Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.


CM IgA2J dimer preparation by size exclusion chromatography
. Preparation of CM IgA2J dimers by size exclusion chromatography. CM IgA2J dimers were generated in two subsequent preparative SECs by fractionating the right side of the dimer peak (vertical dotted lines). The first SEC was conducted three times to obtain sufficient material for the second SEC. Clean CM IgA2J dimers were generated as analyzed by additional analytical SEC (green). Figure A3. Specific antigen binding and ZR-75-1 target cell binding of hPM IgA antibodies were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. (a) Antigen ELISA using TA-MUC1 peptide (red) and non-glycosylated MUC1 peptide as negative control (black). hPM IgA1 specifically binds to the glycosylated TA-MUC1 peptide, less than 10% binding signal for the non-glycosylated peptide at the highest concentration tested. (b) Concentration-dependent binding of hPM IgA2 to ZR-75-1 target cells was shown as percent positive cells (left) and median fluorescence intensity (MFI, right). Mean values of duplicates are shown. One exemplary out of at least two independent experiments is shown.    Figure A7. IgA antibody-mediated activation of granulocytes and monocytes in the presence of target cells. Mean values of duplicates are shown. One exemplary out of two independent experiments is shown. Antibodies and target cells were added to whole blood to investigate the potential of IgA antibodies to mediate effector cell activation. Activation was investigated by measuring the induction of reactive oxygen species (ROS) by flow cytometry. Addition of hPM IgA1 antibody and ZR-75-1 target cells in whole blood resulted in activation of granulocytes and monocytes. With increasing target cell concentrations (indicated by black bar) more granulocytes and monocytes were activated. In the absence of antibody, granulocyte and monocyte activation was low. Granulocytes and monocytes were distinguished by flow cytometry with corresponding gates in forward versus sideward scatter plots. Phorbol 12-myristate 13-acetate (PMA) served as positive control for granulocyte and monocyte activation (+).

Enzyme-linked immunosorbent assays (ELISAs)
Antibodies specific for antibody isotypes, antigens or antibodies specific for a tagged receptor (see below) were coated overnight on Maxisorp 96-well plates (Nunc) at a concentration of 0.5 µ g/mL to 1 µ g/mL. Serial dilutions of antibody samples were prepared and isotype specific HRP-conjugated antibodies were used for detection. ELISAs were conducted according to standard protocols [S1].
IgA antibody concentrations in supernatants were determined using the human IgA ELISA Quantitation Set (Bethyl) according to the manufacturer's protocol.
In case of hPM, a synthetic TA-MUC1 glycosylated peptide was used as antigen. To confirm the mixed glycan-peptide epitope, a non-glycosylated peptide served as negative control. The assay was described previously by Danielczyk et al. [S2].
For FcαRI ELISA, after blocking the plate, each well was incubated with a constant concentration of His-tagged recombinant CD89 prior to sample incubation.
ELISAs were developed using TMB microwell substrate solution (Tebu-bio) and sulfuric acid was used to stop the reaction. For detection at 450 nm and 620 nm an Infinite F200 microplate reader (Tecan) was used.

Determination of the specific production rate
Clones were screened for antibody production per cell per day. Washed cells (4x10 4 cells/mL) were seeded in 0.5 mL medium without selection pressure in 24-well plates. After 3 or 4 days incubation, the volume of the cell suspension was measured, cells were counted and supernatant samples were taken for IgA quantification ELISA (Bethyl). The specific production rate was calculated under the assumption of exponential growth during the 3 or 4 day incubation time using the following formula: Total response: AB + AB2 + AB3 + AB4 + RI

Respiratory burst assay
The Phagoburst kit (Glycotope Biotechnology, Heidelberg, Germany) was used for the respiratory bust assay. The manufacturer's protocol was adapted by the addition of target cells to investigate the dependency on target cells for activation of granulocytes and monocytes.