Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands.

Introduction: The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. Methods: By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. Results: We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Discussion: Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.

Clinical feasibility and treatment outcomes with nonselected autologous tumor-infiltrating lymphocyte therapy in patients with advanced cutaneous melanoma.

Nonselected autologous tumor-infiltrating lymphocytes (TILs) may provide advantages over other treatments for solid tumors, including checkpoint inhibitor-refractory melanoma. This retrospective analysis reports a single-center experience of nonselected autologous TILs derived from digested tumors for compassionate use treatment of advanced cutaneous melanoma, including after programmed cell death protein 1 (PD-1) inhibition. Patients with histologically confirmed metastatic cutaneous melanoma and no standard-of-care treatment options underwent tumor resection for TIL product manufacturing. Patients received lymphodepleting chemotherapy with cyclophosphamide for 2 days and fludarabine for 5 days, followed by a single TIL infusion and post-TIL high-dose interleukin (IL)-2. Safety assessments included clinically significant adverse events (AEs). Efficacy assessments included overall response rate (ORR), complete response (CR) rate, disease control rate (DCR), and overall survival. Between October 2011 and August 2019, 21 patients underwent treatment (median follow-up time, 52.2 months from TIL infusion). Among all treated patients, median age was 45 years, median number of disease sites was 4, 100% had M1c or M1d disease, and 90% received prior checkpoint inhibitor. Twelve patients received TILs after prior PD-1 inhibition. The safety profile among all treated patients and the prior PD-1 inhibitor subgroup was generally consistent with lymphodepletion and high-dose IL-2. No treatment-related deaths occurred. Among all patients, the ORR was 67%, CR rate was 19%, and the DCR was 86%, which was consistent with that observed in the prior PD-1 inhibitor subgroup (58%, 8%, and 75%, respectively). Median overall survival in all treated patients and the prior PD-1 inhibitor subgroup was 21.3 months. In total, 5 patients (24%) had durable ongoing responses (>30 months post-TIL infusion) at data cutoff, and all patients who achieved CR remained alive and disease free. To further illustrate how TIL therapy may integrate into established treatment paradigms, several case studies of patients treated in this series were included. Overall, these data demonstrate that manufacturing of nonselected autologous TILs from tumor digests is feasible and resulted in high rates of durable response in poor-risk patient populations, which may address significant unmet medical need.

A Platform To Enhance Quantitative Single Molecule Localization Microscopy.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Dynamic lateral organization of opioid receptors (kappa, muwt and muN40D ) in the plasma membrane at the nanoscale level.

Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt ), and its naturally occurring isoform (MOPN40D ) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble-averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside-enriched domains and partial association with cholesterol-enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor-specific. KOP-containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D . Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt , whereas this effect was not observed for MOPN40D .

A comparison of the effect of molluscum contagiosum virus MC159 and MC160 proteins on vaccinia virus virulence in intranasal and intradermal infection routes.

Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Molluscum Contagiosum Virus MC159 Abrogates cIAP1-NEMO Interactions and Inhibits NEMO Polyubiquitination.

Molluscum contagiosum virus (MCV) is a dermatotropic poxvirus that causes benign skin lesions. MCV lesions persist because of virally encoded immune evasion molecules that inhibit antiviral responses. The MCV MC159 protein suppresses NF-κB activation, a powerful antiviral response, via interactions with the NF-κB essential modulator (NEMO) subunit of the IκB kinase (IKK) complex. Binding of MC159 to NEMO does not disrupt the IKK complex, implying that MC159 prevents IKK activation via an as-yet-unidentified strategy. Here, we demonstrated that MC159 inhibited NEMO polyubiquitination, a posttranslational modification required for IKK and downstream NF-κB activation. Because MCV cannot be propagated in cell culture, MC159 was expressed independent of infection or during a surrogate vaccinia virus infection to identify how MC159 prevented polyubiquitination. Cellular inhibitor of apoptosis protein 1 (cIAP1) is a cellular E3 ligase that ubiquitinates NEMO. Mutational analyses revealed that MC159 and cIAP1 each bind to the same NEMO region, suggesting that MC159 may competitively inhibit cIAP1-NEMO interactions. Indeed, MC159 prevented cIAP1-NEMO interactions. MC159 also diminished cIAP1-mediated NEMO polyubiquitination and cIAP1-induced NF-κB activation. These data suggest that MC159 competitively binds to NEMO to prevent cIAP1-induced NEMO polyubiquitination. To our knowledge, this is the first report of a viral protein disrupting NEMO-cIAP1 interactions to strategically suppress IKK activation. All viruses must antagonize antiviral signaling events for survival. We hypothesize that MC159 inhibits NEMO polyubiquitination as a clever strategy to manipulate the host cell environment to the benefit of the virus.IMPORTANCE Molluscum contagiosum virus (MCV) is a human-specific poxvirus that causes persistent skin neoplasms. The persistence of MCV has been attributed to viral downregulation of host cell immune responses such as NF-κB activation. We show here that the MCV MC159 protein interacts with the NEMO subunit of the IKK complex to prevent NEMO interactions with the cIAP1 E3 ubiquitin ligase. This interaction correlates with a dampening of cIAP1 to polyubiquitinate NEMO and to activate NF-κB. This inhibition of cIAP1-NEMO interactions is a new viral strategy to minimize IKK activation and to control NEMO polyubiquitination. This research provides new insights into mechanisms that persistent viruses may use to cause long-term infection of host cells.

Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+ T Cells.

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus.

Apoptosis, NF-κB activation, and IRF3 activation are a triad of intrinsic immune responses that play crucial roles in the pathogenesis of infectious diseases, cancer, and autoimmunity. FLIPs are a family of viral and cellular proteins initially found to inhibit apoptosis and more recently to either up- or down-regulate NF-κB. As such, a broad role for FLIPs in disease regulation is postulated, but exactly how a FLIP performs such multifunctional roles remains to be established. Here we examine FLIPs (MC159 and MC160) encoded by the molluscum contagiosum virus, a dermatotropic poxvirus causing skin infections common in children and immunocompromised individuals, to better understand their roles in viral pathogenesis. While studying their molecular mechanisms responsible for NF-κB inhibition, we discovered that each protein inhibited IRF3-controlled luciferase activity, identifying a unique function for FLIPs. MC159 and MC160 each inhibited TBK1 phosphorylation, confirming this unique function. Surprisingly, MC159 coimmunoprecipitated with TBK1 and IKKε but MC160 did not, suggesting that these homologs use distinct molecular mechanisms to inhibit IRF3 activation. Equally surprising was the finding that the FLIP regions necessary for TBK1 inhibition were distinct from those MC159 or MC160 regions previously defined to inhibit NF-κB or apoptosis. These data reveal previously unappreciated complexities of FLIPs, and that subtle differences within the conserved regions of FLIPs possess distinct molecular and structural fingerprints that define crucial differences in biological activities. A future comparison of mechanistic differences between viral FLIP proteins can provide new means of precisely manipulating distinct aspects of intrinsic immune responses.