Abstract
The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 ...(ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG
1
F
C
fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.
There is no cure for HIV infection, and lifelong antiretroviral therapy (ART) is required. N-803 is an IL-15 superagonist comprised of an N72D mutant IL-15 molecule attached to its alpha receptor and ...a human IgG1 fragment designed to increase IL-15 activity. Preclinical studies with both HIV and SIV suggest that the drug has potential to reduce virus reservoirs by activating virus from latency and enhancing effector function. We conducted a phase 1 study of N-803 ( NCT02191098 ) in people living with HIV, the primary objective of which was to assess the safety and tolerability of the drug, with an exploratory objective of assessing the impact on peripheral virus reservoirs. ART-suppressed individuals were enrolled into a dose-escalation study of N-803 in four different cohorts (0.3, 1.0, 3.0 and 6.0 mcg kg
). Each cohort received three doses total, separated by at least 1 week. We enrolled 16 individuals, of whom 11 completed all three doses. The maximum tolerated dose was 6.0 mcg kg
. The primary clinical adverse events (AEs) reported were injection site rash and adenopathy, and four participants experienced a grade 1 or grade 2 QTc prolongation. No significant laboratory AEs attributable to N-803 were observed. In exploratory analyses, N-803 was associated with proliferation and/or activation of CD4
and CD8
T cells and natural killer cells that peaked at 4 d after dosing. IFN-γ, IP-10, MCP-1 and IL-15 increased during treatment. HIV transcription in memory CD4 T cells and intact proviral DNA initially increased after N-803 treatment; however, there was a small but significant decrease in the frequency of peripheral blood mononuclear cells with an inducible HIV provirus that persisted for up to 6 months after therapy. These data suggest that N-803 administration in ART-suppressed people living with HIV is safe and that larger clinical trials are needed to further investigate the effects of N-803 on HIV reservoirs.
Abstract
We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered ...using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity—including mucosal immunity—against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.
Failed T cell-based immunotherapies in the presence of genomic alterations in antigen presentations pathways may be overcome by NK cell-based immunotherapy. This approach may still be limited by the ...presence of immunosuppressive myeloid populations. Here, we demonstrate that NK cells (haNKs) engineered to express a PD-L1 chimeric antigen receptor (CAR) haNKs killed a panel of human and murine head and neck cancer cells at low effector-to-target ratios in a PD-L1-dependent fashion. Treatment of syngeneic tumors resulted in CD8 and PD-L1-dependent tumor rejection or growth inhibition and a reduction in myeloid cells endogenously expressing high levels of PD-L1. Treatment of xenograft tumors resulted in PD-L1-dependent tumor growth inhibition. PD-L1 CAR haNKs reduced levels of macrophages and other myeloid cells endogenously expressing high PD-L1 in peripheral blood from patients with head and neck cancer. The clinical study of PD-L1 CAR haNKs is warranted.
The biology of tumor-derived exosomes (TEX) is only partially understood and much remains to be studied in order to define the effect that the tumor microenvironment or the activation of tumor cells ...exerts on their composition and functions. Increased expression and activity of toll-like receptor 4 (TLR4) in chronic infectious and inflammatory conditions is related with cancer progression: its activation induces an inflammatory signaling that increases the tumorigenic potential of cancer cells promoting their immune evasion. We investigated the immune modulatory properties of TEX released upon cell TLR4 activation, and we found that, although differences were observed depending on the type of the tumor, the treatment influences TEX composition and boosts their immunosuppressive ability. Our results suggest that the activation of TLR4 supports tumor progression by stimulating the release of more effective immunosuppressive exosomes, which allow tumor cells to escape immune surveillance and probably even play a role in the metastatic process.
The identification of extensive genetic heterogeneity in human breast carcinomas poses a significant challenge for designing effective treatment regimens. Significant genomic evolution often occurs ...during breast cancer progression, creating variability within primary tumors as well as between the primary carcinoma and metastases. Current risk allocations and treatment recommendations for breast cancer patients are based largely on characteristics of the primary tumor; however, genetic differences between disseminated tumor cells and the primary carcinoma may negatively impact treatment efficacy and survival. In this review we (1) present current information about genomic variability within primary breast carcinomas, between primary tumors and regional/distant metastases, among circulating tumor cells (CTCs) and disseminated tumor cells (DTCs), and in cell-free nucleic acids in circulation, and (2) describe how this heterogeneity affects clinical care and outcomes such as recurrence and therapeutic resistance. Understanding the evolution and functional significance of the composite breast cancer genome within each patient is critical for developing effective therapies that can overcome obstacles presented by molecular heterogeneity.
Breast cancer intrinsic molecular subtype (IMS) as classified by the expression-based PAM50 assay is considered a strong prognostic feature, even when controlled for by standard clinicopathological ...features such as age, grade, and nodal status, yet the molecular testing required to elucidate these subtypes is not routinely performed. Furthermore, when such bulk assays as RNA sequencing are performed, intratumoral heterogeneity that may affect prognosis and therapeutic decision-making can be missed.
As a more facile and readily available method for determining IMS in breast cancer, we developed a deep learning approach for approximating PAM50 intrinsic subtyping using only whole-slide images of H&E-stained breast biopsy tissue sections. This algorithm was trained on images from 443 tumors that had previously undergone PAM50 subtyping to classify small patches of the images into four major molecular subtypes-Basal-like, HER2-enriched, Luminal A, and Luminal B-as well as Basal vs. non-Basal. The algorithm was subsequently used for subtype classification of a held-out set of 222 tumors.
This deep learning image-based classifier correctly subtyped the majority of samples in the held-out set of tumors. However, in many cases, significant heterogeneity was observed in assigned subtypes across patches from within a single whole-slide image. We performed further analysis of heterogeneity, focusing on contrasting Luminal A and Basal-like subtypes because classifications from our deep learning algorithm-similar to PAM50-are associated with significant differences in survival between these two subtypes. Patients with tumors classified as heterogeneous were found to have survival intermediate between Luminal A and Basal patients, as well as more varied levels of hormone receptor expression patterns.
Here, we present a method for minimizing manual work required to identify cancer-rich patches among all multiscale patches in H&E-stained WSIs that can be generalized to any indication. These results suggest that advanced deep machine learning methods that use only routinely collected whole-slide images can approximate RNA-seq-based molecular tests such as PAM50 and, importantly, may increase detection of heterogeneous tumors that may require more detailed subtype analysis.
Extensive genomic and transcriptomic heterogeneity in human cancer often negatively impacts treatment efficacy and survival, thus posing a significant ongoing challenge for modern treatment regimens. ...State-of-the-art DNA- and RNA-sequencing methods now provide high-resolution genomic and gene expression portraits of individual cells, facilitating the study of complex molecular heterogeneity in cancer. Important developments in single-cell sequencing (SCS) technologies over the past 5 years provide numerous advantages over traditional sequencing methods for understanding the complexity of carcinogenesis, but significant hurdles must be overcome before SCS can be clinically useful. In this review, we: (1) highlight current methodologies and recent technological advances for isolating single cells, single-cell whole-genome and whole-transcriptome amplification using minute amounts of nucleic acids, and SCS, (2) summarize research investigating molecular heterogeneity at the genomic and transcriptomic levels and how this heterogeneity affects clonal evolution and metastasis, and (3) discuss the promise for integrating SCS in the clinical care arena for improved patient care.
To provide a unique global view of the relative potential for evasion of CD8+ and CD4+ T cells by SARS-CoV-2 lineages as they evolve over time, we performed a comprehensive analysis of predicted ...HLA-I and HLA-II binding peptides in Spike (S) and Nucleocapsid (N) protein sequences of all available SARS-CoV-2 genomes as provided by NIH NCBI at a bi-monthly interval between March and December of 2021. A data supplement of all B.1.1.529 (Omicron) genomes from GISAID in early December was also used to capture the rapidly spreading variant. A key finding is that throughout continued viral evolution and increasing rates of mutations occurring at T-cell epitope hotspots, protein instances with worst-case binding loss did not become the most frequent for any Variant of Concern (VOC) or Variant of Interest (VOI) lineage; suggesting T-cell evasion is not likely to be a dominant evolutionary pressure on SARS-CoV-2. We also determined that throughout the course of the pandemic in 2021, there remained a relatively steady ratio of viral variants that exhibit conservation of epitopes in the N protein, despite significant potential for epitope loss in S relative to other lineages. We further localized conserved regions in N with high epitope yield potential, and illustrated heterogeneity in HLA-I binding across the S protein consistent with empirical observations. Although Omicron’s high volume of mutations caused it to exhibit more epitope loss potential than most frequently observed versions of proteins in almost all other VOCs, epitope candidates across its most frequent N proteins were still largely conserved. This analysis adds to the body of evidence suggesting that N may have merit as an additional antigen to elicit immune responses to vaccination with increased potential to provide sustained protection against COVID-19 disease in the face of emerging variants.
ABI-007, an albumin-bound, 130-nm particle form of paclitaxel, was developed to avoid Cremophor/ethanol-associated toxicities in Cremophor-based paclitaxel (Taxol) and to exploit albumin ...receptor-mediated endothelial transport. We studied the antitumor activity, intratumoral paclitaxel accumulation, and endothelial transport for ABI-007 and Cremophor-based paclitaxel. Antitumor activity and mortality were assessed in nude mice bearing human tumor xenografts lung (H522), breast (MX-1), ovarian (SK-OV-3), prostate (PC-3), and colon (HT29) treated with ABI-007 or Cremophor-based paclitaxel. Intratumoral paclitaxel concentrations (MX-1-tumored mice) were compared for radiolabeled ABI-007 and Cremophor-based paclitaxel. In vitro endothelial transcytosis and Cremophor inhibition of paclitaxel binding to cells and albumin was compared for ABI-007 and Cremophor-based paclitaxel. Both ABI-007 and Cremophor-based paclitaxel caused tumor regression and prolonged survival; the order of sensitivity was lung > breast congruent with ovary > prostate > colon. The LD(50) and maximum tolerated dose for ABI-007 and Cremophor-based paclitaxel were 47 and 30 mg/kg/d and 30 and 13.4 mg/kg/d, respectively. At equitoxic dose, the ABI-007-treated groups showed more complete regressions, longer time to recurrence, longer doubling time, and prolonged survival. At equal dose, tumor paclitaxel area under the curve was 33% higher for ABI-007 versus Cremophor-based paclitaxel, indicating more effective intratumoral accumulation of ABI-007. Endothelial binding and transcytosis of paclitaxel were markedly higher for ABI-007 versus Cremophor-based paclitaxel, and this difference was abrogated by a known inhibitor of endothelial gp60 receptor/caveolar transport. In addition, Cremophor was found to inhibit binding of paclitaxel to endothelial cells and albumin. Enhanced endothelial cell binding and transcytosis for ABI-007 and inhibition by Cremophor in Cremophor-based paclitaxel may account in part for the greater efficacy and intratumor delivery of ABI-007.