Unleashing the power of shark variable single domains (VNARs): broadly neutralizing tools for combating SARS-CoV-2

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases

Targeting Serous Epithelial Ovarian Cancer with Designer Zinc Finger Transcription Factors

Ovarian cancer is the leading cause of death among gynecological malignancies. It is detected at late stages when the disease is spread through the abdominal cavity in a condition known as peritoneal carcinomatosis. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. Herein we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines and to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that although Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-TAg mouse model of ovarian cancer resulted in tumor cell growth inhibition, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice accompanied with Maspin re-expression in the treated tumors. Gene expression microarrays of ATF-transduced cells revealed an exceptional specificity for the Maspin promoter. These analyses identified novel targets co-regulated with Maspin in human short-term cultures derived from ascites, such as TSPAN12, that could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of ATFs into tumors with potential clinical applications for metastatic ovarian cancers

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Conotoxin Patenting Trends in Academia and Industry

Sea snails of the genus Conus produce toxins that have been the subjects of numerous studies, projects, publications, and patents over the years. Since Conus toxins were discovered in the 1960s, their biological activity has been thought to have high pharmaceutical potential that could be explored beyond the limits of academic laboratories. We reviewed 224 patent documents related to conotoxins and conopeptides globally to determine the course that innovation and development has taken over the years, their primary applications, the technological trends over the last six years, and the leaders in the field, since the only previous patent review was performed in 2015 and focused in USA valid patents. In addition, we explored which countries/territories protect their inventions and patents and the most relevant collaborations among assignees. We also evaluated whether academia or pharmaceutical companies are the future of conotoxin research. We concluded that the 224 conotoxin patents reviewed in this study have more academic value than industrial value, which was noted by the number of active patents that have not yet been licensed and the contributions to medical research, especially as tools to study neuropathic pain, inflammation, immunology, drug design, receptor binding sites, cancer, neurotransmission, epilepsy, peptide biosynthesis, and depression. The aim of this review is to provide an overview of the current state of conotoxin patents, their main applications, and success based on the number of licensing and products in the market

Preparation of pH-sensitive nanogels bioconjugated with shark antibodies (VNAR) for targeted drug delivery with potential applications in colon cancer therapies.

Cancer is the second leading cause of death worldwide. To combat this disease, novel and specialized therapeutic systems are urgently needed. This is the first study to explore a system that combines shark variable domain (Fv) of new antigen receptor (VNAR) antibodies (hereinafter VNARs), PEGylated nanogels (pH-sensitive poly(N,N-diethylaminoethyl methacrylate, PDEAEM), and the anticancer drug 5-fluorouracil (5-FU) to explore its potential applications in colon cancer therapies. Nanogels were functionalized in a scalable reaction with an N-hydroxysuccinimide (NHS)-terminated polyethylene glycol derivative and bioconjugated with shark antibodies. Dynamic light scattering measurements indicated the presence of monodispersed nanogels (74 to 236 nm). All systems maintained the pH-sensitive capacity to increase in size as pH decreased. This has direct implications for the release kinetics of 5-FU, which was released faster at pH 5 than at pH 7.4. After bioconjugation, the ELISA results indicated VNAR presence and carcinoembryonic antigen (CEA) recognition. In vitro evaluations of HCT-116 colon cancer cells indicated that functionalized empty nanogels are not cytotoxic and when loaded with 5-FU, the cytotoxic effect of the drug is preserved. A 15% reduction in cell viability was observed after two hours of contact with bioconjugated nanogels when compared to what was observed with non-bioconjugated nanogels. The prepared nanogel system shows potential as an effective and site-specific nanocarrier with promising applications in in vivo studies of colon cancer therapies

Synthetic libraries of shark vNAR domains with different cysteine numbers within the CDR3.

The variable domain of New Antigen Receptors (vNAR) from sharks, present special characteristics in comparison to the conventional antibody molecules such as: small size (12-15 kDa), thermal and chemical stability and great tissue penetration, that makes them a good alternative source as therapeutic or diagnostic agents. Therefore, it is essential to improve techniques used for the development and selection of vNAR antibodies that recognize distinct antigens. The development of synthetic antibody libraries offers a fast option for the generation of antibodies with the desired characteristics. In this work three synthetic antibody libraries were constructed; without cysteines (Cys), with one Cys and with two Cys residues within its CDR3, with the objective of determining whether the presence or absence of Cys in the CDR3 favors the isolation of vNAR clones from a synthetic library. The libraries were validated selecting against six mammalian proteins. At least one vNAR was found for each of the antigens, and a clone coming from the library without Cys in the CDR3 was selected with all the antigens. In vitro angiogenesis assay with the isolated anti-VEGF antibodies, suggest that these vNARs are capable of inhibiting in vitro angiogenesis. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival antibodies with affinity maturation by in silico modeling

COVID-19 Neutralizing Antibodies in Breast Milk of Mothers Vaccinated with Three Different Vaccines in Mexico

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the largest pandemic of this century, and all aspects of this virus are being studied. The efforts to mitigate the negative effects associated with the SARS-CoV-2 pandemic have culminated in the development of several vaccines that are effective and safe for use to the general population. However, one aspect that remains relatively underexplored is the efficacy of different vaccines technologies (mRNA and Adenovirus) in providing passive immunity to infants through breastmilk of vaccinated mothers, and whether the antibodies passed through breast milk are functional. In this study, using a Micro-neutralization assay, we evaluate the presence of neutralizing antibodies in breast milk of lactating mothers vaccinated against SARS-CoV-2 with the Pfizer-BioNtech, Johnson & Johnson (J&J)/Janssen, and CanSino Biologics vaccines. Our results show the greatest neutralizing effect in breast milk from mothers vaccinated with Pfizer, followed by mothers vaccinated with J&J. CanSino vaccinations yielded the breast milk with the least neutralizing effects. The results found in this study relating to the neutralizing capacity of breast milk against SARS-CoV-2 highlight the importance of corresponding health authorities recommending vaccination to lactating mothers and of the continuance of breastfeeding to infants due to the potential health benefits

<i>In vitro</i> analysis of VNAR-bioconjugated nanogels (N3).

A) ELISA of VNAR expression. On the horizontal axis: B (blank, Svelty milk 8%-PBS), N3 C- (nanogel without VNAR), C+ (CV043, 175 μg/mL), 1h (sample taken at hour 1 of the coupling reaction), 4h (sample taken at hour 4 of the coupling reaction), 8h (final sample taken at hour 8 of the coupling reaction), and PF (final product after dialysis [N3+VNAR]). B) ELISA assay of CEA recognition. On the horizontal axis: VNAR (CV043, 175 μg/mL), B (blank, Svelty milk 8%-PBS), c) cell viability by aqueous one solution cell proliferation assay (MTS) of empty selected nanogels at 24h, PBS (C-), DMSO 5% (C+), d) cell viability by MTS of 5-FU and nanogels containing 5-FU. On the horizontal axis: B (PBS 7.4), N3 (empty nanogels, 5-FU [20 μg/mL]), N3-VNAR-5FU (20 μg 5FU/mL), and N3-5-FU (20 μg 5FU/mL). ANOVA test, *p < 0.01, **p < 0.001, and ***p < 0.0001 versus N3 and PBS (C-). PDEAEM: poly(N,N-diethylaminoethyl methacrylate), PEGMA: poly(ethylene glycol) methyl ether methacrylate, PEGA-NHS: Acrylate-PEG3500-NHS, and CEA: carcinoembryonic antigen.</p