Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding

Abstract

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.

Keywords: FcγIIA; KD; Pertuzumab; Trastuzumab; VH–VL families; complementary determining region grafting; recombinant antibody production; therapeutic antibodies.

Figure 1

An illustration of the possible permutations of all the Vκ (1–6) and VH (1–7) families with framework regions sequences used in this study. *Denotes the complementarity determining regions (CDRs). For fully grafted sequences with CDRs, refer to Data S1 in Supplementary Material.

Figure 2

The multiple sequence alignment of known commercial antibodies s used for determining the light (top) and heavy chain (middle) complementarity determining regions (CDRs). An example showing Vκ3 framework region determination is as shown (bottom). For the full list, refer to Data S2–S16 in Supplementary Material.

Figure 3

Bubble chart representations of the production of recombinant Pertuzumab antibody variants. The production levels are directly proportional to the area of the circle. The columns show the Vκ family while the rows show the VH families. Each circle in the respective combination shows one of the three plasmid ratio transfections (red, 1:1; purple, 1:3; and green, 3:1) while black solid circles show the original pairings. All circles are benchmarked against the black solid circle which is at 100%. A table of the production rates is also shown in Figure S1 in Supplementary Material.

Figure 4

Bubble chart representations of the production of recombinant Trastuzumab antibody variants. The production levels are directly proportional to the area of the circle. The columns show the Vκ family while the rows show the VH families. Each circle in the respective combination shows one of the three plasmid ratio transfections (red, 1:1; purple, 1:3; and green, 3:1) while black solid circles show the original pairing. All circles are benchmarked against the black solid circle which is at 100%. A table of the production rates is also shown in Figure S2 in Supplementary Material.

Figure 5

The binding kinetics of recombinant Pertuzumab to Her2 and FcγIIa. The recombinant antibody variants are arranged according to VH families and color coded for Vκ (Vκ1—red, Vκ2—green, Vκ3—purple, Vκ4—blue, Vκ5—brown, and Vκ6—orange) while the original pairing is bolded with K_D, K_on, and K_off measurements shown. “Poor response” indicates that the antibody combinations did not yield reliable K_on and K_off during measurements (in triplicates) as determined by the Octet software. “Not produced” indicates that there was no useable amount of the purified product despite several transfection attempts.

Figure 6

The binding kinetics of recombinant Trastuzumab to Her2 and FcγIIa. The recombinant antibody variants are arranged according to VH families and color coded for Vκ (Vκ1—red, Vκ2—green, Vκ3—purple, Vκ4—blue, Vκ5—brown, and Vκ6—orange) while the original pairing is bolded with K_D, K_on, and K_off. “Poor response” indicates that the antibody combinations did not yield reliable K_on and K_off during measurements (in triplicates) as determined by the Octet software. “Not produced” indicates that there was no useable amount of the purified product despite several transfection attempts.