Supplementary Figures 1-12 Legends

Suppl. Table 3. Log2 Fold Changes (FC) of some genes differentially expressed in PTC with BRAFV600E and hTERT mutation vs BRAFV600E-PTC

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell im-munity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vac-cine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, mem-brane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data sug-gest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Suppl. Table 2. Log2 Fold Changes (FC) of some genes differentially expressed in BRAFV600E-PTC vs BRAFWT-PTC

Suppl. Table 4. Log2 Fold Changes (FC) of some genes differentially expressed in PTC wih BRAFV600E and hTERT mutation vs BRAFWT-PTC

Supplementary Table 1 Antibodies list and experimental conditions for immunohistochemistry (IHC)

Suppl. Figure 1 Drug dose-effect analysis in BRAFWT/V600E-KTC1 cells Suppl.Figure 2 Dose-effect analysis of vemurafenib and sorafenib on pericyte viability. Suppl. Figure 3 BRAFWT/WT-TPC1 cells viability assay. Suppl. Figure 4 ELISA analysis of cytokines secretion in pericytes, and PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT. Suppl. Figure 5 Effects of pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 6 Analysis of secreted TSP-1 levels in PTC cells harboring the heterozygous BRAFWT/V600E mutation or with BRAFWT/WT treated with shTSP-1 or shGFP (control) pericyte secretome. Suppl. Figure 7 Effects of shTSP-1 pericyte secretome in BRAFWT/WT-TPC1 cells. Suppl. Figure 8 Viability assay in the presence of SRI31277 in PTC cells and pericytes. Suppl. Figure 9 Effects of the small molecule SRI31277 in PTC cells with BRAFWT/WT. Suppl. Figure 10 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compared to BRAFWT/V600E-PTC samples. Suppl. Figure 11 THBS1 (TSP-1) mRNA expression levels in PTC samples harboring both BRAFWT/V600E and hTERT mutations compared to BRAFWT/WT-PTC samples. Suppl. Figure 12 THBS1 (TSP-1) gene regulatory networks pathways in PTC harboring BRAFWT/V600E and hTERT mutations compared to BRAFWT/WT-PTC clinical samples.

Neoantigens arising from mutations in tumor DNA provide targets for immune-based therapy. Here, we report the clinical and immune data from a Phase Ib clinical trial of a personalized neoantigen-vaccine NEO-PV-01 in combination with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous non-small cell lung cancer (NSCLC). This analysis of 38 patients treated with the regimen demonstrated no treatment-related serious adverse events. Multiple parameters including baseline tumor immune infiltration and on-treatment circulating tumor DNA levels were highly correlated with clinical response. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination. Epitope spread to non-vaccinating neoantigens, including responses to KRAS G12C and G12V mutations, were detected post-vaccination. Neoantigen-specific CD4+ T cells generated post-vaccination revealed effector and cytotoxic phenotypes with increased CD4+ T cell infiltration in the post-vaccine tumor biopsy. Collectively, these data support the safety and immunogenicity of this regimen in advanced non-squamous NSCLC.

Abstract Background: Neoantigens arising from mutations in cancer cell DNA are important targets for T cell-mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides (14-35 amino acids) based on a patient's HLA profile and bioinformatic analysis of tumor neoantigens. We report here relationships between baseline tumor characteristics, immune response, and clinical outcomes from NT-002, a Phase 1b study of NEO-PV-01 with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous NSCLC (NCT03380871). The primary objective of this study was to evaluate the safety of the combination. Materials: Serial blood and tumor biopsies were collected at: i) prior to treatment, ii) after 12 weeks of chemotherapy-pembrolizumab treatment, and iii) after completion of NEO-PV-01 vaccination. Tumor biopsies were characterized by immunohistochemistry for immune and tumor markers, gene expression, whole-exome and TCR sequencing, and single-cell analysis. Antigen-specific responses were measured in blood samples by IFNγ ELISpot, intracellular cytokine staining and functional phenotyping by FACS. Results: A total of 38 patients initiated study treatment (ITT); 21 patients received at least 1 dose of NEO-PV-01 (VAX). The regimen was well-tolerated and consistent with the pembrolizumab plus pemetrexed/carboplatin safety profile. The overall response rate (ORR)/clinical benefit rate (CBR) for the ITT and VAX were 37%/69% and 57%/95%, respectively. Median PFS was 7.2 months (95% CI: 5.6,16.8) for both the ITT and VAX, and median OS 16.8 months (95% CI: 11.6, NR) for both groups. Immune analysis on 12 patients with available samples revealed neoantigen-specific CD4+ and CD8+ T cell responses in all patients tested with an average of 55% of vaccine peptides generating an immune response post-vaccination. Vaccine-induced immune responses were mutant-specific and durable at 52-week treatment timepoint. T cell responses were polyfunctional, as evident by secretion of multiple cytokines (TNFα, IL2, IFNγ), and were activated memory cells with a cytotoxic phenotype. Epitope spread was observed in 7 of 11 patients analyzed thus far. Comprehensive analysis by gene expression, ctDNA and TCR repertoire analysis demonstrated correlations to extended PFS. Additional data on single-cell sequencing of neoantigen-specific T cells and tumor biopsies and correlates to clinical outcomes will be presented. Conclusions: NEO-PV-01 in combination with pembrolizumab and carboplatin/pemetrexed has a good safety profile and induces de novo immune responses in first-line non-squamous NSCLC. The association of baseline disease characteristics to prolonged PFS suggests future patient enrichment strategies for evaluation of this novel regimen in a phase 2 trial. Citation Format: Mark M. Awad, Ramaswamy Govindan, David R. Spigel, Edward B. Garon, Victoria Kohler, Rohit Vyasamneni, Suchitra Ramesh, Tracey E. Sciuto, Melissa A. Moles, Jennifer Tepper, Amy Wanamaker, Zakaria S. Khondkar, John Srouji, Jesse Z. Dong, Kristen N. Balogh, Asaf Poran, Meghan E. Bushway, Mark DeMario, Richard B. Gaynor, Lakshmi Srinivasan. A personal neoantigen vaccine NEO-PV-01 in combination with chemotherapy and pembrolizumab induces broad de novo immune responses in first-line non-squamous NSCLC: Associations with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 73.

Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).

Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient’s neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here, we report relationships between baseline tumor characteristics, immune response and clinical outcome for NT-001, a Phase 1b study of NEO-PV-01 + adjuvant in combination with nivolumab in either first or later line therapy for patients with metastatic melanoma, bladder and non-small cell lung cancer (NCT02897765). This analysis is focused on the melanoma cohort. Methods: Serial blood and tumor biopsies were scheduled for collection: i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Baseline features and immune responses in tumor cells were characterized by immunohistochemistry for multiple immune and tumor markers, gene expression, whole exome and TCR sequencing. Antigen-specific responses by IFNγ ELISpot, intracellular cytokine staining, multi-parameter surface and functional phenotyping by FACS and the presence of cytolytic properties were monitored in serial peripheral blood samples. Durability of immune responses up to 104 weeks post start of treatment will be measured. Results: A cohort of 10 melanoma patients demonstrated CD4 and CD8 T cell responses against 56% of vaccine peptides that were detected primarily in the post-vaccination samples as measured by IFNγ ELISpot. Analysis of additional patients is ongoing. T cell responses were neoantigen-specific for most peptides tested (86%; 12/14). Vaccine-induced immune responses were durable in patients who reached the week 52 treatment timepoint. Most T cell responses were polyfunctional, as evident by secretion of multiple cytokines, exhibited a memory and effector memory phenotype and were cytolytic. Epitope spreading, defined as post-vaccination T cell responses to neoantigens not included in the vaccine, was observed in multiple melanoma patients analyzed and evaluated for association with post vaccine clinical responses. Further, multi-platform assessments of immune and molecular responses including gene expression and TCR repertoire analysis demonstrate extensive responses in patients continuing study past 52 weeks. Additional correlates of clinical outcomes with molecular and immunologic responses will be presented. Conclusions: Treatment with NEO-PV-01 + adjuvant in combination with nivolumab induced broad de novo neoantigen-specific immune responses in metastatic melanoma. Immune responses were specific and correlations with clinical outcomes will be discussed. Citation Format: Siwen Hu-Lieskovan, Patrick A. Ott, Aung Naing, Rana H. Besada, Samantha J. Gates, Victoria R. Kohler, Riley R. Curran, Meghan E. Bushway, Julian Scherer, Kristen N. Balogh, Tracey E. Sciuto, Ying S. Ting, Michael S. Rooney, Dewi Harjanto, Zhengping Huang, Yuting Huang, Yvonne Ware, April Lamb, Lisa D. Cleary, Melissa A. Moles, Richard B. Gaynor, Matthew J. Goldstein, Les H. Brail, Joel Greshock, Lakshmi Srinivasan. The personalized vaccine, NEO-PV-01 with anti-PD1, induces neoantigen-specific de novo immune responses in patients with advanced metastatic melanoma: Association with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 942.

Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient’s neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here we report comprehensive immune-related gene expression analysis of longitudinal tumor biopsies from patients with metastatic melanoma, bladder, and non-small cell lung cancer treated on our NT-001 trial with NEO-PV-01 + adjuvant in combination with nivolumab (NCT02897765) to correlate with clinical outcomes.Methods: Tumor biopsies from all three tumor types were collected i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Targeted gene expression analysis on RNA extracted from FFPE blocks was performed using the NanoString™ nCounter platform. A custom set of 800 genes included markers for immune cell populations, cytolytic markers, immune activation and suppression, and the tumor microenvironment. Gene signatures of key immune features were calculated after normalization with housekeeping genes and used for subsequent analysis.Results: Changes in the immune cell populations and the tumor microenvironment were detected after treatment with nivolumab and NEO-PV-01. Increases in various immune cell subsets, including T cells and B cells, as well as an increase in cytolytic phenotype were observed in tumor biopsies following treatment. Moreover, changes in the tumor microenvironment, consistent with the absence of tumor by histologic evaluation, were detected in many of the post-vaccination biopsies. In addition, these observations were consistent with data from peripheral blood that demonstrated durable de novo neoantigen-specific immune responses after vaccination. Additional exploratory analyses of the data demonstrate differential gene expression in patients’ tumors that align with tumor responses to therapy.Conclusion: Treatment with nivolumab and NEO-PV-01 leads to changes in the tumor microenvironment that are consistent with cell types and phenotypes that could contribute to an anti-tumor response.Citation Format: Meghan E. Bushway, Ying Sonia Ting, Rana H. Besada, Tracey E. Sciuto, Jasmina Prabhakara, Julian Scherer, Kristen N. Balogh, April Lamb, Jennifer A. Kaplan, Lisa D. Cleary, Melissa A. Moles, Sarah E. Church, Yuqi Ren, Xing Ren, Richard B. Gaynor, Matthew J. Goldstein, Les H. Brail, Joel Greshock, Lakshmi Srinivasan. Comprehensive gene expression analysis of the tumor microenvironment in patients with advanced cancer treated with a personalized neoantigen vaccine, NEO-PV-01, in combination with anti-PD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5006.

Abstract Purpose: The BRAFV600E oncogene modulates the papillary thyroid carcinoma (PTC) microenvironment, in which pericytes are critical regulators of tyrosine-kinase (TK)-dependent signaling pathways. Although BRAFV600E and TK inhibitors are available, their efficacy as bimodal therapeutic agents in BRAFV600E-PTC is still unknown. Experimental Design: We assessed the effects of vemurafenib (BRAFV600E inhibitor) and sorafenib (TKI) as single agents or in combination in BRAFWT/V600E-PTC and BRAFWT/WT cells using cell-autonomous, pericyte coculture, and an orthotopic mouse model. We also used BRAFWT/V600E-PTC and BRAFWT/WT-PTC clinical samples to identify differentially expressed genes fundamental to tumor microenvironment. Results: Combined therapy blocks tumor cell proliferation, increases cell death, and decreases motility via BRAFV600E inhibition in thyroid tumor cells in vitro. Vemurafenib produces cytostatic effects in orthotopic tumors, whereas combined therapy (likely reflecting sorafenib activity) generates biological fluctuations with tumor inhibition alternating with tumor growth. We demonstrate that pericytes secrete TSP-1 and TGFβ1, and induce the rebound of pERK1/2, pAKT and pSMAD3 levels to overcome the inhibitory effects of the targeted therapy in PTC cells. This leads to increased BRAFV600E-PTC cell survival and cell death refractoriness. We find that BRAFWT/V600E-PTC clinical samples are enriched in pericytes, and TSP1 and TGFβ1 expression evoke gene-regulatory networks and pathways (TGFβ signaling, metastasis, tumor growth, tumor microenvironment/ECM remodeling functions, inflammation, VEGF ligand–VEGF receptor interactions, immune modulation, etc.) in the microenvironment essential for BRAFWT/V600E-PTC cell survival. Critically, antagonism of the TSP-1/TGFβ1 axis reduces tumor cell growth and overcomes drug resistance. Conclusions: Pericytes shield BRAFV600E-PTC cells from targeted therapy via TSP-1 and TGFβ1, suggesting this axis as a new therapeutic target for overcoming resistance to BRAFV600E and TK inhibitors.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A(164)) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A(164). Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A(164) strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-L-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, Leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor ( VWF ), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.

Objective: Mechanisms for maintenance of endothelial integrity are of enormous biomedical importance. Despite its tenuous structure and constitutive exposure to disruptive strains, the endothelium normally exhibits extremely robust barrier properties. The objective of this study was to elucidate fundamental mechanisms by which the endothelium senses and responds to integrity disruptions in order to maintain its barrier function. Results: We show that in response to ~5-50 micron-scale disruptions in the endothelium, induced by transmigrating leukocytes or a mechanical probe, endothelial cells generate unique ‘ventral lamellipodia' that propagate via integrins toward and across these ‘micro-wounds' to close them and promote re-annealing of the adherens junctions. Experiments combining probe-induced micro-wounding, pharmacologic modulation of contractility & substrate stretching manipulations demonstrate that endothelia 'sense' breaches in its barrier as force imbalance and specifically loss of isometric tension upon rupture of adhesions. Such loss if tension was acutely/locally translated into biomechanical signals for reparative actin remodeling. Indeed, ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of H2O2. Together with Apr2/3, these were functionally required for effective micro-wound healing and maintenance of endothelial barrier function. Conclusion We propose that barrier disruptions are detected as local release of isometric tension, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

Among unicellular organisms, stochastic phenotype switching is a documented strategy for survival. These populations "hedge their bets": while the majority of their cells are adapted to their present environment, a minority remains poised to thrive under drastically different conditions. Bet hedging has also been described in metazoan cells, primarily in vitro. However, its role in tissue homeostasis has yet to be established. Here, we show that von Willebrand factor (vWF) is expressed in a spatially heterogeneous manner in a small fraction of capillary endothelial cells in the heart, skeletal muscle, lung and brain. Moreover, these mosaic patterns are dynamic, in that vWF expression stochastically toggles ON/OFF over time. By contrast, expression of vWF in the aorta and liver is static in time. In cultured primary endothelial cells, biological noise resulted in mosaic vWF heterogeneity through a promoter-level DNA methylation switch. Finally, vWF-/- mice demonstrated extensive endothelial cell damage in capillaries of the heart and impaired cardiac function, but not kidney or aorta. Taken together, these findings suggest that dynamic mosaicism of vWF expression is functionally relevant and that bet hedging represents a previously unrecognized strategy for adaptive, organ-specific homeostasis.