Figure 32: Comparison of IC50 and DAR of Trastuzumab x MMAF ADCs on HCC1954 cell line.
Cytotoxic activity of Trastuzumab ADCs and FDCs conjugated with MMAF on HCC1954. Plotted are the obtained IC50 values against the DAR of the respective construct. Strong cytotoxicity is observed by cleaved ADC and FDC despite the low drug load of approximately 1.
Furthermore, analogous to the clevable antibodies and antibody fragments that were based on Trastuzumab, cell viability was also analyzed with Cetuximab-based ADCs conjugated to MMAE on EGFR-overexpressing cell lines. Cetuximab x MMAE ADCs displayed strong cytotoxicity on EGFR-positive A431 and MDA-MB468 cells, whereas no unspecific cell killing was observed on EGFR-negative MCF-7 cell line.
An overview of the IC50 values of Cetuximab x MMAE ADCs is shown in Appendix 28 and cell viability curves are depicted in Appendix 29.
As cytotoxicity of generated cleavable ADCs and FDCs is maintained, a suitable approach to investigate tumor distribution and penetration of solid tumors is necessary. For evaluation of tumor localization, antibody-fluorophore conjugates will be used for imaging studies.
separation of the Fc part with BHQ-10 from Fab-Alexa Fluor 488. All other formats carry the fluorophore on the light chains and an overview of antibody-fluorophore conjugates is shown in Table 5.
Figure 33: Structure of Trastuzumab CL N297A x AF488 x BHQ-10 using Alexa Fluor 488 as the fluorophore and BHQ-10 as a quencher.
Cleavable antibody-fluorophore conjugate Trastuzumab CL N297A x AF488 x BHQ-10 was generated by sortase A conjugation of Gly3 -C5-AF488 to the light chains of the antibody. BHQ-10 was conjugated to Q295 by transglutaminase reaction (A). The whole intact construct is cleaved in the hinge region, releasing the Fab fragments with the fluorophore attached to it (B).
For analysis of efficient fluorescence activation, measurements were performed with Trastuzumab CL x AF488 x BHQ-10 alone, with enzymes uPA and matriptase, as well as control samples. Control samples comprised Trastuzumab CL x BHQ-10 only with quencher conjugated as well as Trastuzumab natural x AF488. Prior to enzyme cleavage experiments, a calibration curve was carried out by measuring solely increasing AF488 concentrations. The obtained correlation coefficient R2 with 0.9383 was significant enough for analytical evaluation and indicated a proportional increase of the relative fluorescence units (RFU). For the cleavage of Trastuzumab CL x AF488 x BHQ-10 with matriptase a strong increase in RFU was observed from early time points with 320203 RFU to 511733 RFU at 24 h. Compared to the cleavage with matriptase, incubation with uPA resulted in a less increase of fluorescence intensity at 24 h with 382340 RFU (Figure 34).
These results displayed that fluorescence of the Trastuzumab CL x AF488 x BHQ-10 construct is increased upon efficient separation of the Fab fragment containing Alexa Fluor 488 from the Fc portion bearing the BHQ-10 quencher. Moreover, control samples Trastuzumab CL x AF488 x BHQ-10 as well as Trastuzumab natural x AF488 remained unchanged in their RFU over time.
Figure 34: Alexa Fluor 488 calibration and fluorescence intensity over time of different antibody-fluorophore conjugates.
Alexa Fluor 488 was analyzed for an increasing, proportional fluorescence with varying concentrations (A). Alexa Fluor 488 was conjugated to Trastuzumab CL N297A antibody with the quencher BHQ-10 and was incubated with tumor proteases uPA and matriptase. Increasing RFU were observed for both samples over time, indicating a sufficient separation of the Fab fragment with the fluorophore from the Fc portion bearing the quencher (B). Control samples Trastuzumab CL x AF488 x BHQ-10 (without enzyme), Trastuzumab CL x BHQ-10 (only with quencher) and Trastuzumab natural x AF488 (only with fluorophore) were used. Samples were measured in triplicates with EnVision multiplate reader. RFU = Relative Fluorescence Units, AF488 = Alexa Fluor 488, BHQ-10 = Black Hole Quencher-10
As the basic principle for fluorescence activation was confirmed, imaging experiments were carried out with tumor spheroids. Cancer cell lines HCC1954, SKBR-3 and MDA-MB-468 were used to form spheroids, as these cell lines express matriptase. Spheroid formation was checked with IncuCyte S3 Live-Cell Analysis System and was unfortunately only successful with HCC1954 at a cell density of 3000 cells/well, indicated by the presence of an extracellular matrix (Appendix 30). Spheroids had an average diameter size of approximately 300 µm and were grown within 48 h. HCC1954 spheroids were treated for 14 h with antibody-fluorophore conjugates Trastuzumab CL x AF488 x BHQ-10 (with and without matriptase), Trastuzumab nCL x AF488 and Trastuzumab Fab x AF488 to evaluate size-dependent tumor distribution and penetration in an in vitro model.
Live cell imaging was carried out over the time and green fluorescence was detected.
Figure 35: Spheroid distribution and penetration with antibody-fluorophore conjugates.
Depicted are HER2-overexpressing HCC1954 spheroids at a cell density of 3000 cells/well. Antibody-fluorophore conjugates were applied with a concentration of 6.25 nM and cells were imaged over time for 14 h. Trastuzumab CL x AF x BHQ-10 was applied without enzyme to ensure matriptase cleavage is achieved by tumor cells. Control samples that are applied as Fab fragments attached to Alexa Fluor 488 comprised Trastuzumab CL x AF488 x BHQ-10 + MT-SP1 and Trastuzumab Fab x AF488. Trastuzumab nCL x AF488 remains unchanged in the intact IgG form. Scale bar = 400 µm
Imaged time points were 2 h, 6 h, 10 h and 14 h and are illustrated in Figure 35. When comparing the results of Trastuzumab CL x AF488 x BHQ-10 (with and without matriptase) and Trastuzumab Fab x AF488, comparable green fluorescence is observed over the spheroid area at 14 h. This indicates that functional Trastuzumab CL x AF488 x BHQ-10 is cleaved by present tumor protease on tumor cell surface and the cleaved Fab-fluorophore conjugate distributes and penetrates evenly into the spheroid core, reaching almost all cancer cells. In contrast to that, Trastuzumab nCL x AF488 predominantly localizes at exterior fractions compared to the other tested constructs and is not able to reach the central region.
In another experiment with the fluorescence microscope EVOS FL Auto 2 Imaging System, the penetration of the conjugates with fluorophore along the z-stack was monitored and reveales that inside the tumor core a stronger fluorescence is observed for Trastuzumab CL x AF488 x BHQ-10 and Trastuzumab Fab x AF488.
Exemplarily images are shown in Appendix 31.
Taken together, results of cleaved Trastuzumab CL x AF488 x BHQ-10 as well as Trastuzumab Fab x AF488 showed that these constructs significantly better localize across the tumor spheroids and even within the tumor core, leading to an improved tumor distribution and penetration in vitro.
6. Discussion