Production, characterization, and antigen specificity of recombinant 62‐71‐3, a candidate monoclonal antibody for rabies prophylaxis in humans

Abstract

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans.

Current Developments and Future Prospects for Plant-Made Biopharmaceuticals Against Rabies

“…Expression levels of up to 3-4 % TSP (*100 mg/kg fresh tissue weight) were achieved, which were sufficient to purify and characterize the plant-made antibody. In terms of functionality, the plant-made mAb 62-71-3 bound to its antigen and neutralized a rabies pseudotype virus [58]. An important conclusion of this research is the strong neutralizing activity in the FAVN assay (500 IU/mg), which is similar to that of the murine hybridoma-derived counterpart [59].…”

“…A pioneering study conducted by McGarvey et al [35] demonstrated that the G protein can be expressed in tomato Neutralized a rabies pseudotype virus [58,59] plants using a standard nuclear expression approach driven by the 35SCaMV promoter and an Agrobacterium-mediated transformation procedure. Although yields were very low, 1-10 ng/mg soluble protein, the proof of the concept opened important prospects to continue research in this area.…”

Evaluation of Rabies Biologics against Irkut Virus Isolated in China

“…This is particularly important as several virus-neutralizing monoclonal antibodies (MAbs) were offered for replacement of conventional HRIG in human rabies PEP (21)(22)(23)(24)(25)(26)(27)(28). For example, a linear epitope with the key residues LCGV within antigenic site I serves for binding of MAbs CR57 and 62071-3 (22,23). Similarly, conserved conformational antigenic sites II and III contain binding epitopes for several other MAbs (24)(25)(26)(27)(28).…”

Monoclonal antibodies for the prevention of rabies: theory and clinical practice

“…96 Homologous products (recombinant) -fully human intact igG Important parameters that drive selection of the most optimal systems for expression of antibodies include basic structure, protein folding and glycosylation, 97 productivity, ease of purification, 98 product quality and quantity, safety issues, time to clinic and market, and economic considerations such as cost of goods and regulatory issues. 99 In general, eukaryotic systems such as mammalian cells, 99,100 insect cells, 97 yeast, 101 plants, 74,102 green algae, 103 and transgenic animals 104 hold the greatest promise for expressing recombinant human MAbs. However, mammalian cells are predominantly used because they can correctly fold, assemble, glycosylate, and secrete antibodies.…”

“…MAb 62-71-3 was expressed in tobacco leaves and found to be an appropriate candidate MAb in the making of a novel antibody cocktail 74. …”