Figure 25: Comparison of relative internalization of produced antibodies and antibody fragments measured by flow cytometry.
Cellular internalization assay using flow cytometry analysis was carried out by incubation of cancer cell lines MDA-MB-468, MCF-7 and SKBR-3 with antibodies at 10 µg/ml. After incubation, secondary antibody goat anti-human IgG (H+L) Fab Fragment, Alexa Fluor 488 was added and cells were either incubated for 1 h and 48 h at 37°C allowing internalization or at 4°C preventing internalization. Cell surface bound Alexa Fluor 488-labeled antibodies was quenched with an anti-Alexa Fluor® 488 Rabbit IgG Antibody. Percentage of internalization was calculated by comparing MFI of cells incubated at 37°C and those at 4°C. Tmab = Trastuzumab
Table 7: Overview of internalization rates in percentage [%] after 1 h and 48 h incubation.
Different antibody formats were analyzed for internalization rates by flow cytometry. Cancer cell lines MCF-7, SKBR-3 and MDA-MB-468 (HER2-negative) were tested and MDA-MDA-MB-468 is not listed due to no internalization.
MCF-7 SKBR-3
Antibody construct Target 1 h 48 h 1 h 48 h
Herceptin HER2 17 39 5 52
Trastuzumab CL HER2 9 36 1 45
Trastuzumab nCL HER2 13 37 0 44
Trastuzumab natural HER2 13 35 6 40
Trastuzumab Fab His HER2 12 38 3 44
Trastuzumab oa SEED HER2 15 34 0 38
Figure 26: Thermal stability of Trastuzumab-based antibody variants and ADCs measured by nanoDSF.
Melting temperatures (Tm) in °C of Trastuzumab glycosylated antibodies (A), ADCs (B) and deglycosylated antibodies (C) were measured with nanoDSF for thermal unfolding using 15 µl of protein solution per capillary. Values were calculated as means and samples were measured in duplicates.
Values of deglycosylated antibodies are approximately 10°C lower compared to glycosylated IgGs and 60°C is considered as critical Tm for therapeutic antibodies. An overview of the melting temperatures of tested constructs is given in Table 8.
In conclusion, thermal unfolding experiments exhibited no alteration of thermal stability of antibodies as well as ADCs due to modified hinge region. Antibodies with N297A mutation bearing no glycosylation show uniform results with melting temperature 10°C lower than conventional glycosylated antibodies Moreover, sortase toxin-conjugation has also no impact on biophysical properties of ADCs.
Table 8: Overview of melting temperatures (Tm) of antibodies and ADCs.
Listed are melting temperature values of the CH2 domain (first derivate) and Fab domain of unconjugated and deglycosylated antibodies as well as ADCs.
Antibody Tm [°C]
(CH2 domain)
Tm [°C]
(Fab domain)
Herceptin 71,2 81,7
Trastuzumab CL 69,9 79,5
Trastuzumab nCL 70,1 79,8
Trastuzumab natural 69,7 79,5
Trastuzumab oa SEED 67,2 79,5
Trastuzumab Fab His - 80,7
Trastuzumab CL x MMAE 69,0 79,9
Trastuzumab nCL x MMAE 68,9 79,7
Trastuzumab natural x MMAE 68,6 79,7
Trastuzumab Fab His x MMAE - 80,2
Trastuzumab oa SEED x MMAE 66,7 79,6
Kadcyla 65,5 80,9
Trastuzumab CL HC N297A 61,3 80,2
Trastuzumab nCL HC N297A 61,1 80,3
Trastuzumab natural HC N297A 61,4 80,4
Trastuzumab oa SEED HC N297A RF 63,3 79,7
As a next step, the stability of ADC constructs in mouse and human serum will be evaluated.
5.3.1. In vitro mouse and human serum stability of ADCs
Trastuzumab x MMAE constructs were incubated in mouse or human serum at 37°C, 5% CO2 for 96 h to evaluate stability in these matrices due to concerns regarding the stability of the hinge region. Thus, investigations of the ADC constructs in serum from species that are relevant for preclinical testing was achieved.
Sampling time points were 0 h, 2 h, 4 h, 6 h, 24 h, 48 h, 72 h and 96 h. The concentration of ADCs used for incubation was 0.5 mg/ml. The stability of the total antibody was analyzed by ELISA by coating HER2 murine Fc-His and detection with goat-anti human F(ab’)2 x POD.
As a reference value the first time point at 0 h was used and set as 100% at the beginning. Illustrated are the serum stability time curves of the different ADCs as percentage of concentration remaining (Figure 27). The concentration profiles of Trastuzumab CL x MMAE and Trastuzumab nCL x MMAE remained almost constant for the duration of the experiment and show similar results. Therefore, the stability of the ADCs in mouse and human serum indicate that no hinge cleavage is expected.
For mouse serum analysis, nearly all constructs exhibited high stability and stable concentration profiles ranged from 90 to 100 % at 96 h. Apart from that, human serum stability revealed slightly less concentration for IgG-based Trastuzumab CL x MMAE, Trastuzumab nCL x MMAE and Trastuzuman oa SEED x MMAE with values from 78 to 95%, whereas Trastuzumab Fab His x MMAE exhibited a more pronounced decrease in concentration of 54% only in human serum.
Figure 27: Mouse and human serum stability analysis of Trastuzumab x MMAE ADCs.
Samples were incubated in mouse serum (A) or human serum (B) at 37°C, 5% CO2 for 96 h. HER2, murine Fc-His was coated in microtiter plates and detection was achieved with goat-anti human F(ab’)2 x POD antibody. Aliquots were removed at different time points and concentration analysis was performed by ELISA. Depicted are total antibody concentration profiles as triplicates over time.
An overview of the remaining total antibody concentration in percentage of the tested constructs at 96 h is shown in Table 9 and terminal drug load as well as time curves of MMAE for mouse and human serum are shown in Appendix 22 and 23. For drug load experiments, all constructs (except Trastuzumab Fab His x MMAE) remained unchanged in human serum over time and payload loss was not detected by LC-MS. Unlike mouse serum stability, all constructs display MMAE toxin release that is contributed to the well known activity of carboxylesterase 1c, exclusively present in rodent species.
Table 9: Overview of percentage of total antibody concentration in percentage of Trastuzumab MMAE ADCs.
Samples were incubated in mouse or human serum and remaining total antibody concentrations at 96 h are listed.
ADC [%] remaining at 96 h
in mouse serum
[%] remaining at 96 h in human serum
Trastuzumab CL x MMAE 90 95
Trastuzumab nCL x MMAE 100 78
Trastuzumab Fab His x MMAE 100 54
Trastuzumab oa SEED x MMAE 99 88
Taken together, Trastuzumab Fab His x MMAE tended to be less stable in human serum, but stability in mouse serum was similar to all other constructs for total antibody analysis. IgG-based ADCs showed high total antibody concentrations in mouse and human serum, indicating appropriate stability of these constructs that is necessary for exploiting long half-life of antibody molecules.