Internalization

After binding to the cell surface of a target cell, ADCs or ITs have to be internalized to release their cytotoxic payload and kill the cell. The internalization has already been shown for trastuzumab upon HER2 binding and is well documented.²¹⁸ However, no data are available for the bivalent VHH-Fc fusion. Therefore, A549 cells also overexpressing HER1 were treated with 100 nM 7D9G-Fc-Int^N and 7D9G-Gelonin and incubated for 1 h at either 4 °C or 37 °C. At low temperatures the membrane dynamics are inhibited and antibodies are not internalized but bind only to the surface. The staining antibody was later quenched with an α–Alexa488 antibody.

A549 cells incubated at 4 °C show a significantly reduced fluorescence when the surface bound antibodies were quenched, while no reduction was observed for cells incubated at 37 °C (Figure 41 top).

Notice that only the quenched samples are presented for A549 at 37 °C although the colors are depicted for not quenched samples. This clearly shows that the 7D9G antibody shows very good internalization characteristics and confirms the suitability for IT treatment.

Figure 40: Binding of ITs to receptor overexpressing cells is not altered compared to the parental Abs.

MDA-MB-468 cells (HER1⁺) and SK-BR-3 cells (HER2⁺) cells were titrated with the 7D9G and the T4 based ITs, respectively. Binding characteristic and strength were not altered when toxins were attached. Affinities were calculated by sigmoidal curve fitting using GraphPad Prism software (GraphPad Software, Inc.).²⁰²

Results and Discussion 75 Figure 41: Internalization of 7D9G-Gelonin in A549 cells.

Cells displaying the HER1 receptor on their cell surface (A549) and control cells without HER1 (CHO) were incubated with 7D9G-Gelonin and 7D9G-Fc-Int^N at 4 °C and 37 °C, stained with α–hIgG-Fab Alexa488 secondary antibody and analyzed by flow cytometry. Histograms show the relative fluorescence units (RFU) of the detection antibody as measure of receptor binding on a logarithmic scale. In some samples (light colors), surface bound antibodies were quenched with an α–Alexa488 antibody. Note that the green and blue curves in the 37 °C A549 histogram display the quenched samples.

CHO control cells not displaying EGFR on their surface confirmed specific binding to the receptor. Only a slight shift was observed in the 37 °C samples which could also account for unspecific adherence of the secondary antibody to the cell surface.

To investigate the internalization and the time-dependent release of the toxins inside the cell, confocal microscopy was applied. Cells were treated with ITs for one or four hours and the respective route of the IgG and the toxin were followed by staining them with specific antibodies. All surface bound antibodies were removed by stripping them with a low pH washing step. This made sure that the signals obtained during microscopy accounts only for internalized antibodies. The latter were visualized using the Alexa488 labeled -hIgG Fab fragment and the toxins were stained with an Alexa647 labelled -His6

antibody. HER1 positive MDA-MB-468 cells showed endocytic uptake of 7D9G-Gelonin and 7D9G-PE24 after 1h of incubation, marked by a punctate pattern in the 488-channel inside the cell (Figure 42).

Additionally, after 1 h, signals for the antibody and the toxin colocalize for both the gelonin and PE24 IT. After four hours, however, the toxin fluorescence of the PE24 IT is clearly reduced in the vesicles.

Gelonin still seems to be attached to the antibody at this time point as a high degree of colocalization of

Results and Discussion 76 antibody and gelonin is observed. This may be explained by the retrograde transport mechanism of PE24²¹⁹ that may result in a more efficient and faster transport to the cytosol than gelonin. CHO cells confirmed that the VHH did not bind unspecifically and no fluorescence could be detected for both antibody and toxin.

Figure 42: Internalization of 7D9G-ITs and endosomal release of toxins.

MDA-MB-468 (HER1 overexpressing) cells were treated for 1h with 7D9G ITs, washed with low pH glycine buffer to remove surface bound conjugates and stained directly or incubated for another 3h before staining. CHO cells were used as negative cell line. Anti-hIgG Fab-Alexa 488 (1:500) and anti-His6 Alexa 647 antibody (1:1000) were used to specifically visualize the antibody fraction and the toxin fraction of the ITs, respectively. Scale bar = 10 µm.²⁰²

Internalization was also confirmed for the trastuzumab ITs on SK-BR-3 cells (Figure 43). The overall staining intensitites for the Alexa488 and Alexa647 signals were strongly decreased compared those of MDA-MB-469 stained with the VHH ITs. A possible explanation was a different permeabilization of SK-BR-3 cells by triton X-100 which could lead to a decreased penetration and binding of the secondary antibodies. The signal/noise ratio was thus strongly decreased. Binding and internalization could nevertheless be observed and was characterized by a punctuate pattern. Colocalization of toxin and Ab was observed for both 1 and 4 h timepoints. No cytosolic delivery of PE24 could be verified in this cell line with trastuzumab. Since every tumor cell line has different characteristics that also affect organelle dynamics it is possible that maturation of endosomes to late endosomes and lysosomes is slower and also lysosomal proteases like furin occur at a later timepoint. Longer monitoring of the trafficking and specific staining of furin could elucidate if this was the case in SK-BR-3 compared to MDA-MB-468 cells.

Results and Discussion 77 Figure 43: Internalization of trastuzumab ITs and endosomal release of toxins.

SK-BR-3 (HER2 overexpressing) cells were treated for 1h with trastuzumab ITs, washed with low pH glycine buffer to remove surface bound conjugates and stained directly or incubated for another 3h before staining. CHO cells were used as negative cell line. α-hIgG Fab-Alexa 488 (1:500) and α-His6 Alexa 647 antibody (1:1000) were used to specifically visualize the antibody fraction and the toxin fraction of the ITs, respectively. Scale bar = 10 µm.

Unfortunately, unspecific binding of the trastuzumab ITs was observed to the HER2 negative cell line MDA-MB-468 in two independent experiments. In the first one, gelonin was initially marked with the fluorescent tag TAMRA in a SrtA reaction and then ligated to T4 by PTS. When incubated with SK-BR-3 cells, both the WT and the conjugate showed uptake into the cells after 1 h and TAMRA staining confirmed co-localization of the toxin and the Ab (Figure S 3). Unspecific binding of T4-Gelonin-TAMRA was observed, however, on MDA-MB-468 cells. This was confirmed in a second experiment, when the toxin was not labelled with TAMRA but visualized with the α–His6 Alexa647 antibody. The same picture showed that the gelonin conjugate was internalized into the HER2 negative cell line (Figure S 4). On CHO cells, no unspecific binding was detected, though. The phenomenon should be investigated in more detail on more cell lines and with the solitary toxin to see if it binds to the cell surfaces of some cells by its own.²⁰²