The effects of Antibody Engineering CH and CL in Trastuzumab and Pertuzumab recombinant models: Impact on antibody production and antigen-binding

Abstract

Current therapeutic antibodies such as Trastuzumab, are typically of the blood circulatory IgG1 class (Cκ/ CHγ1). Due to the binding to Her2 also present on normal cell surfaces, side effects such as cardiac failure can sometimes be associated with such targeted therapy. Using antibody isotype swapping, it may be possible to reduce systemic circulation through increased tissue localization, thereby minimising unwanted side effects. However, the effects of such modifications have yet to be fully characterized, particularly with regards to their biophysical properties in antigen binding. To do this, we produced all light and heavy chain human isotypes/subtypes recombinant versions of Trastuzumab and Pertuzumab, and studied them with respect to recombinant production and Her2 binding. Our findings show that while the light chain constant region changes have no major effects on production or Her2 binding, some heavy chain isotypes, in particularly, IgM and IgD isotypes, can modulate antigen binding. This study thus provides the groundwork for such isotype modifications to be performed in the future to yield therapeutics of higher efficacy and efficiency.

Role of the IgE variable heavy chain in FcεRIα and superantigen binding in allergy and immunotherapy

“…4 Based on our size exclusion chromatography results of the purified pertuzumab IgE variants, we did not find notable aggregation trends for any of the VH family representatives in our purification conditions, nor was there evidence for natural polymerization as in the case for IgM. 31,54 Our previous work on IgG 1 32 showed the VH family FWRs to have effects on FcgRIIA. To determine whether VH family FWRs have the same effect on IgE to FcεRIa, which has clear implications on type I hypersensitivity and engineering of IgE therapeutics, we found such an effect in 1 of 10 of our recombinant IgEs.…”

“…32 The VH families were joined to the IgE C-region, as previously performed. 31,33 Plasmids were transformed into homemade competent DH5a bacteria, as previously described, 34 and extracted by using our in-house extraction kits. 35 To allow us to minimize differences for future more in-depth investigations, trastuzumab was selected as a cross-checking model because the trastuzumab-pertuzumab pair shared high sequence similarity, had the same VH3 FWR, were both specific against Her2, and were produced by the same company (see Fig E1 in this article's Online Repository at www.jacionline.org).…”

“…Pertuzumab and trastuzumab VH1-VH7 IgE variants were produced by means of cotransfection, as previously performed. [30][31][32]36 Affinity purification was carried out with the Protein L column, followed by size exclusion chromatography fractionation with the Superdex 200-pg 16/600 column (GE Healthcare) to extract monomeric fractions, which were further concentrated by using a 100-kDa Amicon Ultra protein concentrator (catalog no. ACS510024; Merck-Millipore).…”

“…Measurements of pertuzumab IgE variants to Her2 were carried out by loading Her2 on an Ni-NTA biosensor, followed by IgE binding, as previously described. [30][31][32]36,37 Measurements toward FcεRIa were performed in the same manner, as previously described. 32 Measurements of association and dissociation rates of omalizumab to pertuzumab variants were performed with omalizumab preloaded to Anti-hIgG-Fc Capture biosensors (catalog no.…”

“…30 This allosteric effect was also shown when the changing of antibody C-regions affected antigen binding. 31 To investigate the effects of different VH family FWRs on FcεRIa and superantigen binding, as well as for anti-IgE therapeutics, we investigated 7 VH families using pertuzumab antibody as a model for grafting onto the IgE C-region 31 and measured interactions with FcεRIa, superantigen spA, anti-IgE (omalizumab), and Her2 using biolayer interferometry. Trastuzumab VH family variants were also investigated as a cross-check to any possible VH FWR effects found on pertuzumab.…”