Supplemental Figure 1 – Ab033 EGFR binding kinetics. The binding affinity of Ab033 for wild-type EGFR was determined by surface plasmon resonance.

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Supplemental Figure 2 – Number of surface EGF receptors. Labeled Ab033 was added at saturating conditions to A431 and H441 cells on ice for 30 min and the surface levels of EGFR were measured by flow cytometry. Signal values were normalized to receptor counts using a standard curve defined by quantitative flow cytometry beads.

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Supplemental Figure 3 – Workflow for internalization rate determination. (A) To simplify the experimental system, detached cells were labeled on ice to limit endocytosis and produce cells with antibody uniformly bound to the cell surface. (B) The cells were washed to remove unbound antibody and (C) moved to 37 ˚C to initiate internalization of receptor-antibody complex. Time points were taken in one minute increments during the first five minutes of the incubation to monitor initial kinetics and then at spaced intervals up to an hour. (D) After incubation, cells were stained with secondary anti-IgG antibody to mark cell surface bound Ab033. The secondary antibody was also used to quantitate the change in bound EGFR on the cell surface over time. Because the cells were not permeabilized, the secondary antibody only bound Ab033 present on the cell surface and not the internalized portion. The samples were then analyzed by imaging flow cytometry.

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Supplemental Figure 4 – Comparison of Ab033 mAb and ADC internalization rates. Ab033, Ab033-mc-vc-PABC-MMAE, and Ab033-mcMMAF were incubated with A431 cells on ice. The internalization of the monoclonal antibody and the two ADCs was then performed using imaging flow cytometry. The amount remaining on the cell surface was determined using anti-human IgG secondary antibody.

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Supplemental Figure 5 – Visualization of internalization in H441 cells. Representative images from the first five minutes of an internalization assay where chosen to highlight the internalization of Ab033 over time.

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Supplemental Figure 6 – Visualization of full internalization time course in A431 cells. Representative cells from A431 cells were chosen to show the change in Ab033 distribution over the 60 min incubation. ‘Surface’ was the secondary antibody bound to Ab033 on the cellular surface. ‘Total Ab’ was AlexaFluor 488 labeled Ab033. Cells were picked with surface and total antibody signal levels similar to the median intensity values for their respective cell populations.

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Supplemental Figure 7 – Surface localization and internalization scores by imaging flow cytometry. The degree of surface colocalization between Ab033 and a secondary antibody bound to surface Ab033 was calculated using imaging flow cytometry results. A higher surface colocalization score pertains to a greater percentage of surface bound Ab033. The amount of internalization was also assessed by calculating an internalization score for each of the internalization data points for (A) A431 and (B) H441 cells. Higher internalization scores indicate higher degrees of internalization.

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Supplemental Figure 8 – Quenching of AlexaFluor 488 fluorescence. (A) Increasing concentrations of anti-AlexaFluor 488 antibody were added to Ab033-bound A431 cells on ice and the percentage of signal quenching was measured. (B) Images of extracellular Ab033 signal are shown for increasing amounts of anti-AlexaFluor 488 quenching antibody.

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Supplemental Figure 9 – Colocalization of Ab033 to the lysosome. Spatial overlap between Ab033 and a lysosomal marker was calculated for the different pulse-chase samples from A431 cells.

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Supplemental Figure 10 – Uptake and trafficking of Ab033 with pH sensitive dye. (A) A431 and (B) H441 cells were treated with Ab033 containing both AlexaFluor488 and a pH sensitive dye. ‘Total’ is the AlexaFluor signal and ‘Both’ is the composite overlay of ‘Total’ and ‘pH Dye’ signals.

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Supplemental Figure 11 – Preparation of Ab033-E2/F2. (A) Schematic of reducation, conjugation, and purification steps. (B) HIC traces of Ab033-E2 and Ab033-E2/F2 purification steps. (C) RPLC analysis of DTT-reduced Ab033-E2 and Ab033-E2/F2 was performed to monitor the number of drug molecules on each LC and HC. L0 and H0 are abbreviations for LC and HC with no drug while L-E1 and H-E1 are LC and HC with one mc-vc-PABC-MMAE. L-F1 and H-F1 are LC and HC with one mc-MMAF. A minor

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amount of Ab033-E2 had the HC-HC disulfide reduced by TCEP, thereby generating Ab033-E2/F2 with two mc-MMAF conjugated on the HC.

Supplemental Figure 12 – Drug concentration in media. The free MMAE and cys-mc-MMAF media levels for (A) A431 and (B) H441 cells were measured by LC-MS/MS. The data shown is the mean value from 3 replicates, with each replicate measured twice. Error bars are SEM.

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Supplemental Figure 13 – Mechanistic cellular model. A Simbiology model was developed to integrate ADC processing details into a quantitative model system of the cell. ‘NSU’ is an abbreviation for non-specific uptake.

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Supplemental Table 1 - The model system is given by:d (MMA Emedia)

dt=kefflux ,drug×MMA E free+V media×kmediarelease× ADCmedia−kuptake ,drug×MMA Emedia

d ( ADCmedia )dt

= 1V medi a

(−k on× ADCmedia× EGF R extra+koff×complex+kefflux× Ablys−V media×kmedia, release× ADCmedia+knsuout× ADC NSU−k nsu× ADCmedia)

d (EGF Rextra )dt

=knew , EGFR×EGF Rcell−kreceptor , deg×EGF Rextra+k receptor , syn−kon× EGFRextra× ADCmedia+koff×complex+k out ,EGFR×EGF Rcell

d ( complex )dt

=kout× ADC endosome−k¿×complex+kon×EGF R extra× ADCmedia−koff×complex

d ( ADCendosome )dt

=−kout× ADC endosome−k lys× ADCendosome+k¿×complex

d ( ADCNSU )dt

=k nsu× ADCmedia−knsu ,out× ADCNSU−k pino ,traffic× ADCNSU

d ( A blys )dt

=k pino ,traf fic× ADCNSU−kefflux× A blys+k lys× ADCendosome

d (EGF Rcell )dt

=−knew , EGFR×EGF R cell+k lys× ADC endosome−kdeg ,EGFR×EGF Rcell−kout , EGFR×EGF R cell

d (MMA E conj)dt

=DAR×k pino,traffic× ADCNSU−kefflux×MMA Econj−krelease×MMA Econj+DAR×k lys× ADC endosome

d (MMA E free )dt

=−k efflux, drug×MMA Efree+kuptake ,drug×MMA Emedia+krelease×MMA Econj

For the single cell model k nsu=knsu ,single and kuptake , drug=kuptake ,drug ,single while for the all cells model k nsu=knsu ,single×cells and kuptake , drug=kuptake ,drug ,single×cells.Total ADC is calculated as ADC total=ADCendosome+Ablys.Supplemental Table 2:Parameter Units Notation A431 H441EGFR synthesis rate molecules/hour/cell k receptor, syn 36 000 4 500EGFR degradation rate

1/hour k receptor , deg 0.03 0.03

EGFR-Ab complex internalization rate

1/hour k ¿ 2.82 9

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Internalized ADC lysosomal uptake rate

1/hour k lys 0.349 0.380

Intracellular MMAE release rate

1/hour k release 18 1.74

Media MMAE release rate

1/hour kmedia, release 6×10-4 6×10-4

EGFR-Ab binding rate 1/Ms k on 5.6×105 5.6×105

EGFR-Ab disassociation rate

1/s k off 0.0012 0.0012

EGFR signaling rate 1/hour k new ,EGFR 0.001 0.055Non-specific uptake rate

molecules/nM/hour/cell

k nsu, single 2.41×104 3.01×104

Non-specific efflux rate

1/hour k nsu, out 0.01 0.01

ADC efflux rate 1/hour k efflux 0.0832 0.116MMAE efflux rate 1/hour k efflux, drug 0.111 0.117MMAE uptake rate 1/hour/cell kuptake , drug, single 1.38×10-7 2.76×10-8

EGFR-Ab complex recycling rate

1/hour k out 6.6 21

Pinocytosis trafficking rate

1/hour k pino ,traffic 8 8

Number of cells cells cells 1×106 1×106

Intracellular EGFR degradation

1/hour k deg , EGFR 9.3 0.161

EGFR recycling rate 1/hour k out , EGFR 18 1.81Media volume mL V media 1 1

Initial conditions for cell membrane and intracellular EGFR are cell-line dependent: 1.5×105 EGFR per cell membrane for H441 and 1.2×106 for A431 and 2×104 intracellular EGFR per cell for H441 and 4×105 for A431. The initial condition for media ADC was set according to the applied dose of 100 nM and the initial conditions for MMAE were set to be consistent with measurements.

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