DOI: 10.5061/dryad.2280gb5zq
title: Data for: A cell surface-binding antibody atlas nominates a MUC18-directed antibody-drug conjugate for targeting melanoma

description: Recent advances in targeted therapy and immunotherapy have substantially
 improved the treatment of melanoma. However, therapeutic strategies are
 still needed for unresponsive or treatment-relapsed melanoma patients. To
 discover antibody-drug conjugate (ADC)-tractable cell surface targets for
 melanoma, we developed an atlas of melanoma cell surface binding
 antibodies (pAbs) using a proteome-scale antibody array platform (PETAL).
 Target identification of pAbs led to development of melanoma cell killing
 ADCs against LGR6, TRPM1, ASAP1, and MUC18, among others. MUC18 was
 overexpressed in both tumor cells and tumor-infiltrating blood vessels
 across major melanoma subtypes, making it a potential dual-compartment and
 universal melanoma therapeutic target. AMT-253, an MUC18-directed ADC
 based on topoisomerase I inhibitor exatecan and a self-immolative T
 moiety, had a higher therapeutic index compared to its microtubule
 inhibitor-based counterpart and favorable pharmacokinetics and
 tolerability in monkeys. AMT-253 exhibited MUC18-specific cytotoxicity
 through DNA damage and apoptosis and a strong bystander killing effect,
 leading to potent antitumor activities against melanoma cell line and
 patient-derived xenograft models. Tumor vasculature-targeting by a mouse
 MUC18-specific antibody-T1000-exatecan conjugate inhibited tumor growth in
 human melanoma xenografts. Combination therapy of AMT-253 with an
 anti-angiogenic agent generated higher efficacy than single agent in a
 mucosal melanoma model. Beyond melanoma, AMT-253 was also efficacious in a
 wide range of MUC18-expressing solid tumors. Efficient target/antibody
 discovery in combination with the T moiety-exatecan linker-payload
 exemplified here may facilitate discovery of new ADC to improve cancer
 treatment.
description: PETAL monoclonal antibody
 arrays (60,000 mAb printed on 2 x slides) were prepared as previously
 reported. Melanoma cells
 GAK, HMVII and A375 (1-2x10<sup>7</sup>) suspended in 2 ml 1x
 PBS were first labeled with DNA dye Syto-62 (ThermoFisher, S11344) at
 1:10000 ratio for 10 minutes. Labeled cells were then incubated with PETAL
 array for 30 minutes at 37°C without shaking
 (0.5-1x10<sup>7</sup> cells per array). After a gentle washing
 step in 1x PBS buffer to remove unbound cells, the slides were scanned by
 GenePix 4200A (Modecular Device). Images were analyzed by GenePix Pro
 software (GenePix Pro, RRID:SCR_010969).
description: *** # PETAL screening on melanoma cells *** An antibody array platform
 (PETAL) was used to screen overexpressed surface target on melanoma cells
 ## Description of the data and file structure The antibody arrary was
 screened against 3 different cell lines: GAK, HMVII, and A375. For each
 cell line, there was two antibody arrary slides: A1 and A2. See the
 Methods sections for details. ### The list below describes each column of
 data in the Results file: | **Block** | the block number of the feature. |
 | :---- | :------------------------------- | | **Column** | the column
 number of the feature. | | **Row** | the row number of the feature. | |
 **Name** | the name of the feature derived from the Array List (up to 40
 characters long, contained in quotation marks). | | **ID** | the unique
 identifier of the feature derived from the Array List (up to 40 characters
 long, contained in quotation marks). | | **X** | the X-coordinate in µm of
 the center of the feature-indicator associated with the feature, where
 (0,0) is the top left of the image. | | **Y** | the Y-coordinate in µm of
 the center of the feature-indicator associated with the feature, where
 (0,0) is the top left of the image. | | **Dia.** | the diameter in µm of
 the feature-indicator. | | **F635 Median** | median feature pixel
 intensity at wavelength #1 (635 nm). | | **F635 Mean** | mean feature
 pixel intensity at wavelength #1 (635 nm). | | **F635 SD** | the standard
 deviation of the feature pixel intensity at wavelength #1 (635 nm). | |
 **B635 Median** | the median feature background intensity at wavelength #1
 (635 nm). | | **B635 Mean** | the mean feature background intensity at
 wavelength #1 (635 nm). | | **B635 SD** | the standard deviation of the
 feature background intensity at wavelength #1 (635 nm). | | **% &gt;
 B635 + 1 SD** | the percentage of feature pixels with intensities more
 than one standard deviation above the background pixel intensity, at
 wavelength #1 (635 nm). | | **% &gt; B635 + 2 SD** | the percentage of
 feature pixels with intensities more than two standard deviations above
 the background pixel intensity, at wavelength #1 (635 nm). | | **F635 %
 Sat.** | the percentage of feature pixels at wavelength #1 that are
 saturated. | | **F532 Median** | median feature pixel intensity at
 wavelength #2 (532 nm). | | **F532 Mean** | mean feature pixel intensity
 at wavelength #2 (532 nm). | | **F532 SD** | the standard deviation of the
 feature intensity at wavelength #2 (532 nm). | | **B532 Median** | the
 median feature background intensity at wavelength #2 (532 nm). | | **B532
 Mean** | the mean feature background intensity at wavelength #2 (532 nm).
 | | **B532 SD** | the standard deviation of the feature background
 intensity at wavelength #2 (532 nm). | | **% &gt; B532 + 1 SD** | the
 percentage of feature pixels with intensities more than one standard
 deviation above the background pixel intensity, at wavelength #2 (532 nm).
 | | **% &gt; B532 + 2 SD** | the percentage of feature pixels with
 intensities more than two standard deviations above the background pixel
 intensity, at wavelength #2 (532 nm). | | **F532 % Sat.** | the percentage
 of feature pixels at wavelength #2 that are saturated. | | **Ratio of
 Medians** | the ratio of the median intensities of each feature for each
 wavelength, with the median background subtracted. | | **Ratio of Means**
 | the ratio of the arithmetic mean intensities of each feature for each
 wavelength, with the median background subtracted. | | **Median of
 Ratios** | the median of pixel-by-pixel ratios of pixel intensities, with
 the median background subtracted. | | **Mean of Ratios** | the geometric
 mean of the pixel-by-pixel ratios of pixel intensities, with the median
 background subtracted. | | **Ratios SD** | the geometric standard
 deviation of the pixel intensity ratios. | | **Rgn Ratio** | the
 regression ratio of every pixel in a 2-feature-diameter circle around the
 center of the feature. | | **Rgn R²** | the coefficient of determination
 for the current regression value. | | **F Pixels** | the total number of
 feature pixels. | | **B Pixels** | the total number of background pixels.
 | | **Circularity** | a metric based on the variance of the distance of
 each boundary pixel to the centroid of the feature | | **Sum of Means** |
 the sum of the arithmetic mean intensities for each wavelength, with the
 median background subtracted. | | **Log Ratio** | log (base 2) transform
 of the ratio of the medians. | | **F532 Total Intensity** | the sum of all
 pixel intensities in the feature. | | **Flags** | the type of flag
 associated with a feature. | | **Normalize** | the normalization status of
 the feature (included/not included). | | **Autoflag** | reports whether or
 not a feature has been flagged from the Flag Features dialog box. It
 applies to good and bad flags only. | ## Sharing/Access information Not
 applicable.