Non-RBD binding antibody neutralize SARSCoV- 2

Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.

Three-dimensional cell culture approach forin vitroimmunization and the production of monoclonal antibodies.

The generation of monoclonal antibodies using anin vitroimmunization approach is a promising alternative to conventional hybridoma technology. As recently published, thein vitroapproach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. Thisin vitroprocess can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies byin vitroimmunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.

Hybridoma technology: is it still useful?

The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.

Research Progress of Monoclonal Antibody Technology and Analysis of Marketed Drugs

Since the establishment of hybridoma in the 1980s, the antibody technology has achieved great development.Antibody is an immunoglobulin secreted by B lymphocytes, which produces many biological activity, such as blocking, neutralization, activation, kill target cells and regulate immune system via Fc receptor.The development of antibody technology has undergone a long history of mouse monoclonal antibodies, chimeric antibodies, humanized antibodies, and full human monoclonal antibodies.In the transition from mouse antibody to human antibody, a variety of biotechnology breakthroughs have been achieved, such as antibody library technology, humanized mouse technology and B cell cloning technology.Today, antibody drugs have a pivotal position throughout the drug market.Ten years come (2 011.01~2 021.11), 78 monoclonal antibody drugs have been approved for marketing by FDA, are widely distributed in the field of tumor disrases, immune diseases, anti-pathogen infections, nerves, etc.This article reviews monoclonal antibody technologies and antibody drug listing, and provides ideas for the preparation of new antibodies and the choice of drug target.

DEVELOPMENT of COVID-19 MONOCLONAL ANTIBODIES and RECOMBINANT PROTEINS AS REAGENTS for BIOMEDICAL RESEARCH and DIAGNOSTIC TESTS

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.

Rapid and reliable hybridoma screening method that is suitable for production of functional structure-recognizing monoclonal antibody.

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

Establishment of Murine Hybridoma Cells Producing Antibodies against Spike Protein of SARS-CoV-2.

In 2020 the world faced the pandemic of COVID-19 severe acute respiratory syndrome caused by a new type of coronavirus named SARS-CoV-2. To stop the spread of the disease, it is crucial to create molecular tools allowing the investigation, diagnoses and treatment of COVID-19. One of such tools are monoclonal antibodies (mAbs). In this study we describe the development of hybridoma cells that can produce mouse mAbs against receptor binding domain of SARS-CoV-2 spike (S) protein. These mAbs are able to specifically detect native and denatured S proteins in all tested applications, including immunoblotting, enzyme-linked immunosorbent assay, immunofluorescence staining of cells and immunohistochemical staining of paraffin embedded patients' tissue samples. In addition, we showed that the obtained mAbs can efficiently block SARS-CoV-2 infection in in vitro experiments. Finally, we determined the amino acid sequence of light and heavy chains of the mAbs. This information will allow the use of corresponding peptides to establish genetically engineered therapeutic antibodies. To date multiple mAbs against SARS-CoV-2 proteins have been established, however, bigger sets of various antibodies will allow the detection and neutralization of SARS-CoV-2, even if the virus acquires novel mutations.