In Silico Analyses on the Comparative Potential of Therapeutic Human Monoclonal Antibodies Against Newly Emerged SARS-CoV-2 Variants Bearing Mutant Spike Protein

Since the start of the pandemic, SARS-CoV-2 has already infected more than 250 million people globally, with more than five million fatal cases and huge socio-economic losses. In addition to corticosteroids, and antiviral drugs like remdesivir, various immunotherapies including monoclonal antibodies (mAbs) to S protein of SARS-CoV-2 have been investigated to treat COVID-19 patients. These mAbs were initially developed against the wild-type SARS-CoV-2; however, emergence of variant forms of SARS-CoV-2 having mutations in the spike protein in several countries including India raised serious questions on the potential use of these mAbs against SARS-CoV-2 variants. In this study, using an in silico approach, we have examined the binding abilities of eight mAbs against several SARS-CoV-2 variants of Alpha (B.1.1.7) and Delta (B.1.617.2) lineages. The structure of the Fab region of each mAb was designed in silico and subjected to molecular docking against each mutant protein. mAbs were subjected to two levels of selection based on their binding energy, stability, and conformational flexibility. Our data reveal that tixagevimab, regdanvimab, and cilgavimab can efficiently neutralize most of the SARS-CoV-2 Alpha strains while tixagevimab, bamlanivimab, and sotrovimab can form a stable complex with the Delta variants. Based on these data, we have designed, by in silico, a chimeric antibody by conjugating the CDRH3 of regdanivimab with a sotrovimab framework to combat the variants that could potentially escape from the mAb-mediated neutralization. Our finding suggests that though currently available mAbs could be used to treat COVID-19 caused by the variants of SARS-CoV-2, better results could be expected with the chimeric antibodies.

Novel monkey mAbs induced by a therapeutic vaccine targeting the hepatitis B surface antigen effectively suppress hepatitis B virus in mice

Background We have previously obtained a mouse anti-hepatitis B surface antigen (HBsAg) antibody E6F6 with long-lasting serum HBsAg clearance effects. The E6F6 epitope-based protein CR-T3-SEQ13 (HBsAg aa 113–135) vaccination therapy in cynomolgus monkeys induced long-term polyclonal antibodies-mediated clearance of HBsAg in the HBV transgenic (HBV-Tg) mice. Methods We isolated monoclonal antibodies from CR-T3-SEQ13 vaccinated cynomolgus monkeys, compared their therapeutic effects with E6F6, identified their epitopes on HBsAg, determined the pharmacokinetics, and studied their physical property. Results A panel of anti-HBsAg mAbs was generated through memory B cell stimulatory culture. Two lead monkey-human chimeric antibodies, C1–23 and C3–23, effectively suppressed HBsAg and HBV DNA in HBV-Tg mice. The humanized antibodies and humanized-mouse reverse chimeric antibodies of two antibodies exhibited comparable HBsAg clearance and viral suppression efficacy as those versions of E6F6 in HBV-Tg mice. Humanized antibody hu1–23 exhibited more efficacy HBsAg-suppressing effects than huE6F6–1 and hu3–23 in HBV-Tg mice at dose levels of 10 mg/kg and 20 mg/kg. Evaluation of the binding sites indicates that the epitope recognized by hu1–23 is located in HBsAg aa 118–125 and 121–125 for hu3–23. Physical property study revealed that hu1–23 and hu3–23 are stable enough for further development as a drug candidate. Conclusions Our data suggest that the CR-T3-SEQ13 protein is a promising HBV therapeutic vaccine candidate; and hu1–23 and hu3–23 are therapeutic candidates for the treatment of CHB. Moreover, the generation of antibodies from the epitope-based vaccinated subjects may be an alternative approach for novel antibody drug discovery. Statement of Significance Cynomolgus monkey mAbs were generated from an HBsAg-epitope-based-protein vaccination through memory B cell stimulatory culture. The humanized antibodies can efficiently mediate HBsAg clearance in HBV-Tg mice and may serve as anti-HBsAg therapeutic candidates. Generation of mAbs from the epitope-based vaccinated subjects is an alternative approach for novel antibody discovery.

Immunogenicity of biologics used in the treatment of moderate to severe psoriasis

The number of biologic drugs available for the treatment of psoriasis continue to expand. However, being biological proteins and thus potentially immunogenic, there is evidence that anti-drug-antibodies develop against the various therapeutic proteins currently being utilised. Although chimeric antibodies that contain elements of the parental monoclonal antibodies are immunogenic, anti-drug antibodies occur even if the biologic is a fully human protein and these can impact on clinical efficacy and safety. However, there is a wide variation in the reported level of anti-drug-antibodies for the same and different treatments that is highlighting issues with various assays used in anti-drug antibody detection. Here we review the available data on the occurrence of anti-drug antibodies in people with psoriasis treated with biologic agents.

Comparative efficacy and safety of chimeric and recombinant anti-TNF-α mAbs

TNF-α has been known since 1985. It is a multifunctional proinflammatory cytokine, synthesized mainly by monocytes and macrophages. Since its discovery, many studies have been conducted that have proven that it provides homeostatic function and regulates many biological processes in the body. Violation of its regulation in humans is associated with the development of many autoimmune diseases. The intensive studies that led to the understanding of its polyfunctionality and its role in the immunopathogenesis of a number of diseases served as the basis for the development of anti-cytokine therapy with monoclonal antibodies. In 1975, a technique for producing such antibodies was developed. The first antibodies against TNF-α obtained were chimeric, consisting of 30% mouse protein. Because of this feature, drugs based on chimeric antibodies had immunogenicity, which was manifested in the formation of antibodies to the drug, which led to a decrease in their effectiveness. To reduce immunogenicity, scientists in 1990 created the first fully human monoclonal antibody based on a technology called phage display. This is how adalimumab was born, the first fully human multi-clonal antibody to TNF-α. Humira® (adalimumab) is currently considered a widely studied drug from the group of TNF-α inhibitors, with a good safety and efficacy profile. The article presents current data that demonstrate that the drug significantly improves the course of diseases such as rheumatoid and psoriatic arthritis, and will allow for long-term remission in Crohn’s disease.

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

USING OF UNIVERSAL PLASMID CONSTRUCTIONS FOR DESIGN OF RECOMBINANT ANTIBODIES WITH DEFINED SPECIFICITY IN EUKARYOTIC CELLS

Aim. In this study we aimed to develop the methodology to change the antigen specificity of chimeric antibodies by replacing the variable region genes in the previously designed universal plasmid constructions pLK DT-17 and pHG DT-17 encoding the DT-17 antibody against the diphtheria toxin (DT) to the genes of antibody binding to another DT epitope — DT-22.Materials and methods. The genes of the light and heavy chain variable regions of mouse anti-DT antibodies — DT-22 were amplified from the hybridoma producing monoclonal antibodies to DT by reverse transcription and PCR methods. Genetic engineering methods were used to replace the variable regions of DT-17 antibody in the recombinant plasmids pLK DT-17 and pHG DT-17 encoding the light and heavy chains of DT-17 antibody, respectively to the relevant genes of DT-22. Subsequently, a «supervector» pSV DT-22, containing the genes of both chains of the chimeric antibody, was designed. CHO cells were transfected with a «supervector» and a highly productive clone, secreting chimeric antibodies to DT was obtained. Immunochemical and cultural methods were used to evaluate antibody activity. The affinity chromatography was used to purified preparative amounts of antibodies.Results. The yield of purified secreted chimeric DT-22 antibodies was 4 mg from per liter of culture medium. The minimum concentration of chimeric antibodies at which DT was neutralized in the CHO cells was 22 μg/mL of medium.Conclusion. Thus it has been shown how to generate new vector coding synthesis of light and heavy chains of a chimeric DT-22 antibody specific to another DT epitope using previously constructed universal recombinant plasmids pLK DT-17 and pHG DT-17 encoding, light and heavy chains of antibodies against DT DT-17, respectively.