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.

Figure 1

Trastuzumab and Pertuzumab isotype/subtype characterization. Size Exclusion Chromatography profiles of the Trastuzumab and Pertuzumab variants following affinity purification using Superdex size exclusion column on the AKTA Pure system. X-Axis: 40–100 ml time scale. Y-axis: mAU absorption as determined by UV detection. The green lines on the X-axis marks the 70 ml mark which corresponds to ~150 kDa as determined by previous calibrations (see Supplementary Data). Red arrows depict the selected peak fractions of the antibodies used for subsequent analyses while the red horizontal line at the x-axis indicate the fractions collected. SDS-PAGE analyses of concentrated antibodies (reduced) are quantified and shown in the smaller inserts with corresponding band sizes for the light and heavy chain (for gel photos with ladder analysed with GelApp2.0, see Supplementary Data) using GelApp2.0. Graphs are representative of at least 3 independent batches of transfected cells.

Figure 2

Binding affinity measurements of Trastuzumab and Pertuzumab isotype/subtype variants to Her2, using antibodies at 200 nM to 25 nM to pre-loaded Her2 on NTA biosensors. KD, Ka, and Kd were measured and calculated by the the Blitz® software. All experiments were performed with at least three independent occasions in triplicate readings. The X-axis depicts the time in seconds. The Y-axis depicts the binding in nm.

Figure 3

Binding affinity measurements of Trastuzumab and Pertuzumab isotype/subtype variants to Her2 at 100 nM to 6.25 nM concentrations to pre-loaded Trastuzumab and Pertuzumab variants on Protein L (ProL) biosensors. KD, Ka, and Kd were measured and calculated by the Octet QK^e system. All experiments were performed with at least three independent occasions in triplicate readings. The X-axis depicts the time in seconds. The Y-axis depicts the binding in nm.

Figure 4

Trastuzumab and Pertuzumab Cλ light chain isotype characterization. Size Exclusion Chromatography profiles of the Trastuzumab and Pertuzumab variants following affinity purification using Superdex size exclusion column on AKTA Pure. X-Axis: 40–100 ml scale. Y-axis: mAU aborption as determined by UV detection. The green lines on the X-axis marks the 70 ml mark which corresponds to ~150 kDa as determined by previous calibrations (see Supplementary Data). Red arrows depict the selected peak fractions of the antibodies used for subsequent analyses while the red horizontal line at the x-axis indicates the fractions collected. SDS-PAGE analyses of concentrated antibodies (reduced) are quantified and shown in the smaller inserts with corresponding band sizes for the light and heavy chains (for gel photos with ladder analysed with GelApp2.0, see Supplementary Data) using GelApp2.0. Graphs are representative of at least 3 independent batches of transfected cells.

Figure 5

Binding affinity measurements of Trastuzumab and Pertuzumab λ light chain isotype variants to Her2 at 100 nM to 6.25 nM concentrations to Trastuzumab or Pertuzumab variants pre-loaded on AHC biosensors. KD, Ka, and Kd were measured and calculated by the Octet QK^e system. All experiments were performed with at least three independent occasions in triplicate readings., graphs shown were representative of each variant. The X-axis depicts the time in seconds. The Y-axis depicts the binding in nm.