Nanomaterials Upscaling Cell Production and Advancing Exosome-Based Stem Cell Therapies

The COVID-19 pandemic underlined that by investing in both basic and clinical life science research and if there are enough volunteers, it is feasible to have -validated by Phase III clinical trials- vaccines in less than a year. Regarding the treatment options for the people who were infected by COVID-19, we know that it was the large clinical trials - like SOLIDARITY (WHO) and RECOVERY (UK)- that gave the most valid results, and that although hundreds of drugs were repurposed, sadly, most proved to be unsuccessful. Repurposing drugs and compassionate use, were the only options for the first half of 2020. The same applied to the convalescent plasma (CP) approach; however, apart from CP, other cell derived therapeutics were deployed, such as synthetic monoclonal antibodies, which were also tested and given provisional licences by health authorities. Unfortunately, synthetic antibody production comes with problems related to low and slow yield that were not overcome, while SARS-CoV-2 viral mutations may possibly render them less effective. One approach that works and is currently assessed in several clinical trials, is mesenchymal stromal cell (MSCs) and extracellular vesicle (EV) administration for therapy. Interdisciplinarity may prove key here. Easy to produce nanomaterials and biomaterials should be further investigated to increase bioproduction of MSCs, both at the level of therapeutics, as the base substrate for EV production and to upscale synthetic antibody production for therapy.

Eradicating mesothelin-positive human gastric and pancreatic tumors in xenograft models with optimized anti-mesothelin antibody–drug conjugates from synthetic antibody libraries

Mesothelin (MSLN) is an attractive candidate of targeted therapy for several cancers, and hence there are increasing needs to develop MSLN-targeting strategies for cancer therapeutics. Antibody–drug conjugates (ADCs) targeting MSLN have been demonstrated to be a viable strategy in treating MSLN-positive cancers. However, developing antibodies as targeting modules in ADCs for toxic payload delivery to the tumor site but not to normal tissues is not a straightforward task with many potential hurdles. In this work, we established a high throughput engineering platform to develop and optimize anti-MSLN ADCs by characterizing more than 300 scFv CDR-variants and more than 50 IgG CDR-variants of a parent anti-MSLN antibody as candidates for ADCs. The results indicate that only a small portion of the complementarity determining region (CDR) residues are indispensable in the MSLN-specific targeting. Also, the enhancement of the hydrophilicity of the rest of the CDR residues could drastically increase the overall solubility of the optimized anti-MSLN antibodies, and thus substantially improve the efficacies of the ADCs in treating human gastric and pancreatic tumor xenograft models in mice. We demonstrated that the in vivo treatments with the optimized ADCs resulted in almost complete eradication of the xenograft tumors at the treatment endpoints, without detectable off-target toxicity because of the ADCs’ high specificity targeting the cell surface tumor-associated MSLN. The technological platform can be applied to optimize the antibody sequences for more effective targeting modules of ADCs, even when the candidate antibodies are not necessarily feasible for the ADC development due to the antibodies’ inferior solubility or affinity/specificity to the target antigen.

Antibody heavy chain CDR3 length-dependent usage of human IGHJ4 and IGHJ6 germline genes

Background Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. In recent years, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Methods Using VH3–23 as a model framework, we analyzed the naturally and productively rearranged CDR-H3 diversity in human immune repertoire. With homology modeling, we further built VH3–23-based structural models to understand the spatial paratope and its influencing parameters. Results We observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Such CDR-H3 length dependent usage of human germline genes was not impacted by the choices of VH in the V(D)J recombination, ethnic background and health conditions. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Conclusions We quantitatively determined the CDR-H3 length-dependent usage of human germline genes, which makes it possible to design synthetic diversity fully mimicking that of natural immune repertoire. Our observations shed light on the design of next generation synthetic diversity with improved probability of success.

In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

Plastic Antibody of Polypyrrole/Multiwall Carbon Nanotubes on Screen-Printed Electrodes for Cystatin C Detection

This work reports the design of a novel plastic antibody for cystatin C (Cys-C), an acute kidney injury biomarker, and its application in point-of-care (PoC) testing. The synthetic antibody was obtained by tailoring a molecularly imprinted polymer (MIP) on a carbon screen-printed electrode (SPE). The MIP was obtained by electropolymerizing pyrrole (Py) with carboxylated Py (Py-COOH) in the presence of Cys-C and multiwall carbon nanotubes (MWCNTs). Cys-C was removed from the molecularly imprinted poly(Py) matrix (MPPy) by urea treatment. As a control, a non-imprinted poly(Py) matrix (NPPy) was obtained by the same procedure, but without Cys-C. The assembly of the MIP material was evaluated in situ by Raman spectroscopy and the binding ability of Cys-C was evaluated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. The MIP sensor responses were measured by the DPV anodic peaks obtained in the presence of ferro/ferricyanide. The peak currents decreased linearly from 0.5 to 20.0 ng/mL of Cys-C at each 20 min successive incubation and a limit of detection below 0.5 ng/mL was obtained at pH 6.0. The MPPy/SPE was used to analyze Cys-C in spiked serum samples, showing recoveries <3%. This device showed promising features in terms of simplicity, cost and sensitivity for acute kidney injury diagnosis at the point of care.

Recent studies of synthetic antibody-based 3-MCPD determination technology

3-Chloro-1,2-propanediol (3-MCPD) is classified by the International Agency for Research on Cancer as carcinogenic material. 3-MCPD will also become one of the European Union's requirements, proposing the maximum level of the 3-MCPD in palm oil until 2.5 ppm. Although the reported technologies GCMS and HPLC-FLD demonstrated high sensitivity and selectivity on 3-MCPD determination, those technologies invest in chemical and time-consuming sample preparation and analysis. Molecularly imprinted polymer (MIP), or a synthetic antibody, can be used to recognize 3-MCPD. MIP is more robust under extreme environments such as temperature and pH. This paper, therefore, aims to discuss the application of MIP on sample extraction and analysis to detect 3-MCPD. MIP is synthesized by polymerization of functional monomers surrounding 3-MCPD as a template. 3-MCPD is then removed from the MIP, leaving active cavities. Thus, these sites can either covalently or non-covalently rebind to 3-MCPD. Computational or empirical studies could investigate the composition of MIP. MIP can be manufactured as MIP-based solid phase extraction (MIPSPE) and MIP-based sensor. Both applications showed significant analytical parameters, such as recovery higher than 90% and detection limit lower than 2.5 ppm. Therefore, the application of MIP can be flexible for sample preparation and analysis on the 3-MCPD determination. MIP-based technology would be a prospective instrument to detect 3-MCPD. In the future, producing MIP on an industrial scale will be a challenge to monitor the 3-MCPD level in palm oil.

CellectSeq: In silico discovery of antibodies targeting integral membrane proteins combining in situ selections and next-generation sequencing

Synthetic antibody (Ab) technologies are efficient and cost-effective platforms for the generation of monoclonal Abs against human antigens. Yet, they typically depend on purified proteins, which exclude integral membrane proteins that require the lipid bilayers to support their native structure and function. Here, we present an Ab discovery strategy, termed CellectSeq, for targeting integral membrane proteins on native cells in complex environment. As proof of concept, we targeted three transmembrane proteins linked to cancer, tetraspanin CD151, carbonic anhydrase 9, and integrin-α11. First, we performed in situ cell-based selections to enrich phage-displayed synthetic Ab pools for antigen-specific binders. Then, we designed next-generation sequencing procedures to explore Ab diversities and abundances. Finally, we developed motif-based scoring and sequencing error-filtering algorithms for the comprehensive interrogation of next-generation sequencing pools to identify Abs with high diversities and specificities, even at extremely low abundances, which are very difficult to identify using manual sampling or sequence abundances.

Within-Host and Between-Host Evolution in SARS-CoV-2—New Variant’s Source

Some of the newly emerging corona viral variants show high numbers of mutations. This is unexpected for a virus with a low mutation rate due to an inherent proof-reading system. Could such a variant arise under very special conditions occurring in a host where the virus replicates and mutates in a rather unlimited fashion, such as in immune compromised patients? The virus was shown to replicate in an immunosuppressed cancer patient for more than 105 days and might be a source of new variants. These patients are asymptomatic and the virus may therefore escape detection and attention and be high-risk. Similarly, HIV-infected individuals may be immunocompromised and support coronavirus replication with increased mutation rates. The patients may promote “within-host evolution”. Some of the viruses present in such a highly mutagenic swarm or quasispecies within one patient may become founders and cause a pandemic by further “between-host evolution”. B.1.1.7 with 23 mutations may be such a case. Immunosuppressed patients can be identified and treated by the synthetic antibody cocktails as passive immunization and kept under control. Immunosuppressed patients can be easily identified and supervised by healthcare workers—once they become aware of the risk—to avoid new variants with pandemic potential.

An explicitly designed paratope of Amyloid-β prevents neuronal apoptosis in vitro and hippocampal damage in rat brain

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of Amyloid-β...