TCR-T therapy appears feasible and safe and shows preliminary signals of efficacy. This clinical trial is registered at clinicaltrials.gov as NCT03326921.
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T cell reactivity to tumor-specific neoantigens can drive endogenous and therapeutically induced antitumor immunity. However, most tumor-specific neoantigens are unique to each patient (private) and targeting them requires personalized therapy. A smaller subset of neoantigens includes epitopes that span recurrent mutation hotspots, translocations, or gene fusions in oncogenic drivers and tumor suppressors, as well as epitopes that arise from viral oncogenic proteins. Such antigens are likely to be shared across patients (public), uniformly expressed within a tumor, and required for cancer cell survival and fitness. Although a limited number of these public neoantigens are naturally immunogenic, recent studies affirm their clinical utility. In this review, we highlight efforts to target mutant KRAS, mutant p53, and epitopes derived from oncogenic viruses using T cells engineered with off-the-shelf T cell receptors. We also discuss the challenges and strategies to achieving more effective T cell therapies, particularly in the context of solid tumors.
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The discovery and characterization of antigen-specific CD8+ T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8+ T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease.
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Linfócitos T CD8-Positivos , COVID-19 , Humanos , SARS-CoV-2/genética , Antígenos , Epitopos , Peptídeos/genéticaRESUMO
The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.
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Linfócitos T CD8-Positivos , Ácido Linoleico , Ácido Linoleico/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: In the USA, more than 50% of patients with ovarian cancer die within 5 years of diagnosis, highlighting the need for therapeutic innovations. Mesothelin (MSLN) is a candidate immunotherapy target; it is overexpressed by ovarian tumors and contributes to malignant/invasive phenotypes, making tumor antigen loss disadvantageous. We previously showed that MSLN-specific T cell receptor (TCR)-engineered T cells preferentially accumulate within established tumors, delay tumor growth, and significantly prolong survival in the ID8VEGF mouse model that replicates many aspects of human disease. However, T cell persistence and antitumor activity were not sustained. We therefore focused on Fas/FasL signaling that can induce activation-induced cell death, an apoptotic mechanism that regulates T cell expansion. Upregulation of FasL by tumor cells and tumor vasculature has been detected in the tumor microenvironment (TME) of human and murine ovarian cancers, can induce apoptosis in infiltrating, Fas (CD95) receptor-expressing lymphocytes, and can protect ovarian cancers from tumor-infiltrating lymphocytes. METHODS: To overcome potential FasL-mediated immune evasion and enhance T cell responses, we generated an immunomodulatory fusion protein (IFP) containing the Fas extracellular binding domain fused to a 4-1BB co-stimulatory domain, rather than the natural death domain. Murine T cells were engineered to express an MSLN-specific TCR (TCR1045), alone or with the IFP, transferred into ID8VEGF tumor-bearing mice and evaluated for persistence, proliferation, cytokine production and efficacy. Human T cells were similarly engineered to express an MSLN-specific TCR (TCR530) alone or with a truncated Fas receptor or a Fas-4-1BB IFP and evaluated for cytokine production and tumor lysis. RESULTS: Relative to murine T cells expressing only TCR1045, T cells expressing both TCR1045 and a Fas-4-1BB IFP preferentially persisted in the TME of tumor-bearing mice, with improved T cell proliferation and survival. Moreover, TCR1045/IFP+ T cells significantly prolonged survival in tumor-bearing mice, compared with TCR1045-only T cells. Human T cells expressing TCR530 and a Fas-4-1BB IFP exhibit enhanced functional activity and viability compared with cells with only TCR530. CONCLUSIONS: As many ovarian tumors overexpress FasL, an IFP that converts the Fas-mediated death signal into pro-survival and proliferative signals may be used to enhance engineered adoptive T cell therapy for patients.
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Neoplasias Ovarianas , Fator A de Crescimento do Endotélio Vascular , Animais , Carcinoma Epitelial do Ovário , Terapia Baseada em Transplante de Células e Tecidos , Proteína Ligante Fas , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Receptores de Antígenos de Linfócitos T/genética , Microambiente TumoralRESUMO
BACKGROUND: Achieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCRMsln) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state. METHODS: Here, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells. RESULTS: PD-L1 pathway blockade and/or simultaneous blockade of multiple coinhibitory receptors during adoptive cell therapy was insufficient to prevent engineered T cell dysfunction in autochthonous PDA yet resulted in subclinical activity in the lung, without enhancing anti-tumor immunity. Gene expression analysis revealed that ex vivo TCR engineered T cells markedly differed from in vivo primed endogenous effector T cells which can respond to immune checkpoint inhibitors. Early after transfer, intratumoral TCR engineered T cells acquired a similar molecular program to prototypical exhausted T cells that arise during chronic viral infection, but the molecular programs later diverged. Intratumoral engineered T cells exhibited decreased effector and cell cycle genes and were refractory to TCR signaling. CONCLUSIONS: Abrogation of PD-1 signaling is not sufficient to overcome TCR engineered T cell dysfunction in PDA. Our study suggests that contributions by both the differentiation pathways induced during the ex vivo T cell engineering process and intratumoral suppressive mechanisms render engineered T cells dysfunctional and resistant to rescue by blockade of immune checkpoints.
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Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Linfócitos T/metabolismo , Animais , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Neoplasias PancreáticasRESUMO
Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.