Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineered calcium-regulated affinity protein for efficient internalization and lysosomal toxin delivery

doi: 10.1073/pnas.2509081122

Figure Lengend Snippet: Flow cytometric binding analysis of the calcium-dependent interaction between CaRA EGFR and various cancer cell lines. The conditional binding of CaRA EGFR was evaluated on cancer cell lines with high (A-431), intermediate (BxPC-3), and low (Ramos) EGFR-expression. ( A ) For the high EGFR-expressing A-431 cells, a shift in binding signal could be observed for the triplicates of 500 nM CaRA EGFR in the presence of calcium (burgundy) but not in the calcium-free experiment (blue) where the latter is overlapping with the control samples consisting of untreated cells (gray) which have been incubated in the respective buffers either with or without calcium. From the measured median fluorescent intensity of averaged biologically replicate samples with calculated variance shown as error bars, an on-cell affinity curve was graphed for a concentration series of CaRA EGFR ranging from 7.62 pM to 500 nM toward the cancer-derived cell lines with varying EGFR-expression; ( B ) high (A-431), ( C ) intermediate (BxPC-3), or ( D ) low (Ramos). The estimated affinity to the endogenous EGF receptor resulted in around 7 nM. ( E ) The measured median fluorescent intensity for the highest CaRA EGFR concentration tested (500 nM) was plotted for the various cell lines and clearly shows that the obtained signals vary with the described EGFR-expression ( SI Appendix , Table S2 ), indicating a specific target interaction.

Article Snippet: When conjugating the nonconditionally binding Affibody molecule targeting EGFR ( , ) to the same toxin, DM1, it does also not show any cytotoxic effect on cells with low EGFR-expression ( SI Appendix , Fig. S12 ), but when applied to the high EGFR-expressing cells the nonconditionally binding Affibody molecule notably results in a higher IC50 in comparison to CaRA EGFR since it cannot be securely determined within the same concentration interval ( SI Appendix , Fig. S12 ).

Techniques: Binding Assay, Expressing, Control, Incubation, Concentration Assay, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineered calcium-regulated affinity protein for efficient internalization and lysosomal toxin delivery

doi: 10.1073/pnas.2509081122

Figure Lengend Snippet: Cellular localization of CaRA EGFR or Z EGFR:2377 and the targeted EGF receptor and EGFR expression levels after incubation with binders. ( A and B ) A decoupled biological fate is shown for CaRA EGFR and EGFR. For CaRA EGFR , a high intracellular retention is shown compared to the nonconditionally binding Z EGFR:2377 control. The Alexa488 fluorescently labeled CaRA EGFR or Z EGFR:2377 (green) was added to EGFR-expressing A-431 cells and incubated at 37 °C for 15 min or 60 min prior to fixation of the cells and subsequent labeling of EGFR (red, Alexa647) and the cell nuclei (blue, DAPI). In the 15 min time frame, CaRA EGFR -Alexa488 shows colocalization with the Alexa647-labeled EGFR on the membrane as does the nonconditionally interacting Z EGFR:2377 . Internalized CaRA EGFR is already detectable after 15 min of incubation but becomes more pronounced in the 60 min samples, where numerous cells display distinct green vesicles near the nuclei. These vesicles are clearly separated from the red-labeled EGFR, which remains predominantly at the cell surface. In both the 15 min and 60 min time frame, Z EGFR:2377 -Alexa488 shows colocalization with the Alexa647-labeled EGFR on the membrane. When visualizing the colocalization of Alexa647-labeled EGFR and Alexa488-labeled Z EGFR:2377 , the signals largely overlap, indicating strong colocalization between the high-affinity binder and its target receptor even after 60 min. In contrast, internalized CaRA EGFR is clearly visible after 60 min and appears as a distinct population from the labeled EGFR, shown in red in the colocalization graph. In the 2D histograms the red EGFR signal is shown on the x-axis and green CaRA EGFR signal on the y-axis is shown. The colocalization plot shows that Z EGFR:2377 colocalizes with after 60 min, also shown in yellow in the cell image. However, a discrete localization of binder without EGFR is shown for CaRA EGFR after 60 min. This is also visible as solid green vesicle-like structures in the cell image. ( C ) Quantitative flow cytometry of NCI-H292 cells incubated for 15 min to 24 h with EGF, BTC, CaRA EGFR , Z EGFR:2377 , or Cetuximab shows that CaRA EGFR does not downregulate EGFR surface expression within the tested time frame, whereas clear downregulation is observed for the natural ligands, Z EGFR:2377 , and Cetuximab. Data are shown as mean out of triplicates.

Article Snippet: When conjugating the nonconditionally binding Affibody molecule targeting EGFR ( , ) to the same toxin, DM1, it does also not show any cytotoxic effect on cells with low EGFR-expression ( SI Appendix , Fig. S12 ), but when applied to the high EGFR-expressing cells the nonconditionally binding Affibody molecule notably results in a higher IC50 in comparison to CaRA EGFR since it cannot be securely determined within the same concentration interval ( SI Appendix , Fig. S12 ).

Techniques: Expressing, Incubation, Binding Assay, Control, Labeling, Membrane, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineered calcium-regulated affinity protein for efficient internalization and lysosomal toxin delivery

doi: 10.1073/pnas.2509081122

Figure Lengend Snippet: Confocal microscopy images of A-431 cells expressing EGFR exposed to CaRAEGFR show lysosomal accumulation and in vitro cytotoxicity assay results in an IC50 of 0.8 nM. (A-F) Alexa488 fluorescently labeled CaRA EGFR is shown in green, lysosome maker in blue, and colocalization appears as turquoise. ( A – C ) A-431 cells incubated at 37 °C with Alexa488 fluorescently labeled CaRA EGFR for 0 min, 1 h, and 2 h. ( D – F ) A-431 cells incubated at room temperature with Alexa488 fluorescently labeled CaRA EGFR for 10 min before washout and increased temperature to 37 °C to initiate internalization of the EGFR. Confocal micrographs from the center of the cells were captured after 30 min ( D ), 2 h ( E ), and 18 h ( F ). CaRA EGFR is initially localized at the membrane, where EGFR is commonly expressed. The images show colocalization of signals of CaRA EGFR and the lysosome dye as early as after 30 min, with a following time-dependent increase ( G ). ( H ) The cytotoxicity of CaRA EGFR conjugated to the toxin DM1 was determined by incubating serial dilutions of the conjugate or controls with the cell line A-431 as indicated. Ztaq-DM1 was used as a nontargeting control conjugated to DM1 and CaRA EGFR is the targeting molecule without the cytotoxin. The cell viability was measured after 48 h. The viability of untreated cells in complete medium was used for normalization to 100% viability. The data points represent the average of triplicates in the same assay with error bars showing the SD. The IC50 of 0.8 ± 0.3 nM was calculated from three independent experiments.

Article Snippet: When conjugating the nonconditionally binding Affibody molecule targeting EGFR ( , ) to the same toxin, DM1, it does also not show any cytotoxic effect on cells with low EGFR-expression ( SI Appendix , Fig. S12 ), but when applied to the high EGFR-expressing cells the nonconditionally binding Affibody molecule notably results in a higher IC50 in comparison to CaRA EGFR since it cannot be securely determined within the same concentration interval ( SI Appendix , Fig. S12 ).

Techniques: Confocal Microscopy, Expressing, In Vitro, Cytotoxicity Assay, Labeling, Incubation, Membrane, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineered calcium-regulated affinity protein for efficient internalization and lysosomal toxin delivery

doi: 10.1073/pnas.2509081122

Figure Lengend Snippet: Calcium-dependent binding analysis of CaRA EGFR with the recombinant EGFR. ( A ) Biolayer interferometry was used to assess the binding to the target EGFR in solution (twofold dilution series from 2,000 nM to 63 nM) by capturing CaRA EGFR -ABD on HSA-immobilized sensors. Binding kinetics was evaluated in buffer supplemented with 1 mM calcium at pH 7.4. Kinetic fits to a 1:1 binding model are shown in black. Averages of the association rate constant k a , the dissociation rate constant k d , and the dissociation equilibrium constant K D resulted in k a = 5.8 * 10 3 M −1 s −1 , k d = 1.0 * 10 −4 s −1 and a K D of 20 nM. ( B ) Evaluation of calcium-dependent release of the target EGFR (at 500 nM) in 1 mM calcium and dissociation in either 1 mM calcium (burgundy) or 1 μM calcium (beige). ( C ) The calcium affinity was estimated by conducting the same experiment as in Fig. 2 B with associating to EGFR in 1 mM calcium and dissociating in different calcium concentrations ranging from 1 mM to 0.48 μM. The response change after 30 s dissociation was normalized against the maximum binding response for the individual curves at the different release conditions. The data were fitted to a sigmoidal curve, from which the calcium affinity can be estimated to be around 6.4 μM. ( D ) Thermal stability of CaRA EGFR was quantified with CD spectroscopy at 221 nm by exposing the protein to a temperature gradient from 4 °C to 95 °C. The experiment was performed in a buffer supplemented with various calcium concentrations (1 mM, 500 μM, 250 μM, 50 μM, and 1 μM) at pH 7.4. The melting temperature increased by 10 °C following an increase in calcium concentration from 1 μM (beige) to 1 mM (burgundy), highlighting the secondary structure’s dependence on calcium.

Article Snippet: When conjugating the nonconditionally binding Affibody molecule targeting EGFR ( , ) to the same toxin, DM1, it does also not show any cytotoxic effect on cells with low EGFR-expression ( SI Appendix , Fig. S12 ), but when applied to the high EGFR-expressing cells the nonconditionally binding Affibody molecule notably results in a higher IC50 in comparison to CaRA EGFR since it cannot be securely determined within the same concentration interval ( SI Appendix , Fig. S12 ).

Techniques: Binding Assay, Recombinant, Circular Dichroism, Concentration Assay

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: As shown in , , and binding of Cetuximab blocks binding of Z EGFR 1907’ affibody to sEGFR 1–621.

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: SDS-PAGE analysis showing the purity of the proteins used for the Biacore analyses. Expected molecular weights: sEGFR 1–501 56kDA, sEGFR 1–621 69kDa, Z EGFR 1907’ 11kDa and Cetuximab 152kDa. (B) Binding kinetics for the Z EGFR 1907’ and Cetuximab interacting with sEGFR 1–501 and sEGFR 1–621 , used to determine K d values using Biacore S200. (C) Competitive binding kinetics of Z EGFR 1907’ and Cetuximab detected under various conditions shows Cetuximab and Z EGFR 1907’ appear to bind to the same EGFR binding site. Solid and dotted line graphs represent raw and fitted data respectively.

Article Snippet: As shown in , , and binding of Cetuximab blocks binding of Z EGFR 1907’ affibody to sEGFR 1–621.

Techniques: SDS Page, Binding Assay