Generation of human IgG1 and IgG3 TA99 mAb with histidine-arginine rearrangements in Fc domains at amino acid position 435.

<p>Human IgG1 was mutated to contain an arginine at position 435 (IgG1 H435R), whereas the arginine at position 435 in human IgG3 was changed to histidine (IgG3 R435H). (<b>A</b>) Specific anti-human IgG1 or (<b>B</b>) anti-human IgG3 ELISA confirmed the correct isotype of mAbs. Staining of B16F10-gp75 with (<b>C</b>) different human TA99 mAb and (<b>D</b>) mouse TA99 IgG2a confirmed binding to surface gp75 and equal binding efficiency to gp75 of all human TA99 mAb. Concentration curves of human IgG1 and IgG3 mAb (<b>E</b>) and mouse IgG2a TA99 (<b>F</b>) on B16F10-gp75. Of note, scales of human (<b>E</b>) and mouse (<b>F</b>) antibodies are different.</p

Cytotoxicity assays using murine macrophages and B16F10-gp75 tumour cells.

<p>(<b>A</b>) Remaining B16F10-gp75 cells after a 24 hour incubation with macrophages and 2 μg/ml TA99 mAbs (different isotypes). (<b>B</b>) FACS analysis of co-cultures of DiO labelled murine macrophages (FL1) and DiI labelled B16F10-gp75 (FL2) tumour cells after 24 hours of treatment with 1 μg/ml mouse IgG2a or human IgG1 or IgG3 TA99 mAb. Macrophages, which have phagocytosed B16F10-gp75 tumour cells are encircled in FACS plots. (<b>C</b>) Percentage of remaining viable tumour cells and (<b>D</b>) increase in number of macrophages, which have phagocytosed B16F10-gp75 tumour cells after treatment of co-cultures with different concentrations of mAb. Percentages of tumour cells after culture with isotype antibodies were set at 100%. Double-positive macrophages were depicted relative to the co-cultures with isotype antibodies (set to 1), as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177736#pone.0177736.ref004" target="_blank">4</a>]. Mouse MG4 or human HEPC mAb were used as isotypes controls, which were set to 100%. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001.</p