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The immune phenotype of a tumour is a key predictor of its response to immunotherapy1–4. Patients who respond to checkpoint blockade generally present with immune-inflamed5–7 tumours that are highly infiltrated by T cells. However, not all inflamed tumours respond to therapy, and even lower response rates occur among tumours that lack T cells (immune desert) or that spatially exclude T cells to the periphery of the tumour lesion (immune excluded)8. Despite the importance of these tumour immune phenotypes in patients, little is known about their development, heterogeneity or dynamics owing to the technical difficulty of tracking these features in situ. Here we introduce skin tumour array by microporation (STAMP)—a preclinical approach that combines high-throughput time-lapse imaging with next-generation sequencing of tumour arrays. Using STAMP, we followed the development of thousands of arrayed tumours in vivo to show that tumour immune phenotypes and outcomes vary between adjacent tumours and are controlled by local factors within the tumour microenvironment. Particularly, the recruitment of T cells by fibroblasts and monocytes into the tumour core was supportive of T cell cytotoxic activity and tumour rejection. Tumour immune phenotypes were dynamic over time and an early conversion to an immune-inflamed phenotype was predictive of spontaneous or therapy-induced tumour rejection. Thus, STAMP captures the dynamic relationships of the spatial, cellular and molecular components of tumour rejection and has the potential to translate therapeutic concepts into successful clinical strategies.

Supplementary Figure 5: Control spectrum obtained via quantitative LC-MS for the anti-HER2-VC(S)-MMAE ADC after 3 hours with in buffer with no proteolytic solution.

PDF file - 233K, Fig S4A. Validation and application of a compacted 22-gene VDV subset in human clinical samples. Fig S4B and C. Stratification of NO16966 patients by VEGF and CD31 expression levels.

PDF file - 154K, Fig S5. Most proxVDV genes are not obviously up-regulated by rVEGF in vitro.

Figure S5,6. Effects of Nrf2 knockdown in different growth conditions

S1. Sequences of RNAi reagents S2. Data from siRNA screens S3. Genes decreased following Nrf2 KD and increased in KEAP1 mutant lung cancer cells S4. Data from CRISPR screens

Supplementary Methods

Supplementary methods and figure legends

PDF file - 83K, Fig S6. Effect of VDV gene expression and bevacizumab treatment in OS.

PDF file - 113K, Fig S1. Kinetics of anti-VEGF effects on proliferation (ki67) and tumor burden in the RIP-TβAg model.

Antibody–drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027–37. ©2017 AACR.

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients.Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy.Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies.Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

PDF file - 63K, Fig S3. Enrichment of VDV markers Tumor-Associated Endothelial Cells.

PDF file - 180K, Table S1. Genes in the VDV signature, ordered by anti-VEGF response in human IBC trial. Table S2. Gene Ontology terms over-represented in VDV signature. Table S3. Summary of tumors models tested.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non–small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors.Significance:This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non–small cell lung cancer.

PDF file - 708K, Fig S2A. Histological evidence for the in vivo activity of VEGF pathway inhibitors in MDA-MB-231 tumors. Fig S2B. In vivo VEGF blockade or VEGFR-2 downstream signaling inhibition induces consistent downregulation of proxVDV genes. Fig S2C. The proxVDV gene ESM1 is an in vivo VEGF target specifically expressed in tumor-associated vasculature

Figure S1. Consequences of KEAP1 mutations on Nrf2 expression and ubiquitination Figure S2-4. Inducible knockdown of Nrf2 in KEAP1 mutant and WT cells and tumors

Figures S9-11. Effects of knocking down genes involved in oxidative stress

PDF file - 95K

PDF file - 6626K, Validation of IHC antibody specificity for LDHB, LDHA, MCT1, and MCT4

PDF file - 963K, Additional top targets from RNAi screen that are specifically required for triple negative breast cancer

XLSX file - 25K, RNAi optimization and screening conditions

XLSX file - 59K, Genes that comprise the glycolysis and oxidative phosphorylation gene signatures

PDF file - 663K, Glycolysis signature genes that correlate to LDHB expression in basal-like tumors

PDF file - 257K, Animal weight change and tumor volume of individual xenograft tumors

PDF file - 791K, mRNA expression profiles of lactate dehydrogenase enzymes in breast cancer subtypes

XLSX file - 31K, Results from the RNAi screen and identification of hits

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1β, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.

SummaryStrategies for maximizing the potency and specificity of cancer immunotherapies have sparked efforts to identify recurrent epitopes presented in the context of defined tumor-associated neoantigens. Discovering these “neoepitopes” can be difficult owing to the limited number of peptides that arise from a single point mutation, a low number of copies presented on the cell surface, and variable binding specificity of the human leukocyte antigen (HLA) class I complex. Due to these limitations, many discovery efforts focus on identifying neoepitopes from a small number of cancer neoantigens in the context of few HLA alleles. Here we describe a systematic workflow to characterize binding and presentation of neoepitopes derived from 47 shared cancer neoantigens in the context of 15 HLA alleles. Through the development of a high-throughput neoepitope-HLA binding assay, we surveyed 24,149 candidate neoepitope-HLA combinations resulting in 587 stable complexes. These data were supplemented by computational prediction that identified an additional 257 neoepitope-HLA pairs, resulting in a total of 844 unique combinations. We used these results to build sensitive targeted mass spectrometry assays to validate neoepitope presentation on a panel of HLA-I monoallelic cell lines engineered to express neoantigens of interest as a single polypeptide. Altogether, our analyses detected 84 unique neoepitope-HLA pairs derived from 37 shared cancer neoantigens and presented across 12 HLA alleles. We subsequently identified multiple TCRs which specifically recognized two of these neoantigen-HLA combinations. Finally, these novel TCRs were utilized to elicit a T cell response suggesting that these neoepitopes are likely to be immunogenic. Together these data represent a validated, extensive resource of therapeutically relevant neoepitopes and the HLA context in which they can be targeted.

Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Purification of human CD4

CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell-lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with a new, optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a novel, synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts.