CellCelector™ as a platform in isolating primary B cells for antibody discovery

Background The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming, and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. Methods In the current study, we present a method to isolate individual antigen specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR specific B-cells from 1,189 antibody secreting cells (ASCs) and isolated 94 by CellCellector. Results We identified a diverse set of heavy chain CDR sequences and cloned and expressed 20 into a standard human IgG1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400,000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1,196 unique EGFR-specific antibodies could be discovered. Conclusions CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Design and Immunological Properties of the Novel Subunit Virus-like Vaccine against SARS-CoV-2

The COVID-19 pandemic is ongoing, and the need for safe and effective vaccines to prevent infection and to control spread of the virus remains urgent. Here, we report the development of a SARS-CoV-2 subunit vaccine candidate (Betuvax-CoV-2) based on RBD and SD1 domains of the spike (S) protein fused to a human IgG1 Fc fragment. The antigen is adsorbed on betulin adjuvant, forming spherical particles with a size of 100–180 nm, mimicking the size of viral particles. Here we confirm the potent immunostimulatory activity of betulin adjuvant, and demonstrate that two immunizations of mice with Betuvax-CoV-2 elicited high titers of RBD-specific antibodies. The candidate vaccine was also effective in stimulating a neutralizing antibody response and T cell immunity. The results indicate that Betuvax-CoV-2 has good potential for further development as an effective vaccine against SARS-CoV-2.

Monoclonal Antibody Cocktail therapy for COVID-19: A Pharmacological innovation

The novel SARS-CoV-2 infection has ripped through international health systems and protocols causing unprecedented mortality, morbidity and global trade deficits amounting to billions. Various monoclonal antibodies have been proposed for use in the treatment of COVID-19 infections. One such drug is LY-CoV555 which in an ongoing phase two trial study conducted by Chen P et al, showed to have an elimination of 99.97% of the viral RNA. The monoclonal antibody 47D11 discovered by Wang et al, binds to SARS-CoV-2. The 47D11 has been reconfigured into a human IgG1 isotope. It has shown that the 47D11 mAb effectively neutralizes the SARS-COV-2 virus. The stance and development however for the treatment of COVID-19 with monoclonal antibodies has shifted from a monotherapy to a so-called monoclonal antibody “cocktail” therapy. REGN-COV2 is such a cocktail developed with the use of two monoclonal antibodies REGN10987 and REGN10933 which have subsequently been named Imdevimab and Casirivimab. REGN-COV2 is currently under study in four phase 2 and 3 trial studies. These studies are multicentric in nature and are being conducted to evaluate the drug’s efficacy, dosing and clinical use as compared to the placebo. The mechanism of action of such monoclonal antibodies is related chiefly to the inhibition of the virus’s ability to perform its invasion and multiplication within the human body. The severity coupled with the sheer novelty of the SARSCoV-2 virus demands the use of newer therapies to both decrease the mortality and morbidity in patients suffering from the infection. The use of a combination of monoclonal antibodies is thereby well established and evident to both decrease the viral infection load, but is also useful in disrupting the virus’s life cycle and thus decreases the replication and viral shedding. It is therefore poignant that a combination of monoclonal antibodies, a “cocktail” therapy is employed so as to attack the virus at its various stages and thus this multifaceted approach may enhance the patient’s prognosis.

An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, natural antibodies isolated from convalescent patients are vulnerable to SARS-CoV-2 Spike mutations. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb dimer, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern or interest. Deep-mutational scanning experiments identify the potential binding epitopes of the monomeric Nb1 and Nb2 on the RBD and demonstrate that bivalent Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that Nb1-Nb2 broadly neutralizes SARS-CoV-2, including variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to the biparatopic Nb (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0*10E-12 M) and neutralizing activity (IC50 = 0.0017 nM). Together, we developed a biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Bispecific Antibodies for IFN-β Delivery to ErbB2+ Tumors

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells.

Anti-virulence Bispecific Monoclonal Antibody Mediated Protection Against Pseudomonas aeruginosa Ventilator-Associated Pneumonia in a Rabbit Model

Ventilator-associated pneumonia is an important clinical manifestation of the nosocomial pathogen Pseudomonas aeruginosa . We characterized the correlates of protection of MEDI3902, a bispecific human IgG1 mAb that targets the P. aeruginosa type-3-secretion PcrV protein and the Psl exopolysaccharide, in a rabbit model of ventilator-associated pneumonia using lung-protective, low-tidal volume mechanical ventilation. Rabbits infused with MEDI3902 prophylactically were protected, whereas those pretreated with irrelevant isotype-control IgG (c-IgG) succumbed between 12 and 44 hours post infection [100% (8/8) vs. 0% (8/8) survival, P <0.01 by log-rank test]. Lungs from rabbits pretreated with c-IgG, but not those with MEDI3902, had bilateral, multifocal areas of marked necrosis, hemorrhage, neutrophilic inflammatory infiltrate, diffuse fibrinous edema in alveolar spaces. All rabbits pretreated with c-IgG developed worsening bacteremia that peaked at the time of death, whereas only 38% (3/8) rabbits pretreated with MEDI3902 developed such high-grade bacteremia (two-sided Fisher’s exact test, P =0.026). Biomarkers associated with acute respiratory distress syndrome were evaluated longitudinally in blood samples collected every 2-4 hours to assess systemic pathophysiological changes in rabbits pretreated with MEDI3902 or c-IgG. Biomarkers were sharply increased or decreased in rabbits pretreated with c-IgG, but not those pretreated with MEDI3902, including ratio of arterial oxygen partial pressure to fractional inspired oxygen PaO 2 /FiO 2 <300, hypercapnia or hypocapnia, severe lactic acidosis, leukopenia and neutropenia. Cytokines and chemokines associated with ARDS were significantly downregulated in lungs from rabbits pretreated with MEDI3902 compared with c-IgG. These results suggest that MEDI3902 prophylaxis could have potential clinical utility for decreasing severity of P. aeruginosa ventilator-associated pneumonia.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral receptor decoy that targets the vulnerable site on SARS-CoV-2 spike (S). Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbored structurally validated mutations that enhance S binding and remove enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, the lead variant prevented or delayed lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

CoV-RBD121-NP Vaccine Candidate Protects against Symptomatic Disease Following SARS-CoV-2 Challenge in K18-hACE2 Mice and Induces Protective Responses That Prevent COVID-19-Associated Immunopathology

We developed a SARS-CoV-2 vaccine candidate (CoV-RBD121-NP) comprised of a tobacco mosaic virus-like nanoparticle conjugated to the receptor-binding domain of the spike glycoprotein of SARS-CoV-2 fused to a human IgG1 Fc domain. CoV-RBD121-NP elicits strong antibody responses in C57BL/6 mice and is stable for up to 12 months at 2–8 or 22–28 °C. Here, we showed that this vaccine induces a strong neutralizing antibody response in K18-hACE2 mice. Furthermore, we demonstrated that immunization protects mice from virus-associated mortality and symptomatic disease. Our data indicated that a sufficient pre-existing pool of neutralizing antibodies is required to restrict SARS-CoV-2 replication upon exposure and prevent induction of inflammatory mediators associated with severe disease. Finally, we identified a potential role for CXCL5 as a protective cytokine in SARS-CoV-2 infection. Our results suggested that disruption of the CXCL5 and CXCL1/2 axis may be important early components of the inflammatory dysregulation that is characteristic of severe cases of COVID-19.

Development of a SARS-CoV-2 Vaccine Candidate Using Plant-Based Manufacturing and a Tobacco Mosaic Virus-like Nano-Particle

Stable, effective, easy-to-manufacture vaccines are critical to stopping the COVID-19 pandemic resulting from the coronavirus SARS-CoV-2. We constructed a vaccine candidate CoV-RBD121-NP, which is comprised of the SARS-CoV-2 receptor-binding domain (RBD) of the spike glycoprotein (S) fused to a human IgG1 Fc domain (CoV-RBD121) and conjugated to a modified tobacco mosaic virus (TMV) nanoparticle. In vitro, CoV-RBD121 bound to the host virus receptor ACE2 and to the monoclonal antibody CR3022, a neutralizing antibody that blocks S binding to ACE2. The CoV-RBD121-NP vaccine candidate retained key SARS-CoV-2 spike protein epitopes, had consistent manufacturing release properties of safety, identity, and strength, and displayed stable potency when stored for 12 months at 2–8 °C or 22–28 °C. Immunogenicity studies revealed strong antibody responses in C57BL/6 mice with non-adjuvanted or adjuvanted (7909 CpG) formulations. The non-adjuvanted vaccine induced a balanced Th1/Th2 response and antibodies that recognized both the S1 domain and full S protein from SARS2-CoV-2, whereas the adjuvanted vaccine induced a Th1-biased response. Both adjuvanted and non-adjuvanted vaccines induced virus neutralizing titers as measured by three different assays. Collectively, these data showed the production of a stable candidate vaccine for COVID-19 through the association of the SARS-CoV-2 RBD with the TMV-like nanoparticle.

Anti-Drug Antibodies in Pigtailed Macaques Receiving HIV Broadly Neutralising Antibody PGT121

Broadly neutralising antibodies (bNAbs) may play an important role in future strategies for HIV control. The development of anti-drug antibody (ADA) responses can reduce the efficacy of passively transferred bNAbs but the impact of ADA is imperfectly understood. We previously showed that therapeutic administration of the anti-HIV bNAb PGT121 (either WT or LALA version) controlled viraemia in pigtailed macaques with ongoing SHIV infection. We now report on 23 macaques that had multiple treatments with PGT121. We found that an increasing number of intravenous doses of PGT121 or human IgG1 isotype control antibodies (2-4 doses) results in anti-PGT121 ADA induction and low plasma concentrations of PGT121. ADA was associated with poor or absent suppression of SHIV viremia. Notably, ADA within macaque plasma recognised another human bNAb 10E8 but did not bind to the variable domains of PGT121, suggesting that ADA were primarily directed against the constant regions of the human antibodies. These findings have implications for the development of preclinical studies examining multiple infusions of human bNAbs.