Proteolytic degradation of monoclonal antibodies produced in Nicotiana tabacum

Interest in using plants for the production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, which leads to a reduction in protein yield, a loss of functionality and complications in downstream processing, is a significant problem. Typically, fully assembled antibody expressed in the plant is accompanied by additional immunoglobulin species of lower molecular weight, which are predominantly generated by the enzymatic action of plant proteases. As plant proteases are pivotal to maintaining metabolic functions, expression of fully assembled antibodies, alien to the plant proteomic system, remains a challenging task. The present study examined two monoclonal antibodies expressed in transgenic Nicotiana tabacum; the murine IgG 1 K, Guy's 13, which recognises an epitope on the surface protein SAIIII of Streptococcus mutans; and the human IgG 1 K 2G 12, which targets high mannose structures within the HIV-l gp120 envelope glycoprotein. The stability of plant-expressed antibodies during downstream processing was investigated. These studies demonstrated that proteolytic degradation is predominantly an in planta event, taking place prior to extraction. Intracellular antibody trafficking was investigated in plant protoplasts to gain a further understanding of the site and mechanism of degradation. The results obtained indicated that the initial proteolytic cleavage occurred intracellularly. Antibody integrity was affected at an early stage in the secretory pathway (ER/Golgi) and also by transport to the vacuole. The apoplastic space is not a major site of antibody degradation. The composition of each lower molecular weight band from protein A/G affinity purified antibody was determined using western blotting, N-terminal sequencing and mass spectrometry. The latter two techniques revealed that, although no conserved target sequences were evident, the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to interdomain or solvent exposed regions in the antibody. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy chain and light chain mutants were expressed transiently in combination in Nicotiana tabacum and demonstrated alterations in fragmentation pattern. Some mutant combinations, (particularly Serll3Thr/Ala1l4Gly) in the heavy • 11 3 chain together with changes in the light chain (Leu104Ile/Glu105 Asp, Lysl03Ser or Ile106Leu) resulted in a significant increase in the ratio of assembled antibody to antibody fragments, compared to the non-mutated Guy's 13. Overall this thesis strengthens understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to reduce degradation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo