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.
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.
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.
Generating mammalian cells with specific mitochondrial DNA (mtDNA)-nuclear DNA (nDNA) combinations is desirable but difficult to achieve and would be enabling for studies of mitochondrial-nuclear ...communication and coordination in controlling cell fates and functions. We developed 'MitoPunch', a pressure-driven mitochondrial transfer device, to deliver isolated mitochondria into numerous target mammalian cells simultaneousl
. MitoPunch and MitoCeption, a previously described force-based mitochondrial transfer approach, both yield stable isolated mitochondrial recipient (SIMR) cells that permanently retain exogenous mtDNA, whereas coincubation of mitochondria with cells does not yield SIMR cells. Although a typical MitoPunch or MitoCeption delivery results in dozens of immortalized SIMR clones with restored oxidative phosphorylation, only MitoPunch can produce replication-limited, non-immortal human SIMR clones. The MitoPunch device is versatile, inexpensive to assemble, and easy to use for engineering mtDNA-nDNA combinations to enable fundamental studies and potential translational applications.
We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia ...factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.
Poly (ADP-ribose) polymerase inhibitors (PARPi) prevent single-stranded DNA repair. Olaparib is a PARPi approved for the treatment of BRCA mutant ovarian and breast carcinoma. Emerging clinical data ...suggest a benefit of combining olaparib with immunotherapy in prostate cancer patients both with and without somatic BRCA mutations.
We examined if olaparib, when combined with IgG
antibody-dependent cellular cytotoxicity (ADCC)-mediating monoclonal antibodies (mAbs) cetuximab (anti-EGFR), or avelumab (anti-PD-L1), would increase tumor cell sensitivity to killing by natural killer (NK) cells independently of BRCA status or mAb target upregulation. BRCA mutant and BRCA wildtype (WT) prostate carcinoma cell lines were pretreated with olaparib and then exposed to NK cells in the presence or absence of cetuximab or avelumab.
NK-mediated killing was significantly increased in both cell lines and was further increased using the ADCC-mediating mAbs. Pre-exposure of NK cells to recombinant IL-15/IL-15Rα further increased the lysis of olaparib treated tumor cells. In addition, olaparib treated tumor cells were killed to a significantly greater degree by engineered high-affinity NK cells (haNK). We show here for the first time that (a) olaparib significantly increased tumor cell sensitivity to NK killing and ADCC in both BRCA WT and BRCA mutant prostate carcinoma cells, independent of PD-L1 or EGFR modulation; (b) mechanistically, treatment with olaparib upregulated death receptor TRAIL-R2; and (c) olaparib significantly enhanced NK killing of additional tumor types, including breast, non-small cell lung carcinoma, and chordoma.
These studies support the combined use of NK- and ADCC-mediating agents with correctly timed PARP inhibition.
The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the ability to escape existing humoral protection conferred by previous infection and/or ...immunization necessitates the discovery of broadly reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identify a set of antibodies against SARS-CoV-2 spike (S) proteins and characterize the structures of nAbs that recognize epitopes in the S1 subunit of the S glycoprotein. These structural studies reveal distinct binding modes for several antibodies, including the targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interact with angiotensin-converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. Further, we engineer a potent ACE2-blocking nAb to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is an approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.
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•Broadly reactive neutralizing antibodies (nAbs) may overcome SARS-CoV-2 variant escape•mRNA display is used to rapidly identify SARS-CoV-2 spike (S)-protein-directed nAbs•Structural studies reveal distinct binding modes for several identified antibodies•An engineered nAb sustains binding to variant Gamma E484K and Delta L452R spikes
Tanaka et al. identify a set of SARS-CoV-2 spike (S)-targeted potentially neutralizing antibodies (nAbs) by mRNA display. Structural analyses reveal distinct binding modes, including the targeting of rare cryptic S receptor-binding domain epitopes. A further engineered ACE2-blocking nAb shows sustained binding to S RBD with the E484K and L452R substitutions.
BackgroundCombination immune therapy has demonstrated curative potential in multi-agent pre-clinical studies and early clinical studies. We have created a single-agent combination immune therapy ...platform, dubbed virus-like DNA (VLD), to activate multiple anti-cancer immune signals leveraging the robust T cell responses elicited by viral infection. VLD was designed to avoid neutralizing antibody responses, immunotherapy-mediated toxicities, and extensive manufacturing cost and time. VLD comprises of a) antigen, composed of a polytope selected based on genomic variants to activate CD4+ and CD8+ T cells, b) a proprietary protein that activates three independent T helper 1 and cytotoxic T cell signaling pathways, and c) a proprietary dual CTLA-4/PD-1 engaging agent. We hypothesized that a rationally-designed combination of different classes of immune modulating factors will elicit tumor antigen-specific effector T cell responses and eradicate established tumors.MethodsMice were implanted with tumor cells prior to treatment with VLD. VLD expressing immunomodulators and predicted T cell epitopes was administered intravenously to tumor-bearing mice. Tumor measurements and animal health were monitored for the ensuing 2 months, upon which, surviving animals were rechallenged with tumor cells.ResultsIntravenous administration of VLD to animals with established tumors completely eradicated or significantly delayed tumors in recipient animals. Combining VLD with anti-PD-L1 far outperformed anti-PD-L1 alone which was not significantly better than untreated controls. Furthermore, rechallenge of tumor-free animals with tumor cells demonstrated complete protection 60 days after initial challenge, suggesting VLD-mediated formation of immunological memory. VLD’s combination of immunomodulators uniquely activated splenic effector memory and antigen-reactive CD8+ T cells.ConclusionsThese data suggest that VLD provides a scalable, modular, rapid, robust, and cost-effective means of inducing a durable anti-tumor host response by harnessing critical components of vaccines and gene therapies into one immunogene product to eradicate cancer by activating antigen-reactive T cells.
BackgroundHepatitis B virus (HBV) infection is associated with approximately 25% of liver cancer cases in the US and 56% worldwide. Low rates of HBV vaccination in people 50 years or older combined ...with the fact that HBV infection is highest amongst the 40–49 year old demographic (i.e. individuals born prior to 1986) is a cause for concern as the median age for liver cancer diagnosis is 66 years of age. Despite the impact of immunotherapies in liver cancer over the past decade, durable responses remain elusive. Our approach to significantly improving outcomes is to activate and mobilize tissue-resident and systemic cancer-specific T effector memory cells (TEM), especially for the treatment of cancer in organs with low T cell count such as the liver.MethodsWe synthesized a novel capsid-modified adenovirus, SynBAd, for use as a therapeutic cancer vaccine targeting systemic immunity without causing commonly-reported adenovirus-mediated hepatotoxicity. We further engineered SynBAd to express a polytope of HBV antigens to activate the elimination of HBV+ tumor cells. SynBAd, administered intravenously (IV) for systemic and liver-specific T cell activation, was evaluated in a tumor-protection mouse model of liver cancer.ResultsIV administration of SynBAd expressing a polytope comprised of HBV epitopes significantly extended the lives of mice with a liver-localized tumor bearing the same epitopes in contrast to control animals. Moreover, recipients of SynBAd expressing an irrelevant antigen did not demonstrate comparable levels of protection. SynBAd further induced antigen specific CD8 T cell immunity in mice and a population of effector memory T cells at levels equivalent with those induced by human Adenovirus serotype 5 (Ad5). The virus predominantly localized to the liver and spleen and induced antigen-specific T cells though more robustly in the latter than Ad5. Although SynBAd is a potent immunogen, antibodies generated in mice immunized with Ad5 only weakly neutralized SynBAd infection in vitro, suggesting that preexisting Ad5 antibodies will not dramatically influence SynBAd efficacy.ConclusionsThe model used is representative of colon metastases to the liver as well as HBV-driven hepatocellular carcinoma. Localization of the vaccine after administration, activation of antigen specific T cells, mobilization of effector memory T cells, and efficacy in the liver cancer model suggest that this platform promotes a systemic tumor controlling immune response with wide-ranging clinical potential beyond liver cancer.
We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation ...Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 E1-, E2b-, E3-, previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 10
6
TCID
50
) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.
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