Supplemental Information for

Theranostic targeting of CUB domain containing protein 1 in pancreatic cancer

Anna Moroz1,2, Yung-hua Wang1, Jeremy M. Sharib3, Junnian Wei1, Ning Zhao1, Yangjie Huang1, Zhuo Chen1, Alex J. Martinko4, Jie Zhuo4, Shion A. Lim4, Lydia Zhang4, Youngho Seo1, Sean D. Carlin5, Kevin K. Leung4, Eric A. Collisson6,7, Kimberly S. Kirkwood3,7, James A. Wells4,7, Michael J. Evans1,4,7,*

1Department of Radiology and Biomedical Imaging, University of California San Francisco, 505 Parnassus Ave, San Francisco CA 94143

2Skolkovo Institute of Science and Technology, Skolkovo Innovation Center, 3 Nobel Street, Moscow 143026 , Russia.

3Department of Surgery, University of California San Francisco, 505 Parnassus Ave, San Francisco CA 94143

4Department of Pharmaceutical Chemistry, University of California San Francisco, 505 Parnassus Ave, San Francisco CA 94143

5Department of Radiology, University of Pennsylvania, 3400 Spruce Street, 1 Silverstein Philadelphia, PA 19104

6Department of Medicine, University of California San Francisco, 505 Parnassus Ave, San Francisco CA 94143

7Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, 505 Parnassus Ave, San Francisco CA 94143

Corresponding author:

Michael J. Evans, PhDTel: 415-514-1292Michael.evans@ucsf.edu

Supplemental Methods:

General Methods

Unless otherwise noted, all chemicals were acquired from commercial vendors and used without

further purification. HPAC, Capan-1, Panc10.05, Panc2.03, and HPAFII were purchased from

American Type Culture Collections and cultured according to manufacturer’s recommendations.

A polyclonal antibody targeting CDCP1 (cat. no. 4115) was purchased from Cell Signaling

Technologies. The monoclonal antibody AC15 was purchased from Sigma to probe for actin

levels on immunoblot. Para-isothiocyanatobenzyl-desferrioxamine and 1,4,7,10-

tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester were

obtained from Macrocyclics (Dallas, TX) and used without further purification. 89ZrCl4 was

purchased from 3D Imaging, LLC (Maumelle, AR). 177LuCl3 and 225AcCl3 were purchased from

Oak Ridge National Laboratory. A non-targeting human IgG1, isolated from human myeloma

plasma (cat. no. 400120), was acquired from Millipore Sigma (Burlington, MA).

Antibody expression and purification

The 4A06 IgG was identified and cloned from Fab-phage into the pFUSE-hIgG1-Fc vector

(InvivoGen) as previously described1. It was expressed as a secreted protein using the Expi293

Expression system (Thermo) according to manufacture protocol. Briefly, 30µg of heavy and

light chain DNA was mixed and transiently transfected into 75 million Expi293 cells using the

Expifectamine kit. Enhancers were added 20 hours after transfection and cells were incubated for

4-6 additional days at 37˚C in a 8% CO2 shaker before the supernatant was harvested

by centrifugation. Protein was then purified using HiTrap Protein A HP columns (GE

Healthcare) and assayed by SDS-PAGE.

Antibody bioconjugation

To conjugate DOTA to 4A06, the antibody (400 L at a concentration of 7.65 mg/mL) was

dispersed in 500 L of 0.1 M sodium bicarbonate buffer (pH 9.0). The final reaction mixture was

adjusted to a total volume of 1 mL by adding a sufficient amount of 0.1 M sodium bicarbonate

buffer. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide

ester (10 mg/mL in DMSO, 40-60 molar excess) was added to the antibody solution dropwise

while mixing vigorously. The final concentration of DMSO was kept below 2% (v/v) to avoid

any precipitation. The reaction incubated for 90 min at 37oC, whereupon the reaction mixture

was purified with a PD-10 column using an ammonium acetate mobile phase (0.2 M sodium

acetate, pH 7.0). The DOTA-4A06 solution was aliquoted and stored at -20o C until time of use.

To conjugate DFO to 4A06, the antibody (261L at a concentration of 7.65 mg/mL) was

dispersed in 200 L of 0.1 M sodium bicarbonate buffer (pH 9.0). The final reaction mixture was

adjusted to a total volume of 0.5 mL by adding a sufficient amount of 0.1 M sodium bicarbonate

buffer. Para-Isothiocyanatobenzyl-desferrioxamine (p-Df-Bz-NCS, 30 mM in DMSO, 4 eq.) was

added to the antibody solution drop wise while mixing vigorously. The final concentration of

DMSO was kept below 2% (v/v) to avoid any precipitation. The reaction was allowed to

incubate for 30 min at 37oC, whereupon the reaction mixture was purified with a G25 column

using an ammonium acetate mobile phase (0.2 M sodium acetate, pH 7.0). The solution was

aliquoted and stored at -20o C until time of use.

Kinetic constants for 4A06, DFO-4A06, DOTA-4A06 against human CDCP1 were

determined using an Octet RED384 instrument (ForteBio). Six concentrations of the

recombinant human ectodomain of CDCP1 (250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM)

and 7.812 nM were tested for binding to the respective antibody, which was immobilized on

Anti-Human IgG Fc Capture biosensors (Fortebio). All measurements were performed at room

temperature in 384-well microplates and the running buffer was PBS with 0.5% (w/v) bovine

serum albumin (BSA) and 0.05% (v/v) Tween 20. The antibody was loaded for 180 sec from a

solution of 300 nM, baseline was equilibrated for 60 s, and then the antigen was associated for

600 s followed by 1200 sec disassociation. Between each sample, the biosensor surfaces were

regenerated three times by exposing them to 10 mM glycine, pH 1.5 for 5 sec followed by PBS

for 5 sec. Data were analyzed using a 1:1 interaction model on the ForteBio data analysis

software 8.2.

Radiolabeling

A solution of 89Zr-oxalic acid (5 mCi; 40 μl) was neutralized with 2 M Na2CO3 (18 μl). After 3

min, 0.30 ml of 0.5 M HEPES (pH 7.1–7.3) and 1.5 mg of DFO-4A06 (pH = 7) were added into

the reaction vial. After incubation for 60 min at 37oC, the reaction progress was monitored by

instant thin layer chromatography (ITLC) using a 20 mM citric acid (pH 4.9–5.1) mobile phase.

The product was purified with a PD10 column. The decay corrected radiochemical yield was

consistently > 95%.

A solution of 177LuCl3 (8mCi; 40 l) was diluted with 200L 0.2M ammonium acetate. 2

mg in 1.4 mL of DOTA-4A06 (pH = 7) were added into the reaction vial. After incubation for 60

min at 37oC, the reaction progress was monitored by ITLC using a 20 mM citric acid (pH 4.9–

5.1) mobile phase. The product was purified with a PD10 column. The decay corrected

radiochemical yield was consistently > 95%.

A solution of 225Ac-nitrate (120 uCi) dissolved in 0.2 M HCl was added to an Eppendorf

vial, and the accurate activity was measured by using a dose calibrator. To this were added 2 M

tetramethyl ammonium acetate buffer (25 uL), L-ascorbic acid (150 g/L; 10 uL), and the 4A06-

DOTA (120 ug). The pH of the reaction was determined by pH paper; pH of a typical reaction

was 5.8. The mixture was incubated in 37.0 oC for 1.5h. After this, the reaction was then

quenched with 2M DTPA (10 uL) and purified using an G25 desalting column that had been

equilibrated previously with 1% human serum albumin in 0.5M HEPES. The product was eluted

in approximately 2 mL of 1% human serum albumin and analyzed by ITLC to determine the

radiochemical purity. The decay corrected radiochemical yield was consistently > 95%.

Cell internalization assay

Approximately 1x106 cells/well were plated in a 12 well dish with complete media. Prior to

starting the uptake assay, cells were washed thoroughly with PBS and covered in 0.5 mL of PBS. 89Zr-4A06 (0.5 L/well, ~0.5 Ci/well) was added to each well, and the plates were immediately

transferred to a cell culture incubator maintained at 37o C or a 4o C refrigerator. After one hour,

the unbound radioactivity was removed and reserved for analysis. The cells were washed with

PBS (2 x 5 mL), and all of the PBS fractions were combined. The surface bound antibody was

stripped using a 60 sec incubation with 0.5 mL volume of 10% citric acid. The acid fraction was

collected and reserved for analysis. The internalized activity was collected by lysing the cells in

0.5 M NaOH (aq.). The cell lysate was collected and reserved for analysis. The fractions were

counted using a Hidex Automatic gamma counter. The internalized activity was expressed as a

percentage of the total activity recovered from each well.

Immunoblot

Actively proliferating cells were washed with PBS and lysed with RIPA Lysis and Extraction

Buffer (Millipore, Temecula, CA) supplemented with Protease and phosphatase Inhibitor

Cocktail (Thermo Scientific) at 4°C for 10 min. Immunoblotting was performed using antibodies

to CDCP1 (1:1000) and actin (1:5000). Protein concentration was determined by the Bradford

absorbance assay, and 15 µg of lysate were resolved with 1D SDS-PAGE using 7% Tris acetate

precast gels (Invitrogen).

Flow cytometry

Approximately 1x106 cells per sample were lifted with Cellstripper (Corning, Manassas, VA),

washed twice with PBS pH 7.4, and subsequently blocked with flow cytometry buffer (PBS, pH

7.4, 3% BSA). Anti-CDCP1 (10 mg/mL) or commercial antibodies were added to cells which

were kept for 30 minutes on ice. Antibodies were detected by adding Protein A – Alexafluor-647

or Alexafluor-488 conjugate (Life Technologies; 1:1000). Cells were extensively washed, and

fluorescence was quantified using a FACSCalibur on FL4 single channel analysis (BD

Biosciences). All flow cytometry data were analysed using FlowJo software (FlowJo LLC,

Oregon). Data were normalized for living cells only.

DNA and RNA Genetic Analysis

DNA and RNA extraction as well as whole exome and RNA sequencing was performed on

primary tumors and PDX tissue by GENEWIZ (GENEWIZ Inc.  South Plainfield, NJ).  In brief,

DNA and RNA were extracted from tumor tissue using DNA Miniprep and RNeasy mini kit,

respectively (Qiagen, Germany).  Nucleic acid concentration was quantified using Qubit 2.0

Fluorometer (Life Technologies, Carlsbad, CA).  Library preparation was by NEB NextUltra

DNA library preparation kit (Illumina, San Diego, CA) and CleanTag Small RNA Library

Preparation Kit (TriLink BioTechnologies, San Diego, CA) following the manufacturers

recommendations.  Whole exome DNA sequencing and RNA-seq next-generation sequencing

was then performed on an Illumina HiSeq 2500 (Illumina, San Diego, CA) with 100 nt read

single indexing sequencing protocol.  Sequencing data was de-multiplexed with CASAVA

software 1.8.2 (Illumina, San Diego, CA USA).  

Adapters were trimmed using Trimmomatic 0.38.  For the PDX samples, raw read from

the FASTQ files were classified as mouse or human using Xenome 1.0.0 and the mouse reads

were removed from further processing.  For the RNA-seq analysis, all samples were processed

using the CGL RNA-seq pipeline from UC Santa Cruz.  We chose this pipeline in order to

facilitate comparisons between the samples in our study and public datasets such as Genotype-

Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), both of which have already

been processed using the CGL RNA-seq pipeline.  To filter out low expression genes, we kept

genes with at least 10 reads in at least two samples.  Differential gene expression was performed

using the DESEQ2 package and functional enrichment was performed using the ToppGene

Suite.  PCA was performed on the 5,000 most variable genes (IQR)

after rlogtransformation.  Tumor purity was estimated using the ESTIMATE package and added

as a covariate in the differential expression analysis.  For the PCA plot, we adjusted for tumor

purity using linear regression.  The whole-exome data was processed using the bcbio-nextgen

cancer variant calling pipeline with the tumor-only configuration and the following variant

callers: freebayes, vardict, varscan.  bcbio uses a majority voting ensemble approach to combine

calls from multiple variant callers and flattens structural variant calls into a combined

representation. Likely germline background mutations were filtered by their presence in public

databases such as 1000 genomes and ExAC.

Supplemental Figure 1. Biolayer interferometry (BLI) data showing that functionalization of

4A06 with chelators does not impact antibody affinity. Two experiments using different

concentrations of antibody are shown. The traces are representative of the outcome of three

independent experiments.

Supplemental Figure 2. The complete repertoire of biodistribution data for 89Zr-4A06 after

injection in nu/nu mice bearing HPAC xenografts. Each treatment arm consisted of n = 5 mice.

The data represent the mean ± standard deviation.

Supplemental Figure 3. The complete repertoire of biodistribution data for mice treated with 89Zr-4A06 alone, or a co-injection of 89Zr-4A06 with 20x excess unlabeled 4A06. The data were

collected at 48 hours post injection in nu/nu mice bearing HPAC xenografts. Each treatment arm

consisted of n = 5 mice. The data represent the mean ± standard deviation.

Supplemental Figure 4. High magnification (40x) images of digital autoradiography of 89Zr-

4A06 overlaid on PDX tissues (stained with H&E) are shown above standard magnification (4x)

images. At right is shown the original ImageJ file showing the relative intensities of radiotracer

uptake in either PDX.

Supplemental Figure 5. The complete repertoire of biodistribution data for 177Lu-4A06 after

injection in nu/nu mice bearing HPAC xenografts. Each treatment arm consisted of n = 4 mice.

The data represent the mean ± standard deviation.

Supplemental Figure 6. In vitro uptake data showing that 89Zr-4A06 is internalized in two

PDAC cell lines. The raw data represent the cell internalized portion of 89Zr-4A06, which was

collected with 0.5 M NaOH after removing the unbound and surface bound 89Zr-4A06. The data

were collected at the indicate time point. The % of activity internalized was subsequently

normalized between treatment arms exposed to 37o C or 4o C, a negative control for cellular

internalization. *P<0.01. The data are representative of two independent experiments.

Supplemental Table 1. A summary of the receptor density data calculated for CDCP1 in human

pancreatic cancer cell lines using 125I-labeled 4A06. These data were previously reported. For

comparison, we have also reported receptor density data for HER2 and PSMA, which were

previously calculated by other labs.

Cell line Histology Receptor Receptor per cell PMID

HPAC PDAC CDCP1 2.9 x 10-6 29359686

SW620 PDAC CDCP1 1.2 x 10-6 29359686

MiaPaCa2 PDAC CDCP1 1.9 x 10-6 29359686

BT474-M3 Breast cancer HER2 1.9 x 10-6 24035511

SKOV3 Ovarian cancer HER2 1.3 x 10-6 24035511

MDA-MD-453 Breast Cancer HER2 3.9 x 10-6 24035511

MDA-PCa-2b Prostate Cancer PSMA 1.5 x 10-5 21750220

LNCaP Prostate Cancer PSMA 1.2 x 10-5 21750220

C4-2 Prostate Cancer PSMA 1.0 x 10-5 21750220

Supplemental Table 2. A summary of the biodistribution data collected at 72 hours post

injection of 89Zr-4A06 and the P values from an unpaired, two tailed Student’s t test to determine

significant changes compared to HPAC tracer uptake values. The data are reported as mean ±

standard deviation and are representative of two independent experiments.

Tumor Radiotracer uptake in tumors

(%ID/g)

P value compared to HPAC

HPAC 15.21 ± 2.2 N/A

Panc02.03 5.25 ± 1.2 0.0008

Panc10.05 6.09 ± 0.5 0.002

Capan-1 6.81 ± 1.2 0.001

HPAF II 7.78 ± 4.8 0.09

Supplemental Table 3. A summary of the mutations identified from UCPDAC-187 and

UCPAsC-208 using the UCSF 500 Cancer gene panel test.

PDX ID Gene Mutation Reads Mutant allele

frequency

(%)

Pathogenic?

UCPDAC-187 KRAS p.G12V 871 52 Yes

TP53 c.919+1G>A 845 39 Yes

U2AF1 p.S34F 840 16 Yes

ARID1B p.G386R 150 9 Unknown

CDKN2A p.A118D 342 37 Unknown

TSHZ3 p.E693K 1054 4 Unknown

UCAsC-208 KRAS p.G12D 388 40 Yes

CDKN2A p.H83Y 200 44 Yes

KMT2D p.Q2811fs 669 37 Yes

SMAD4 p.S232fs 216 37 Yes

ARID2 p.Q1823* 742 32 Likely

TP53 p.F113del 421 35 Likely

APC p.R1589H 537 4 Unknown

ARID2 p.A1812D 767 32 Unknown

FGF6 p.R17H 597 5 Unknown

NFE2L2 p.S597R 502 24 Unknown

References

1. Martinko, A.J., et al. Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins. Elife 7(2018).