Holistic investigation of Pertuzumab and Trastuzumab grafted variable heavy families (VH1-7) and heavy chain constant (IgAs & IgM) antibodies and their effects on receptor (FcαRI & FcμR), antigen (Her2) and super antigen (protein A, G & L) binding

Abstract

Antibodies are highly specific immune proteins made up of two heavy and light chains assembled in a “Y” shape. The heavy and light chains are further segmented into regions, mainly the variable and constant regions. Based on the sequence classifications, there are six kappa variables (Vκ1-6), eleven lambda variables (Vλ1-11), two light constants (Cκ & Cλ); and seven heavy variables (VH1-7) and nine heavy constants (Gamma 1-4, γ1-4; Alpha 1 & 2, α1 & 2; Mu, μ; Epsilon, ε & Delta, δ) families. Given the importance of these regions in therapeutics and diagnostics, there are protein engineering opportunities in these regions to finetune antibody functions. To study these regions and effects systematically, this work investigated the relationship of mRNA expression of antibody-related genes, the contribution from the variety of signal peptides (SP), the influence of variable (V-) and constant (C-) regions across IgGs and IgAs on both the Pertuzumab and Trastuzumab models for a holistic comparison. The research measured IgG4/ IgE and IgD/ IgM mRNA expression in peripheral blood morphonuclear cells and performed signal peptide manipulations, finding that essential (EAA) and non-essential amino acid (NEAA) residues were limiting factors in recombinant production with the supplementation of EAA alleviating nutritional constraints. Investigating the whole antibody, antigen (Her2) and receptors (FcαRI & FcμR) engagements were found to be holistically affected by the V- and C- regions. In conclusion, the thesis has revealed findings of connectivity and influence between the various regions of the antibody with influence on function and recombinant production relevant for therapeutic development and diagnostic use.