Site‐Specific Encoding of Photoactivity in Antibodies Enables Light‐Mediated Antibody–Antigen Binding on Live Cells

Abstract Antibodies have found applications in several fields, including, medicine, diagnostics, and nanotechnology, yet methods to modulate antibody–antigen binding using an external agent remain limited. Here, we have developed photoactive antibody fragments by genetic site‐specific replacement of single tyrosine residues with photocaged tyrosine, in an antibody fragment, 7D12. A simple and robust assay is adopted to evaluate the light‐mediated binding of 7D12 mutants to its target, epidermal growth factor receptor (EGFR), on the surface of cancer cells. Presence of photocaged tyrosine reduces 7D12‐EGFR binding affinity by over 20‐fold in two out of three 7D12 mutants studied, and binding is restored upon exposure to 365 nm light. Molecular dynamics simulations explain the difference in effect of photocaging on 7D12‐EGFR interaction among the mutants. Finally, we demonstrate the application of photoactive antibodies in delivering fluorophores to EGFR‐positive live cancer cells in a light‐dependent manner.

Construction of pSANG10_7D12-32TAG, pSANG10_7D12-109TAG, and pSANG10_7D12-113TAG plasmids: For construction of pSANG10_7D12-32TAG mutant, the pSANG10_7D12 plasmid was digested with XbaI and SalI. For construction of pSANG10_7D12-109TAG and pSANG10_7D12-113TAG mutants, the pSANG10_7D12 plasmid was digested with SalI and HindIII. After digestion, the plasmid backbone was purified on 1% agarose and the DNA extracted using QIAquick Gel Extraction Kit (QIAGEN). The mutations in 7D12 were introduced by cloning short gene fragments (IDT) with the corresponding mutations into the digested plasmids. Gibson cloning (NEB) was used for this purpose. After cloning the sequence of mutated plasmids was confirmed by Sanger Sequencing.

Construction of pULTRA_PylRS/PyltRNACUA plasmid:
In order to construct pULTRA_PylRS/PyltRNACUA plasmid, pULTRA_MjCNFRS/MjtRNACUA plasmid was digested with PstI and XhoI (New England Biolabs). After digestion, the reaction mixture was run on 1% agarose gel. The band corresponding to pULTRA_MjCNFRS backbone (without tRNA and flanking region) was excised and extracted using QIAquick Gel Extraction Kit (QIAGEN). The PyltRNACUA having the same promoter and terminator as for MjtRNACUA (procured as gene fragment from Integrated DNA Technologies (IDT)) was subsequently cloned into the pULTRA_MjCNFRS backbone using Gibson cloning (New England Biolabs). After cloning the sequence of pULTRA_MjCNFRS/PyltRNACUA was confirmed by Sanger Sequencing. In order to replace MjCNFRS in pULTRA_MjCNFRS/PyltRNACUA, the plasmid was digested with NotI (New England Biolabs). PylRS was PCR amplified from another high copy plasmid. PCR product and the digested plasmid were run on 1% agarose gel, the corresponding bands excised and DNA extracted using QIAquick Gel Extraction Kit (QIAGEN). PylRS (PCR product) was cloned into the pULTRA_ PyltRNACUA backbone using Gibson cloning (New England Biolabs). After cloning the sequence of pULTRA_PylRS/PyltRNACUA was confirmed by Sanger Sequencing.

Expression of wt7D12:
For periplasmic expression of wt7D12, pSANG10_7D12 plasmid was transformed into BL21(DE3)pLysS chemically competent cells. After transformation, cells were recovered in 1 ml SOB medium for one hour at 37°C. 50 µl of recovered cells were transferred onto LB-agar plates supplemented with 50 µg ml -1 kanamycin. The plates were incubated overnight (37°C, 16 h). A single colony from this plate was used to inoculate 10 ml of 2xTY-GK media (2xTY media with 4% glucose, 50 µg ml -1 kanamycin) and incubated overnight (37°C, 220rpm, 16 h). Next day, this culture was used to subculture 500 ml 2xTY-GK, such that OD600= 0.1. This was then incubated until OD600 reached 0.4-0.6 (37°C, 220rpm, 2-3 h). IPTG (1 mM final concentration) was added to induce the expression of 7D12 and the culture was incubated overnight (30°C, 160rpm, 16 h). Next day, cells were pelleted (3,200g, 4°C, 10 minutes). The supernatant was discarded and the cells were resuspended in 25 ml periplasmic extraction buffer-1 (20% sucrose, 100 mM Tris-HCl, 1 mM EDTA, pH 8.0). The resuspended cells were incubated on ice for 30 minutes and then centrifuged (10,000g, 4°C, 10 minutes). The supernatant was removed and stored at 4°C-this is "periplasmic fraction-1". The resulting pellet was resuspended in 25 ml periplasmic extraction buffer-2 (5 mM MgCl2) and incubated on ice for 20 minutes. The samples were centrifuged (10,000g, 4°C, 10 minutes) and the supernatant was collected-this is "periplasmic fraction-2". Both the periplasmic fractions were combined, passed through a 0.2 μm filter, and dialyzed overnight at 4°C against 1x phosphate buffered saline (PBS). For purification of 7D12 antibody fragment from the periplasmic mixture, 1 ml of Ni-NTA resin was added to the dialyzed fractions and mixed gently on a rocker (4°C, 1 h). This was transferred into a gravity-flow column and washed with 10 ml PBS buffer. Resin was then washed twice with 8 ml Ni-NTA wash buffer (50 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole, pH 8.0). To elute the bound 7D12, 500 µL Ni-NTA elution buffer (50 mM Tris-HCl, 300 mM NaCl and 200 mM imidazole, pH 8.0) was added and incubated for 15 minutes at room temperature, this was repeated 8 times. Elution fractions were pooled and dialyzed overnight at 4°C against 1xPBS buffer. Dialyzed fractions were then concentrated by Vivaspin 500 columns with 3 kDa molecular weight cut-off (GE Healthcare) and the yields were determined using a calorimetric Pierce BCA protein assay (ThermoFisher Scientific) measured at 562 nm. The samples were subsequently analysed by SDS-PAGE. Nu-PAGE LDS loading buffer (Invitrogen) was added to samples and heated to 95°C for 15 minutes, then centrifuged (13,000 g, 15 minutes, 4°C) and loaded on a 4-12% Bis-Tris gel (Invitrogen) along with BIO-RAD pre-stained protein ladder (Precision Plus All Blue Protein Standards) as a marker. The gel was then stained with Coomassie Blue (InstantBlue, Expedeon) ( Figure S2a) and the identity of the protein was further confirmed by electrospray ionization mass spectrometry coupled with liquid chromatography, LC-ESI-MS ( Figure S2b). The molecular weights observed on SDS-PAGE gel and mass spectrometry were in good agreement with expected molecular weight of 7D12.

Expression and purification of amber mutants of 7D12 with site-specifically incorporated photocaged tyrosine (pcY):
Chemically competent BL21(DE3)pLysS cells containing pULTRA_MjpcYRS/MjtRNACUA were transformed with pSANG10_7D12, pSANG10_7D12-32TAG, pSANG10_7D12-109TAG, or pSANG10_7D12-113TAG plasmids. After transformation, cells were recovered in 1 ml SOB medium for one hour at 37°C. 50 µl of recovered cells were transferred onto LB-agar plates supplemented with 50 µg ml -1 kanamycin and 75 µg ml -1 spectinomycin. The plates were incubated overnight (37°C, 16 h). A single colony from each plate was used to inoculate 50 ml of 2xTY-GKS media (2xTY media with 4% glucose, 50 µg ml -1 kanamycin and 75 µg ml -1 spectinomycin) and incubated overnight (37°C, 220rpm, 16 h). Next day, this culture was used to subculture 500 ml 2xTY-GKS media, such that OD600= 0.1. This was then incubated until OD600 reached 0.4-0.6 (37°C, 220rpm, 2-3 h). IPTG (1 mM final concentration) was added to induce the expression of 7D12 and the culture was supplemented with 4 mM photocaged tyrosine (pcY) for expression with unnatural amino acid (positive samples in Figure 1). The culture was incubated overnight (30°C, 160rpm, 16 h). Next day, cells were pelleted (3,200g, 4°C, 10 minutes). The supernatant was discarded and the cells were resuspended in 25 ml periplasmic extraction buffer 1 (20% sucrose, 100 mM Tris-HCl, 1 mM EDTA, pH 8.0). The resuspended cells were incubated on ice for 30 minutes and then centrifuged (10,000g, 4°C, 10 minutes). The supernatant was removed and stored at 4°C-this is "periplasmic fraction-1". The resulting pellet was resuspended in 25 ml periplasmic extraction buffer 2 (5 mM MgCl2) and incubated on ice for 20 minutes. The samples were centrifuged (10,000g, 4°C, 10 minutes) and the supernatant was collected-this is "periplasmic fraction-2". Both the periplasmic fractions were combined, passed through a 0.2-μm filter, and dialyzed overnight at 4°C against 1x phosphate buffered saline (PBS). 1 ml of Ni-NTA resin was added to the dialyzed solution and mixed gently on a rocker (4°C, 1 h). This was transferred into a gravity-flow column and washed with 10 ml PBS buffer. Resin was then washed twice with 8 ml Ni-NTA wash buffer (50 mM Tris-HCl, 300 mM NaCl and 20 mM imidazole, pH 8.0). To elute the bound 7D12, 500 µL Ni-NTA elution buffer (50 mM Tris-HCl, 300 mM NaCl and 200 mM imidazole, pH 8.0) was added and incubated for 15 minutes at room temperature, this was repeated 8 times. Elution fractions were pooled and dialyzed overnight at 4°C against PBS buffer. Dialyzed fractions were then concentrated by Vivaspin 500 columns with 3 kDa molecular weight cut-off (GE Healthcare) and the yields were determined using a calorimetric Pierce BCA protein assay (Thermo Fisher Scientific) measured at 562 nm. After protein purification and concentration, the samples were subsequently resolved by SDS-PAGE. Nu-PAGE LDS loading buffer (Invitrogen) was added to 20 µl of protein samples and heated at 95°C for 15 minutes, then centrifuged (13,000 g, 15 minutes, 4°C) and loaded on a 4-12% Bis-Tris gel (Invitrogen) along with BIO-RAD pre-stained protein ladder (Precision Plus All Blue Protein Standards) as a marker. The gel was then stained with Coomassie Blue (InstantBlue, Expedeon) and the identity of the protein was further confirmed by electrospray ionization mass spectrometry coupled with liquid chromatography, LC-MS. The molecular weights observed on SDS-PAGE gel and mass spectrometry were in good agreement with expected molecular weight of 7D12 and its mutants with site-specifically incorporated photocaged tyrosine ( Figure 1B and Figure S5).

On-cell assay for measuring the binding of His-tagged antibody fragments:
All cell lines were grown in Dulbecco's modification of Eagle medium (DMEM; Gibco, Invitrogen) containing L-glutamine, 4.5 g/L D-Glucose, 110 mg/L Sodium pyruvate, 10% (v/v) foetal bovine serum (FBS), and 1% (v/v) cocktail of penicillin and streptomycin (PEN/STREP, Sigma-Aldrich), which will be referenced as "complete medium" in the text. A431 and MDA-MB-231 cells were grown in a T-75 flask in complete medium (DMEM, 10% FBS, 1% PEN/STREP) using standard tissue culture procedures until 80-90% confluence. After washing with 1x phosphate buffered saline (PBS) and trypsinising, cells were pelleted (300g, 5 minutes) and resuspended in 10 ml fresh complete medium. The cells were then counted on a hemocytometer and diluted to 200,000 cells/ml. 200 µl of this solution was dispensed into each well (40,000 cells/well) of a 96-well plate and grown overnight. The medium was replaced with 200 µl of complete medium supplemented with 7D12 or its photocaged mutants at the desired concentration. The plate was incubated for 10 minutes (37 o C, 5% CO2). After removing medium, the cells were fixed using formaldehyde; 150 µl of 3.7% formaldehyde solution in sterile Mili-Q water was added to each well and incubated for 20 min at room temperature. The formaldehyde solution was removed and cells were washed three times (200 µl, 5 minutes, gentle rocking) with PBST (1X PBS supplemented with 1% Tween-20). After removing the wash buffer, 100 µl of blocking buffer (10% milk in PBST) was added and cells were incubated at room temperature for 1 h with gentle rocking. The blocking buffer was removed. 50 µl solution containing primary anti-6x-His tag antibody was added to each well and the plate was incubated at room temperature for 1 h. The primary antibody solution contained mouse anti-6x-His tag antibody (ThermoFisher Scientific) at 1:500 dilution and 1% milk in PBST. After incubation with the primary antibody, cells in each well were washed three times with PBST (200 µl, 5 minutes, gentle rocking). Subsequently, 50 µl of HRP-linked secondary antibody solution was applied to each well and incubated at room temperature for 1 h. The HRP-linked secondary antibody solution contained anti-mouse-HRP-linked antibody (Cell Signaling) at 1:1000 dilution and 1% milk in PBST. After incubation with secondary antibody, the cells were washed five times with PBST (200 µl, 5 minutes, gentle rocking). Finally, 200 µl of SuperSignal chemiluminescent Substrate (ThermoFisher Scientific) was added to each well and the plate was imaged using BIORAD GelDoc XR+. The chemiluminescence intensity in each well was further quantified by measuring the signal using a CLARIOstar plate reader (BMG labtech).

Conditions for light-mediated decaging of photoactive antibodies in on-cell assay:
In the on-cell assay, wt7D12, 7D12pcY32, 7D12pcY109 and 7D12pcY113 were irradiated with 365 nm light using a UV transilluminator (GelDocMega; BioSystematica).The photon flux and the intensity of 365 nm light from this UV transilluminator, measured using a laser power meter (FieldMate; Coherent), were estimated to be ~33 mW/cm 2 and 14 mW, respectively. 7D12 samples were loaded onto an 18 mm glass coverslip (surface area of coverslip= 2.54cm 2 ) and placed over the transilluminator, followed by irradiation with 365 nm light for 4 min. After irradiation, samples were transferred to 1.5 ml tube and the concentration of the protein was measured using calorimetric Pierce BCA protein assay (Thermo Fisher Scientific).

Cell viability experiments under irradiation conditions for on-cell assay:
2 ml of A431 cells were seeded (200,000 cell/ml) on 18 mm cover glass placed in a 6-well plate (Corning, product code: 3471). This plate was incubated overnight (37 o C, 5% CO2). The cover glass with the attached cells was removed and placed on a UV transilluminator (GelDocMega; BioSystematica), followed by irradiation with 365 nm light for 4 min. This cover glass was transferred back to complete medium and the cells were allowed to proliferate for 48 h (37 o C, 5% CO2). Subsequently, the cells were detached from the cover glass using trypsin, resuspended in complete media and mixed with resazurin in a 96 well plate. Resazurin (0.1 mg/mL in PBS; Sigma-Aldrich) was added to the cells in a 1:10 dilution for 2 h at 37 °C. Resazurin exhibits a blue colour and low fluorescence in metabolically inactive cells but is converted to a highly fluorescent product (resorufin) upon metabolism by viable cells. Fluorescence was then measured on a Flexstation 3 plate reader (Molecular Devices; laser excitation, 570 nm; emission detection, 600 nm).

Computational methods:
The crystal structure for 7D12-EGFR domain III complex (PDB ID: 4KRL) [6] was used as the starting structure for wt7D12-EGFR domain III simulations. This crystal structure was also used to generate the three complexes with the mutants, 7D12pcY32, 7D12pcY109, and 7D12pcY113: pcY was modelled in to substitute Y32, Y109, and Y113, respectively. The CHARMM36 force field [7] with CMAP corrections [8] was adopted for proteins and ions, and CGENFF [9] parameters were used for pcY. Parameter assignment and optimization for pcY, was done using ffTK, [10] following the CGENFF methodology. Quantum mechanical target data for parameter optimization was generated using GAUSSIAN09. [11] Using VMD, [12] each of the four complexes was solvated with water molecules modelled as TIP3P, [13] and Na + and Cl − ions were added to neutralize the system with a net concentration of 150 mM. All the simulations were performed using NAMD 2.12. [14] The systems were minimized for 10,000 steps with all heavy atoms of the protein restrained with a spring constant of k = 5 kcal/mol/Å 2 . The restraints on the side chains were then removed and the systems were again minimized for 10,000 steps, followed by equilibration for 0.2 ns using the NVT ensemble, with stepwise heating up to 310 K, to allow the side chains to relax. Next, all the restraints were removed and the systems were again minimized for 10000 steps, then equilibrated for 0.2 ns in the NVT ensemble with stepwise heating to 310K. Finally, the system was equilibrated in the NPT ensemble for 0.5 ns, followed by the data collection runs for 300 ns using the NPT ensemble and periodic boundary conditions. Temperature was maintained at 310 K using Langevin dynamics [15] with a damping coefficient of 1 ps −1 . Pressure was kept at 1 atm using the Nosé-Hoover Langevin piston method [15][16] with a piston period of 100 fs and a piston decay of 50 fs. Short-range interactions were cut off at 12 Å with a switching applied at 10 Å. Long-range electrostatic forces were calculated using the particle mesh Ewald (PME) [17] method at a grid density of >1 Å −3 . Bonded, non-bonded, and PME calculations were performed at 2-, 2-, and 4-fs intervals, respectively.

Labelling of antibody fragments with BODIPY-TMR-X:
To a 37.5 µl solution of wt7D12 (100 µM) or 7D12pcY32 (100 µM), 51 µl of water, 1.5 µl of 10 mM BODIPY-TMR-X SE in DMF (ThermoFisher Scientific) and 10 µl of 1 M NaHCO3 (pH 8) were added. The reaction mixture was incubated at 25°C for 1 h with shaking (600rpm). To remove excess fluorophore, the labelled wt7D12 and 7D12pcY32 samples were applied to Zeba desalting columns (MWCO 7000 Da, ThermoFisher Scientific). To ensure that all the unbound fluorophore is removed, desalting was performed three times for each sample. The concentration of wt7D12-TMR-X and 7D12pcY32-TMR-X were determined using a calorimetric Pierce BCA protein assay (ThermoFisher Scientific) measured at 562 nm.

Microscopy set-up:
For all live cell imaging experiments, a physiological imaging medium was used to provide better clarity and reduced background. This imaging medium contained 120 mM NaCl, 5 mM KCl, 2 mM CaCl2.2H2O, 1 mM MgCl2.6H2O, 1 mM NaH2PO4, 1 mM NaHCO3, 25 mM HEPES and adjusted to pH 7.2. Once the pH was adjusted, medium was supplemented with 11 mM Glucose, 2.5 mM myo-Inositol, 2 mM Glutamine and BME amino acids (50X solution from Sigma-Aldrich). This medium will be referenced as "imaging medium" in the text. 2 ml of A431 cells (400,000 cells/well) suspended in complete medium were seeded in each well of an ultra-low attachment 6-well plate (Corning, 3471) containing 18 mm cover glass. This plate was incubated overnight (37 o C, 5% CO2). Once 80-90% confluence was observed, cover glass was washed three times with imaging medium and mounted into a microscope chamber (Luidin chamber, Life imaging services). A peristaltic pump was connected to the microscope chamber and allowed for a constant flow of imaging media over live cells at a rate of 1 ml/min. Time-lapse was set up to take brightfield and TexasRed (500ms exposure) images every 30 seconds over a 42 min time period. Cells were imaged on a Zeiss Axiovert 200M microscope at 37ºC using Zeiss AxioVision software. Brightfield and fluorescence images were captured using a 40x (1.3 NA) Plan-Neofluar, oil-immersion objective lens and a Zeiss AxioCam MRm CCD camera. BODIPY-TMR-X fluorescence was excited at 572 ± 14 nm and emission collected at 629 ± 28 nm. 500nM dilutions of wt7D12 and 7D12pcY32 were made up in imaging medium and passed over A431 cells at different time points. First, 2 ml of 7D12pcY32 was passed over with no 365 nm light, followed by 10 ml wash of imaging medium. Then another 2 ml of 7D12pcY32 was passed over while being irradiated with 365 nm for 1 minute. After another wash of 10 ml imagine medium, 2 ml of wt7D12 was passed, followed by the final wash. Microscopy images were processed by ImageJ (Fiji).

Conditions for light-mediated decaging of photoactive antibodies in microscopy experiments:
Light-mediated activation of 7D12pcY32 on the microscope was achieved by opening DAPI channel for 1 minute. The photon flux and the laser power of 365 nm light from DAPI channel, measured using laser power meter (FieldMate; Coherent), were estimated to be ~0.25 mW/cm 2 (40x 1.3 NA Plan-Neofluar objective area of 3.06mm 2 ) and 8 mW, respectively.
Cell viability experiments under irradiation conditions for microscopy experiments: 2 ml of A431 cells were seeded (400,000 cell/well) on 18 mm cover glass placed in a 6-well plate (Corning, product code: 3471). This plate was incubated overnight (37 o C, 5% CO2). The cover glass with the attached cells was removed and placed under the microscope, followed by irradiation with 365 nm light for 1 min. This cover glass was transferred back to complete medium and the cells were allowed to proliferate for 48 h (37 o C, 5% CO2). Subsequently, the cells were detached from the cover glass using trypsin, resuspended in complete media and mixed with resazurin in a 96 well plate. Resazurin (0.1 mg/mL in PBS; Sigma-Aldrich) was added to the cells in a 1:10 dilution for 2 h at 37 °C. Resazurin exhibits a blue colour and low fluorescence in metabolically inactive cells but is converted to a highly fluorescent product (resorufin) upon metabolism by viable cells. Fluorescence was then measured on a Flexstation 3 plate reader (Molecular Devices; laser excitation, 570 nm; emission detection, 600 nm). Figure S1. Sequence of gene block inserted into pSANG10 plasmid for generation of pSANG10_7D12 plasmid. Red -Gibson overhang, and Underlined -Restriction sites used for cloning. Protein sequence is shown below the DNA sequence, Greenleader peptide sequence, Purple -wt7D12 sequence, Blue -His6-tag, and black Y* -sites for insertion of pcY Figure S3. Screening of pSUP_MjCNFRS/6xMjtRNACUA, pEvolv_2xMjCNFRS/MjtRNACUA and pULTRA_MjCNFRS/MjtRNACUA plasmids. Comparison of band intensities in the western blot demonstrates unnatural amino acid dependent expression of 7D12 and that the pULTRA suppressor plasmid is most efficient for incorporation of 4-Azido-L-phenylalanine (AzF) using MjRS/MjtRNA pair in 7D12. Figure S4. Genetic site-specific incorporation of N6-(tert-butoxycarbonyl)-L-lysine (BocK) and N6-[(2-propynyloxy)carbonyl]-L-lysine (CAK) in 7D12. Comparison of band intensities in the Coomassie stained gel demonstrates that pCDF suppressor plasmid is more efficient than the pULTRA suppressor plasmid at incorporating unnatural amino acids using PylRS/PyltRNA pair. Figure S5. ESI-MS of the wt7D12, 7D12pcY32, 7D12pcY109, and 7D12pcY113 confirms their identity. The observed and the calculated molecular weights are in good agreement with each other. Figure S6. On-cell assay reports specific binding between 7D12 antibody fragment and EGFR on cell surface. Low chemiluminescence signal observed when wt7D12 was incubated with MDA-MB-231 cells, a cell line with low levels of EGFR, compared to the signal from A431 cancer cell line, that has higher levels of EGFR. Figure S8. On-cell binding experiments to assess the interaction of EGFR with mutants of 7D12 containing site-specifically incorporated pcY were performed in triplicates. On-cell assays performed on the surface of A431 cells demonstrates that the presence of pcY at positions 32 and 113 in 7D12 inhibits its binding to EGFR. However, 7D12pcY109 mutant shows binding affinity similar to wt7D12. The binding to 7D12pcY32 and 7D12pcY113 mutants is restored upon irradiation with 365 nm light. These experiments were performed in triplicates, REP1, REP2 and REP3, to ensure reproducibility of data. Figure S9. Quantitative assessment of light-mediated binding of photoactive 7D12 to EGFR on the surface of A431 cancer cells. A) Chemiluminescence intensities obtained from on-cell binding experiments for wt7D12, 7D12pcY32, 7D12pcY109 and 7D12pcY113, before and after irradiation with 365 nm light, were quantified using CLARIOstar plate reader and concentration of 7D12, where X-axis is in log scale. The lines show the trace obtained after fitting the data to a sigmoidal nonlinear equation using GraphPad. Some error bars are too small to be clearly visible. B) The KD and IMAX values obtained for 7D12 and its mutants containing pcY after fitting the data. KD is the concentration of the 7D12, where chemiluminescence intensity is half of the maximum chemiluminescence intensity. IMAX is the maximum chemiluminescence intensity at saturation. The value of IMAX for decaged 7D12pcY32 and 7D12pcY113 is 86% and 76%, respectively, of the IMAX for wt7D12; indicating recovery of binding to these levels.  Figure S10. Measurement of cell viability of A431 cells after irradiation with 365 nm light on UV transilluminator. A431 cells were irradiated with 365 nm light for 4 minutes on a UV transilluminator; similar irradiation conditions were used for decaging 7D12 in on-cell experiments shown in Figure 2. Cells were allowed to proliferate for 48 hours after irradiation with 365 nm light, and viability of these cells was measured using Resazurin cell viability assay. A decrease of ~12% in A431 cell viability is observed after irradiation with 365 nm light. This experiment was performed in duplicates. Figure S11. The number of 7D12-EGFR domain III contacts during 300ns of simulations, measured as the number of 7D12 heavy atoms within 3.5 Å of EGFR heavy atoms, show that 7D12pcY32 and 7D12pcY113 form lesser contacts with EGFR for long periods, compared to the complexes with wt7D12 and 7D12pcY109. These observations suggest that the presence of pcY at positions 32 and 113 destabilizes the 7D12-EGFR domain III complex. Figure S12. The binding of 7D12 to EGFR is affected by presence of BODIPY-TMR-X label. Comparison of binding of labelled and unlabelled wt7D12 to EGFR assessed using on-cell assay on the surface of A431 cells shows a reduction in binding by ~1.5-fold due to the presence of BODIPY-TMR-X label. The chemiluminescence counts for wt7D12 and labelled-7D12 at 200 nM concentration are 6838468 and 4436744, respectively. Figure S13. Measurement of cell viability of A431 cells after irradiation with 365 nm light on the microscope. A431 cells were irradiated for 1 minute by opening the DAPI channel on microscope; similar irradiation conditions were used in microscopy experiments shown in Figure 4. Cells were allowed to proliferate for 48 hours after irradiation with 365 nm light, and viability of these cells was measured using Resazurin cell viability assay. A decrease of ~13% in A431 cell viability is observed after irradiation with 365 nm light.