The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to ...combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
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•A subset of neoantigen-expressing pancreas cancer evades immune surveillance•Markers of T cell exhaustion typify pancreas cancer tumor-infiltrating lymphocytes•The CD155/TIGIT axis promotes immune evasion in pancreas cancer•TIGIT/PD-1 co-blockade plus CD40 agonism reinvigorates tumor-reactive T cells
Freed-Pastor et al. identify the CD155/TIGIT axis as a key driver of immune evasion in pancreas cancer. Neoepitope prediction reveals a subset of human pancreas cancer patients with predicted high-affinity neoepitopes and functional interrogation using preclinical models identifies a combination immunotherapy approach (TIGIT/PD-1 co-blockade plus CD40 agonism) capable of eliciting profound anti-tumor responses.
DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not ...durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.
Genetically engineered mouse models only capture a small fraction of the genetic lesions that drive human cancer. Current CRISPR-Cas9 models can expand this fraction but are limited by their reliance ...on error-prone DNA repair. Here we develop a system for in vivo prime editing by encoding a Cre-inducible prime editor in the mouse germline. This model allows rapid, precise engineering of a wide range of mutations in cell lines and organoids derived from primary tissues, including a clinically relevant Kras mutation associated with drug resistance and Trp53 hotspot mutations commonly observed in pancreatic cancer. With this system, we demonstrate somatic prime editing in vivo using lipid nanoparticles, and we model lung and pancreatic cancer through viral delivery of prime editing guide RNAs or orthotopic transplantation of prime-edited organoids. We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.
Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g., those with microsatellite ...instability (MSI)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) colorectal cancer (CRC) express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in MSI CRC. Using a versatile platform for modulating neoantigen expression in CRC organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross priming and drives immediate T cell dysfunction. Strikingly, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response.
Immunity genes have repeatedly experienced natural selection during mammalian evolution. Galectins are carbohydrate-binding proteins that regulate diverse immune responses, including maternal-fetal ...immune tolerance in placental pregnancy. Seven human galectins, four conserved across vertebrates and three specific to primates, are involved in placental development. To comprehensively study the molecular evolution of these galectins, both across mammals and within humans, we conducted a series of between- and within-species evolutionary analyses. By examining patterns of sequence evolution between species, we found that primate-specific galectins showed uniformly high substitution rates, whereas two of the four other galectins experienced accelerated evolution in primates. By examining human population genomic variation, we found that galectin genes and variants, including variants previously linked to immune diseases, showed signatures of recent positive selection in specific human populations. By examining one nonsynonymous variant in Galectin-8 previously associated with autoimmune diseases, we further discovered that it is tightly linked to three other nonsynonymous variants; surprisingly, the global frequency of this four-variant haplotype is ∼50%. To begin understanding the impact of this major haplotype on Galectin-8 protein structure, we modeled its 3D protein structure and found that it differed substantially from the reference protein structure. These results suggest that placentally expressed galectins experienced both ancient and more recent selection in a lineage- and population-specific manner. Furthermore, our discovery that the major Galectin-8 haplotype is structurally distinct from and more commonly found than the reference haplotype illustrates the significance of understanding the evolutionary processes that sculpted variants associated with human genetic disease.
BackgroundAberrant translation of the non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I; HLA-I in humans) and these ...non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies. While emerging evidence suggests that translation of unannotated open reading frames (uORFs) can give rise to MHC class I-associated peptides (MAPs) across a range of malignancies, it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting non-canonical MAPs (ncMAPs) can elicit a T cell response.MethodsWe leveraged twelve pancreatic cancer (PDAC) patient-derived organoids (PDOs) to purify the malignant compartment from low tumor cellularity tumor specimens. We developed a cutting-edge proteogenomics pipeline, coupled with high-depth immunopeptidomics to identify pancreatic cancer MAPs derived from somatic mutations, retained introns, and uORFs. To investigate the cancer-specificity of ncMAPs, we developed a translation-centric analysis pipeline that examines translation of uORFs encoding ncMAPs across a range of healthy tissues, including healthy thymus. To evaluate for immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand ncMAP-specific cytotoxic T lymphocytes (CTLs) and evaluate cytolytic potential.ResultsWe demonstrate that ncMAPs are abundant and predominate over mutation-derived peptides in the pancreatic cancer immunopeptidome, establishing a novel class of recurrent cancer-restricted epitopes available for immune recognition. We observed widespread translation and MHC-I presentation of numerous ncMAPs across many healthy tissues, highlighting the importance of our translation-centric approach to assess cancer-restriction. Excitingly, we nominated over 500 ncMAPs that exhibit cancer-specific translation patterns. Approximately 30% of ncMAPs exhibited bona fide cancer-restricted translation patterns, and a substantial subset of these were shared among patients. We next interrogated immunogenicity using a highly sensitive ex vivo vaccination platform and demonstrated that the majority of cancer-restricted ncMAPs evaluated were highly immunogenic. Remarkably, the proportion of ncMAPs harboring immunogenic potential was substantially higher than mutation-derived neoepitopes and tumor-associated antigens, underscoring their therapeutic potential relative to traditional immunotherapy targets.ConclusionsThese findings demonstrate that aberrant translation in pancreatic cancer can give rise to recurrent cancer-restricted ncMAPs capable of recognition by cytotoxic T lymphocytes. Collectively, our findings furnish a novel set of recurrent, cancer-restricted immunotherapy targets not subject to central tolerance. We believe these findings will prompt translation-centric investigations in other solid tumors. We envision that these novel antigens will augment ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies.Ethics ApprovalInformed consent was obtained from patients at least 18 years old with pancreatic cancer under Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB)-approved protocols 11–104, 17–000, 03–189, and/or 14–408 for tissue collection, molecular analysis, and organoid generation.
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical ...management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.
Pancreatic cancer is a lethal malignancy recalcitrant to immune checkpoint blockade and other immunotherapies. A subset of tumors is computationally predicted to harbor potentially immunogenic ...peptides for MHC class I (MHC-I) presentation, but the nature, expression, and immunogenicity of these peptides has yet to be determined. The only prior study of the pancreatic cancer immunopeptidome focused on profiling MHC-I-associated peptides (MAPs) from canonical proteins in bulk tumor samples; however, non-malignant cell populations comprise most of the pancreatic tumor mass, obscuring the identity of MAPs that derive specifically from cancer cells. In the second chapter of this thesis, I resolve this challenge through extensive profiling of patient-derived organoids with whole-genome sequencing, RNA sequencing, and immunopeptidomics. These data enable a proteogenomics approach that tailors MAP identification to each individual patient sample. Harnessing this platform, my colleagues and I uncovered a diverse cohort of MAPs derived from somatic mutations and transcript isoforms that are functionally unexpressed in most or all healthy tissues. These include MAPs derived from novel, unannotated open reading frames (nuORFs) present within long noncoding RNAs, processed transcripts, and 5’ and 3’ untranslated regions. We found that cytotoxic T cells specific to nuORF-derived MAPs can be readily generated from peripheral blood mononuclear cells of healthy donor individuals. This result highlights the immunogenicity of nuORF-derived MAPs and establishes them as promising targets for immunotherapies in pancreatic cancer.;
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In Chapter 3, I report the development of a genetically engineered mouse model (GEMM) for performing prime editing in vivo. This system represents a rapid alternative to traditional cancer mouse models, which often take months or years to develop. Through a Creinducible prime editor enzyme encoded in the mouse germline, prime editor GEMMs can mediate rapid and precise engineering of most cancer mutations, including many that are challenging or infeasible to achieve with other CRISPR technology. We demonstrate the utility of this system by mediating secondary Kras mutations and common Trp53 hotspot mutations in model-derived pancreatic organoids. Finally, we model lung and pancreatic cancer in vivo using lentiviral delivery of prime editing guide RNAs or orthotopic transplantation of prime edited organoids. We anticipate that prime editing GEMMs will accelerate preclinical functional studies of cancer-associated alleles that are challenging to model by traditional approaches.
Ph.D.
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