Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy.

Neoadjuvant immune checkpoint blockade has shown promising clinical activity. Here, we characterized early kinetics in tumor-infiltrating and circulating immune cells in oral cancer patients treated with neoadjuvant anti-PD-1 or anti-PD-1/CTLA-4 in a clinical trial (NCT02919683). Tumor-infiltrating CD8 T cells that clonally expanded during immunotherapy expressed elevated tissue-resident memory and cytotoxicity programs, which were already active prior to therapy, supporting the capacity for rapid response. Systematic target discovery revealed that treatment-expanded tumor T cell clones in responding patients recognized several self-antigens, including the cancer-specific antigen MAGEA1. Treatment also induced a systemic immune response characterized by expansion of activated T cells enriched for tumor-infiltrating T cell clonotypes, including both pre-existing and emergent clonotypes undetectable prior to therapy. The frequency of activated blood CD8 T cells, notably pre-treatment PD-1-positive KLRG1-negative T cells, was strongly associated with intra-tumoral pathological response. These results demonstrate how neoadjuvant checkpoint blockade induces local and systemic tumor immunity.

Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein.

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis.

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. We find that the Wnt receptor Frizzled2 (Fzd2) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3. A gene signature regulated by this pathway predicts metastasis and overall survival in patients. We have developed an antibody to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b.

Sequential application of anticancer drugs enhances cell death by rewiring apoptotic signaling networks.

Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis-using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling-revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.

MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM.

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.

Phase 1 dose escalation study of seribantumab (MM-121), an anti-HER3 monoclonal antibody, in patients with advanced solid tumors.

Background Overactivation of human epidermal growth factor receptor 3 (HER3) triggers multiple intracellular pathways resulting in tumor cell survival. This Phase 1 study assessed the safety, efficacy, and pharmacokinetics (PK) of seribantumab, a fully human anti-HER3 monoclonal antibody. Methods Adult patients with advanced or refractory solid tumors were treated in six dose cohorts of seribantumab: 3.2, 6, 10, 15, or 20 mg/kg weekly, or 40 mg/kg loading dose followed by 20 mg/kg weekly maintenance dose (40/20 mg/kg) using a modified 3 + 3 dose escalation strategy with cohort expansion. Primary objectives were identification of a recommended Phase 2 dose (RP2D) and determination of objective response rate. Secondary objectives were assessment of safety, dose-limiting toxicities, and PK. Results Forty-four patients (26 dose escalation; 18 dose expansion) were enrolled. Seribantumab monotherapy was well tolerated with most adverse events being transient and mild to moderate (grade 1 or 2) in severity; maximum tolerated dose was not reached. The highest dose, 40/20 mg/kg, was identified as RP2D. Best response was stable disease, reported in 24% and 39% of patients during the dose escalation and expansion portions of the study, respectively. Seribantumab terminal half-life was ≈100 h; steady state concentrations were reached after 3-4 weekly doses. Conclusions Seribantumab monotherapy was well tolerated across all dose levels. Safety and PK data from this study support further seribantumab investigations in genomically defined populations.Clinical trial registration NCT00734305. August 12, 2008.

A phase 1 study combining the HER3 antibody seribantumab (MM-121) and cetuximab with and without irinotecan.

Background HER3/EGFR heterodimers have been implicated as a mode of resistance to EGFR-directed therapies. Methods This Phase 1 trial assessed the tolerability, maximum tolerated dose (MTD) and pharmacokinetic (PK) properties of the HER-3 antibody seribantumab in combination with cetuximab (Part I) or cetuximab and irinotecan (Part II) in patients with EGFR-dependent cancers. In Part I, escalating doses of seribantumab and cetuximab were administered. In Part II of the trial, escalating doses of seribantumab/cetuximab were combined with irinotecan 180 mg/m2 administered every two weeks. Results 34 patients were enrolled in Part I (seribantumab/cetuximab) and 14 patients were enrolled in Part II (seribantumab/cetuximab/irinotecan). Common toxicities of seribantumab/cetuximab included acneiform rash, diarrhea, stomatitis, and paronychia. The MTD of Part I was seribantumab 40 mg/kg bolus, then 20 mg/kg weekly combined with cetuximab 400 mg/m2 bolus, then 250 mg/m2 IV weekly. Common toxicities reported in the seribantumab/cetuximab/irinotecan combination were similar to the Part I portion. However, toxicities were more frequent and severe with the triplet combination. There was one treatment-related death in Part II secondary to Grade 4 neutropenia and grade 3 diarrhea. Other dose-limiting toxicities in Part II were Grade 3 mucositis and Grade 3 diarrhea. A cholangiocarcinoma patient, previously untreated with EGFR-directed therapy, had a confirmed partial response (PR). One colorectal cancer patient, previously treated with EGFR-directed therapy, had an unconfirmed PR. Conclusions Seribantumab/cetuximab was well tolerated and patients experienced toxicities typical to EGFR inhibition. Unlike the seribantumab/cetuximab doublet, seribantumab/cetuximab/irinotecan was difficult to tolerate in this heavily pretreated population. There was limited efficacy of the combination therapy.

Systematic analysis of bacterial effector-postsynaptic density 95/disc large/zonula occludens-1 (PDZ) domain interactions demonstrates Shigella OspE protein promotes protein kinase C activation via PDLIM proteins.

Diseases caused by many Gram-negative bacterial pathogens depend on the activities of bacterial effector proteins that are delivered into eukaryotic cells via specialized secretion systems. Effector protein function largely depends on specific subcellular targeting and specific interactions with cellular ligands. PDZ domains are common domains that serve to provide specificity in protein-protein interactions in eukaryotic systems. We show that putative PDZ-binding motifs are significantly enriched among effector proteins delivered into mammalian cells by certain bacterial pathogens. We use PDZ domain microarrays to identify candidate interaction partners of the Shigella flexneri effector proteins OspE1 and OspE2, which contain putative PDZ-binding motifs. We demonstrate in vitro and in cells that OspE proteins interact with PDLIM7, a member of the PDLIM family of proteins, which contain a PDZ domain and one or more LIM domains, protein interaction domains that participate in a wide variety of functions, including activation of isoforms of protein kinase C (PKC). We demonstrate that activation of PKC during S. flexneri infection is attenuated in the absence of PDLIM7 or OspE proteins and that the OspE PDZ-binding motif is required for wild-type levels of PKC activation. These results are consistent with a model in which binding of OspE to PDLIM7 during infection regulates the activity of PKC isoforms that bind to the PDLIM7 LIM domain.

Heregulin and HER3 are prognostic biomarkers in oropharyngeal squamous cell carcinoma.

BACKGROUND: Although heregulin and human epidermal growth factor receptor 3 (HER3) are frequently expressed at high levels in patients with head and neck cancer, their prognostic value remains unclear. The authors explored the prognostic significance of heregulin/HER3 expression in patients with oropharyngeal squamous cell carcinoma (OPSCC), taking into account other HER family members as well as p16 status. METHODS: Ninety-six primary tumor specimens from patients with OPSCC were retrospectively collected and analyzed for heregulin messenger RNA (mRNA) using in situ hybridization and for HER3, epidermal growth factor receptor, and human epidermal growth factor receptor 2 (HER2) using quantitative immunohistochemistry. Heregulin and HER3 mRNA levels were also examined among different tumor types using The Cancer Genome Atlas database. RESULTS: High heregulin mRNA (> the median) correlated significantly with poor overall survival (OS) (hazard ratio [HR], 8.48; 95% confidence interval [95% CI], 2.17-33.17 [P =.002]) but not disease-free survival (HR, 1.52; 95% CI, 0.64-3.65 [P =.341]) in patients with OPSCC. Heregulin mRNA correlated negatively with OS in both patients with p16-positive (P =.049) and p16-negative (P =.091) OPSCC on univariate analysis. High HER3 (> the median) also correlated with poor OS (HR, 4.68; 95% CI, 1.47-14.90 [P =.009]) on multivariate analysis. Epidermal growth factor receptor levels independently correlated with disease-free survival (P =.025) and inversely correlated with p16 status (P =.012). In addition, The Cancer Genome Atlas data demonstrated that head and neck squamous cell carcinoma exhibits higher heregulin expression compared with other solid tumor types examined. CONCLUSIONS: High heregulin mRNA and high HER3 protein levels were found to independently correlate with poor OS in patients with OPSCC. These data support targeting HER3 in patients with heregulin-high OPSCC and warrant further clinical investigation.

Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected.

Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.

Cross-talk between receptor tyrosine kinase and tumor necrosis factor-α signaling networks regulates apoptosis but not proliferation.

Although many of the signaling networks activated by receptor tyrosine kinases (RTKs) and cytokine receptors are well understood, how these networks interconnect is much less clear. We set out to determine how cells respond to simultaneous exposure to opposing signals and how their downstream networks process this information. Using six isogenic cell lines, each stably transfected with a different RTK, we found that, in each case, the cognate growth factor induced proliferation, whereas TNFα induced apoptosis. Surprisingly, when the cells were treated simultaneously with growth factor and TNFα, the growth factor enhanced, rather than antagonized, TNFα-induced cell death. In contrast, TNFα had no effect on growth factor-induced proliferation, suggesting that cross-talk between these networks is unidirectional. A quantitative, system-wide study of signaling at early and late time points corroborated this observation: proteins in the RTK networks were not affected by TNFα treatment, but proteins in the TNFα network were affected by growth factors. These studies also highlighted the stress mitogen-activated protein kinase proteins p38 and c-Jun N-terminal kinase as the key nodes of signal integration, and their activation states at an early time point correlated well with subsequent measurements of apoptosis. Knocking down cRaf reduced the growth factor enhancement of TNFα-induced apoptosis, highlighting its role as a regulator of network cross-talk upstream of p38 and c-Jun N-terminal kinase. Overall, we found that when cells encounter conflicting stimuli, their phenotypic response is determined not by the sum of isolated processes, but by how their signaling networks interconnect. This underscores the need to build mechanistic models of network integration as a first step in predicting cellular behavior in complex settings and in rationally designing combination therapies.

Tyrosine-phosphorylated caveolin-1 blocks bacterial uptake by inducing Vav2-RhoA-mediated cytoskeletal rearrangements.

Certain bacterial adhesins appear to promote a pathogen's extracellular lifestyle rather than its entry into host cells. However, little is known about the stimuli elicited upon such pathogen host-cell interactions. Here, we report that type IV pili (Tfp)-producing Neisseria gonorrhoeae (P(+)GC) induces an immediate recruitment of caveolin-1 (Cav1) in the host cell, which subsequently prevents bacterial internalization by triggering cytoskeletal rearrangements via downstream phosphotyrosine signaling. A broad and unbiased analysis of potential interaction partners for tyrosine-phosphorylated Cav1 revealed a direct interaction with the Rho-family guanine nucleotide exchange factor Vav2. Both Vav2 and its substrate, the small GTPase RhoA, were found to play a direct role in the Cav1-mediated prevention of bacterial uptake. Our findings, which have been extended to enteropathogenic Escherichia coli, highlight how Tfp-producing bacteria avoid host cell uptake. Further, our data establish a mechanistic link between Cav1 phosphorylation and pathogen-induced cytoskeleton reorganization and advance our understanding of caveolin function.

Lysate microarrays enable high-throughput, quantitative investigations of cellular signaling.

Lysate microarrays (reverse-phase protein arrays) hold great promise as a tool for systems-level investigations of signaling and multiplexed analyses of disease biomarkers. To date, however, widespread use of this technology has been limited by questions concerning data quality and the specificity of detection reagents. To address these concerns, we developed a strategy to identify high-quality reagents for use with lysate microarrays. In total, we tested 383 antibodies for their ability to quantify changes in protein abundance or modification in 20 biological contexts across 17 cell lines. Antibodies yielding significant differences in signal were further evaluated by immunoblotting and 82 passed our rigorous criteria. The large-scale data set from our screen revealed that cell fate decisions are encoded not just by the identities of proteins that are activated, but by differences in their signaling dynamics as well. Overall, our list of validated antibodies and associated protocols establish lysate microarrays as a robust tool for systems biology.

Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells.

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell's membrane and subsequently release surface-bound molecules directly into the cell's cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology's potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

Protein microarrays and proteomics.

The system-wide study of proteins presents an exciting challenge in this information-rich age of whole-genome biology. Although traditional investigations have yielded abundant information about individual proteins, they have been less successful at providing us with an integrated understanding of biological systems. The promise of proteomics is that, by studying many components simultaneously, we will learn how proteins interact with each other, as well as with non-proteinaceous molecules, to control complex processes in cells, tissues and even whole organisms. Here, I discuss the role of microarray technology in this burgeoning area.

A quantitative protein interaction network for the ErbB receptors using protein microarrays.

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.

Linear combinations of docking affinities explain quantitative differences in RTK signaling.

Receptor tyrosine kinases (RTKs) process extracellular cues by activating a broad array of signaling proteins. Paradoxically, they often use the same proteins to elicit diverse and even opposing phenotypic responses. Binary, 'on-off' wiring diagrams are therefore inadequate to explain their differences. Here, we show that when six diverse RTKs are placed in the same cellular background, they activate many of the same proteins, but to different quantitative degrees. Additionally, we find that the relative phosphorylation levels of upstream signaling proteins can be accurately predicted using linear models that rely on combinations of receptor-docking affinities and that the docking sites for phosphoinositide 3-kinase (PI3K) and Shc1 provide much of the predictive information. In contrast, we find that the phosphorylation levels of downstream proteins cannot be predicted using linear models. Taken together, these results show that information processing by RTKs can be segmented into discrete upstream and downstream steps, suggesting that the challenging task of constructing mathematical models of RTK signaling can be parsed into separate and more manageable layers.

System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.

The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.