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.

Chimeric Peptides from Californiconus californicus and Heterodontus francisci with Antigen-Binding Capacity: A Conotoxin Scaffold to Create Non-Natural Antibodies (NoNaBodies).

Research into various proteins capable of blocking metabolic pathways has improved the detection and treatment of multiple pathologies associated with the malfunction and overexpression of different metabolites. However, antigen-binding proteins have limitations. To overcome the disadvantages of the available antigen-binding proteins, the present investigation aims to provide chimeric antigen-binding peptides by binding a complementarity-determining region 3 (CDR3) of variable domains of new antigen receptors (VNARs) with a conotoxin. Six non-natural antibodies (NoNaBodies) were obtained from the complexes of conotoxin cal14.1a with six CDR3s from the VNARs of Heterodontus francisci and two NoNaBodies from the VNARs of other shark species. The peptides cal_P98Y vs. vascular endothelial growth factor 165 (VEGF165), cal_T10 vs. transforming growth factor beta (TGF-ß), and cal_CV043 vs. carcinoembryonic antigen (CEA) showed in-silico and in vitro recognition capacity. Likewise, cal_P98Y and cal_CV043 demonstrated the capacity to neutralize the antigens for which they were designed.

First pan-specific vNAR against human TGF-ß as a potential therapeutic application: in silico modeling assessment.

Immunotherapies based on antibody fragments have been developed and applied to human diseases, describing novel antibody formats. The vNAR domains have a potential therapeutic use related to their unique properties. This work used a non-immunized Heterodontus francisci shark library to obtain a vNAR with recognition of TGF-ß isoforms. The isolated vNAR T1 selected by phage display demonstrated binding of the vNAR T1 to TGF-ß isoforms (-ß1, -ß2, -ß3) by direct ELISA assay. These results are supported by using for the first time the Single-Cycle kinetics (SCK) method for Surface plasmon resonance (SPR) analysis for a vNAR. Also, the vNAR T1 shows an equilibrium dissociation constant (KD) of 9.61 × 10-8 M against rhTGF-ß1. Furthermore, the molecular docking analysis revealed that the vNAR T1 interacts with amino acid residues of TGF-ß1, which are essential for interaction with type I and II TGF-ß receptors. The vNAR T1 is the first pan-specific shark domain reported against the three hTGF-ß isoforms and a potential alternative to overcome the challenges related to the modulation of TGF-ß levels implicated in several human diseases such as fibrosis, cancer, and COVID-19.

Neutralizing Ability of a Single Domain VNAR Antibody: In Vitro Neutralization of SARS-CoV-2 Variants of Concern.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causal pathogen of coronavirus disease 2019 (COVID-19). The emergence of new variants with different mutational patterns has limited the therapeutic options available and complicated the development of effective neutralizing antibodies targeting the spike (S) protein. Variable New Antigen Receptors (VNARs) constitute a neutralizing antibody technology that has been introduced into the list of possible therapeutic options against SARS-CoV-2. The unique qualities of VNARs, such as high affinities for target molecules, capacity for paratope reformatting, and relatively high stability, make them attractive molecules to counteract the emerging SARS-CoV-2 variants. In this study, we characterized a VNAR antibody (SP240) that was isolated from a synthetic phage library of VNAR domains. In the phage display, a plasma with high antibody titers against SARS-CoV-2 was used to selectively displace the VNAR antibodies bound to the antigen SARS-CoV-2 receptor binding domain (RBD). In silico data suggested that the SP240 binding epitopes are located within the ACE2 binding interface. The neutralizing ability of SP240 was tested against live Delta and Omicron SARS-CoV-2 variants and was found to clear the infection of both variants in the lung cell line A549-ACE2-TMPRSS2. This study highlights the potential of VNARs to act as neutralizing antibodies against emerging SARS-CoV-2 variants.

Neutralizing antibodies levels are increased in individuals with heterologous vaccination and hybrid immunity with Ad5-nCoV in the north of Mexico.

The coordinated efforts to stop the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) include massive immunization of the population at a global scale. The humoral immunity against COVID-19 is conferred by neutralizing antibodies (NAbs) that occur during the post-infection period and upon vaccination. Here, we provide robust data showing that potent neutralizing antibodies are induced in convalescent patients of SARS-CoV-2 infection who have been immunized with different types of vaccines, and patients with no previous history of COVID-19 immunized with a mixed vaccination schedule regardless of the previous infection. More importantly, we showed that a heterologous prime-boost in individuals with Ad5-nCoV (Cansino) vaccine induces higher NAbs levels in comparison to a single vaccination scheme alone.

Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders.

The shark-derived autonomous variable antibody domains known as VNARs are attractive tools for therapeutic and diagnostic applications due to their favorable properties like small size (approximately 12 kDa), high thermal and chemical stability, and good tissue penetration. Currently, different techniques have been reported to generate VNAR domains against targets of therapeutic interest. Here, we describe methods for the preparation of an immune VNAR library based on bacteriophage display, and for the preparation of a synthetic library of VNAR domains using a modified protocol based on Kunkel mutagenesis. Finally, we describe procedures for in silico maturation of a VNAR using a bioinformatic approach to obtain higher affinity binders.

Distribution of pathogenic vibrios and Vibrio spp. in the water column and sediment samples from the southern Gulf of Mexico.

This study quantified the distribution of Vibrio spp. by qPCR and pathogenic vibrio species by metagenomics, during 2 oceanographic cruises-XIXIMI-04 and XIXIMI-05 -in the southern Gulf of Mexico (GoMex). A total of 708 samples from various levels of the water column and 22 sediment samples were analyzed, according to a designed net of sampling lines. Sampling was focused on reported water masses with distinctive characteristics, to detect the presence-absence of vibrios. The results indicated that the genus Vibrio was detected along the entire water column and in sediments. Pathogenic vibrios, such as V, campbellii, V. parahaemolyticus, V. vulnificus or V. cholerae were also detected in the water column and in sediments, in both oceanographic cruises. Thus, the ecological conditions of the GoMex permit the growth of Vibrio spp. in deep water environments of the GoMex, despite continuous oil input from natural and anthropogenic sources.