1 2 3 1 , Hans-Peter Gerber , Frank Loganzo...2017/10/20 · 1 Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1) Matthew Sung1, Xingzhi

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Caveolae-mediated endocytosis as a novel mechanism of resistance to trastuzumab emtansine (T-DM1)

Matthew Sung1, Xingzhi Tan1, Bingwen Lu1, Jonathan Golas1, Christine Hosselet1, Fang Wang1, Laurie

Tylaska2, Lindsay King2, Dahui Zhou3, Russell Dushin3, Jeremy S. Myers1, Edward Rosfjord1, Judy

Lucas1, Hans-Peter Gerber4, Frank Loganzo1

1Pfizer Inc., Oncology Research & Development, Pearl River, NY 2Pfizer Inc., Biomedicine Design, Groton, CT 3Pfizer Inc., Worldwide Medicinal Chemistry, Groton, CT 4Maverick Therapeutics Inc., Brisbane, CA

Running title:

Altered trafficking as mechanism of ADC resistance

Keywords:

T-DM1, Drug Resistance, Biomarker, Caveolin-1, Antibody-Drug Conjugate

Abbreviations:

T-DM1, trastuzumab emtansine;

ADC, antibody-drug conjugate;

T, trastuzumab;

T-ADC, trastuzumab antibody-drug conjugate;

mc, maleimidocaproyl;

vc, mcValCitPABC;

N87-TM, T-DM1-resistant N87 cells;

CAV1, caveolin-1;

Aur0101, auristatin analog;

Aur8261, auristatin analog;

mg/kg, milligrams per kilogram;

Financial Support:

No public funds were used in support of this work.

Corresponding author:

Matthew Sung, Pfizer Oncology, 401 North Middletown Road, 200/4714A, Pearl River, NY 10965.

Phone: 845-602-3731; Email: [email protected]

on July 2, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 20, 2017; DOI: 10.1158/1535-7163.MCT-17-0403

mailto:[email protected]://mct.aacrjournals.org/

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ABSTRACT

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical

benefit for patients with HER2+ metastatic breast cancer, however its clinical activity is limited by

inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1,

especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-

DM1 resistance utilizing a cyclical dosing schema in which cells received T-DM1 in an “on-off” routine

until a T-DM1 resistant population was generated. T-DM1 resistant N87 cells (N87-TM) were cross-

resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells

do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g. MDR1)

expression compared to parental N87 cells. Intriguingly, T-ADCs utilizing auristatin payloads attached

via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-

TM cells implanted into athymic mice formed T-DM1 refractory tumors which remain sensitive to T-

ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment

in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells

internalize T-ADCs into intracellular caveolin-1 (CAV1) positive puncta and alter their trafficking to the

lysosome compared to N87 cells. T-DM1 colocalization into intracellular CAV1 positive puncta

correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest

caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response

to T-DM1.



http://mct.aacrjournals.org/

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INTRODUCTION

Antibody-drug conjugates (ADCs) are emerging therapeutic modalities strategically designed to

selectively deliver chemotherapeutic agents to tumors while limiting non-tumor tissue toxicities. ADCs

are biotherapeutics compromised of a tumor-targeting antibody and a cytotoxic payload attached via a

chemical linker. Once bound to their target antigen, ADCs are internalized and release their cytotoxic

payload. Since the early 2000’s, three ADCs have received FDA-approval and over 50 more are currently

in clinical development (1,2).

Despite encouraging signs of early clinical benefit, the sustained efficacy of many targeted anti-

cancer therapies is limited by either inherent or acquired tumoral drug resistance. The molecular

mechanisms that drive clinical drug resistance are multi-factorial and ultimately depend upon the

therapeutic modality and cancer indication (3). Modifications of the drug target (e.g. kinase mutations)

(4,5), drug efflux protein expression (e.g. ABCB1 in AML) (6-8), and shifts in the expression ratio of

pro- versus anti-apoptotic proteins (e.g. BCL-2 expression in AML) (9) are examples of tumor alterations

that correlate with reduced clinical response to anti-cancer therapies. However, the molecular

mechanisms that drive clinical resistance to ADCs remain largely unknown.

Trastuzumab emtansine (T-DM1, Kadcyla®) currently represents the only FDA-approved ADC

for the treatment of a solid tumor indication. T-DM1 is composed of the trastuzumab antibody

chemically bound to the microtubule-disrupting DM1 maytansinoid via a thioether linker (10). In the

phase III EMILIA clinical trial, T-DM1 showed significantly improved progression free and overall

survival versus lapatinib plus capecitabine in HER2+ metastatic breast cancer patients who had failed

previous treatment with trastuzumab and a taxane (11). The overall response rate for HER2+ patients

treated with T-DM1 was 43.6% (11). On the basis of these results, T-DM1 was granted FDA approval for

treatment of this patient population. However, these data showed that, despite high tumoral HER2-

positivity, over 50% of patients treated with T-DM1 did not show clinical benefit. In the phase II/III

GATSBY clinical trial in HER2+ gastric cancer patients, T-DM1 did not show enhanced clinical response

versus standard chemotherapy (12). Thus, HER2 expression does not solely predict clinical response to




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T-DM1 and the need exists to more accurately define the patient sub-population most likely to respond to

T-DM1.

While data are still emerging from T-DM1-treated patient samples, multiple pre-clinical models

of T-DM1 resistance have been generated to characterize the changes at the tumor cell level that may

mediate clinical resistance to T-DM1. While the models of T-DM1 resistance in these studies were

generated with different dosing schemas, the following are mechanisms responsible for T-DM1 resistance

seen across multiple models: (1) decreased HER2 expression (13,14), (2) increased expression and

activity of drug efflux proteins (13,14), and (3) increased heregulin expression (15).

Herein, we used an ADC structurally similar to T-DM1 (13) to generate T-DM1 resistant cells

in vitro. We report the generation and characterization of three models of T-DM1 resistance, including

one with a unique trafficking defect that has not been previously associated with T-DM1 acquired

resistance and may provide a novel biomarker for clinical response to T-DM1.




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MATERIALS & METHODS

Cell Lines

NCI-N87 gastric carcinoma, HCC1954 breast carcinoma, and BT474 breast carcinoma cells were

obtained from ATCC (Manassas, VA) and maintained in RPMI media supplemented with 10% fetal

bovine serum (FBS), 1% L-glutamine, and 1% sodium pyruvate. H69 and H69AR cell lines were

obtained from ATCC and were maintained in RPMI/25mM HEPES media supplemented with 10% FBS,

1% L-glutamine, and 1% sodium pyruvate. KB, KB8.5, and KBVI cells were generously provided by Dr.

Michael Gottesman (National Cancer Institute). Parental N87 cells were purchased from ATCC (CRL-

5822, lot 58033347) in June 2010. N87 and N87-TM cells were authenticated as NCI-N87 in 2014

(IDEXX BioResearch, Columbia, MO), when the cells were being characterized for this work.

Mycoplasma testing was conducted approximately quarterly using MycoAlert (Lonza, Allendale, NJ), and

results were consistently negative; the most recent testing was conducted in October 2016.

Bioconjugations

ADCs and chemical structures of disclosed payloads described herein were prepared as previously

described (13). The syntheses and structures of the novel auristatin linker-payloads as well as preparation

and characterization of ADCs presented herein are described in the supplementary methods.

Generation of T-DM1 Resistant Cells

Cells were passaged into two separate flasks and each flask was treated identically with respect to the

resistance-generation protocol to enable biological duplicates. Cells were exposed to five cycles of T-

DM1 conjugate at 10-fold IC50 concentrations (10 nmol/L payload concentration; ~388 ng/mL antibody

concentration) for 3 days, followed by approximately 4 to 11 days recovery without treatment. After five

cycles at 10 nmol/L of the T-DM1 conjugate, the cells were exposed to six additional cycles of 100

nmol/L T-DM1 in a similar fashion. The procedure was intended to simulate the chronic, multi-cycle

(on/off) dosing at maximally tolerated doses typically used for cytotoxic therapeutics in the clinic,




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followed by a recovery period. Parental cells derived from NCI-N87 are referred to as N87, and cells

chronically exposed to T-DM1 are referred to as N87-TM. Moderate- to high-level drug resistance

developed within 4 months for N87-TM cells. Drug selection pressure was removed after ~3 - 4 months

of cycle treatments when the level of resistance no longer increased after continued drug exposure.

Responses and phenotypes remained stable in the cultured cell lines for approximately 3 – 6 months.

Thereafter, a reduction in the magnitude of the resistance phenotype as measured by cytotoxicity assays

was occasionally observed, in which case early passage cryo-preserved T-DM1 resistant cells were

thawed for additional studies. All reported characterizations were conducted after removal of T-DM1

selection pressure for at least 2 - 8 weeks to ensure stabilization of the cells. Data were collected from

various thawed cryopreserved populations derived from a single selection, over approximately 1 – 2 years

after model development to ensure consistency in the results. Clonal populations derived from single

N87-TM cells using a limited dilution technique. Briefly, single cells were plated into each well of a 96-

well plate and cultured in the presence of 100 nmol/L T-DM1 until clonal populations were established.

N87 and N87-TM cells were genetically authenticated in 2014 (Idexx Bioresearch, Columbia, MO).

Cytotoxicity Assay

Cellular responses to ADCs and free drugs were measured with a cytotoxicity assay as previously

described (13). Briefly, cells were seeded into 96-well plates and treated with varying concentrations of

ADCs or free drugs for 96 hours. Cell viability was measured using CellTiter® 96 AQueous One MTS

Solution (Promega, Madison, WI). IC50 values were calculated using a four-parameter logistic model

with XLfit (IDBS, Bridgewater, NJ).

Proteomic profiling and immunoblot analyses

Proteomic profiling of membrane fractions was prepared and analyzed as previously described (13).

Whole cell lysates were prepared in RIPA buffer supplemented with protease inhibitor cocktail (Sigma,

St. Louis, MO). Lysates were fractionated on 4-20% SDS-PAGE gels (Bio-Rad, Hercules, CA),




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transferred onto membranes and probed with primary and secondary antibodies further detailed in the

supplementary methods.

Immunofluorescence and Live-Cell Imaging

For immunofluorescence experiments, cells were seeded onto #1.5 coverslips (Electron Microscopy

Services, Hatfield, PA) and fixed with 4% paraformaldehyde. Cells were stained with primary antibodies

at room temperature (RT) for 1 hour and secondary antibodies at RT for 30 min. Coverslips were

mounted onto slides with ProLong® Diamond Antifade Mountant (ThermoFisher Scientific). Images

were acquired with a Zeiss LSM 710 (Carl Zeiss Inc., Thornwood, NY). For imaging cytometry, cells

were incubated with PE-labeled trastuzumab (2 µg/mL) on ice for 90 minutes or at 37°C for time points

indicated. At designated time points, internalization was stopped by addition of cold PBS and cells were

fixed with Cytofix/Cytoperm solution (BD Biosciences) for 20 minutes. Cells were immunostained with

LAMP1-AF647 (BD Biosciences; cat# 562622) for 30 minutes or CAV1 antibody for 30 minutes

followed by an anti-rabbit IgG Fab2 AF647 secondary antibody (ThermoFisher Scientific; cat# A-21246)

for 30 minutes. Cells were analyzed on the ImageStreamX Mark II Imaging Flow Cytometer (AMNIS,

EMD Millipore, Seattle, WA). For live-cell imaging experiments, cells were seeded into 4-well

CELLview dishes (Grenier Bio-One, Austria) and imaged with an ImagEM camera (Hamamatsu

Photonics K.K., Hamamatsu City, Japan) mounted onto a CSU-X1 spinning disk head (Yokogawa

Electric Corporation, Inc., Tokyo, Japan) on an Eclipse Ti microscope stand (Nikon Instruments Inc.,

Melville, NY) equipped with a 60x oil objective (NA1.4, Nikon Instruments Inc.). Image analysis was

performed using Volocity 3D Image software (PerkinElmer, Waltham, MA).

In vivo efficacy studies

All procedures performed on animals in this study were in accordance with established guidelines and

regulations, and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee.

Pfizer animal care facilities that supported this work are fully accredited by AAALAC International.




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Female NOD scid gamma (NSG) immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were

obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were injected subcutaneously in the right

flank with suspensions of either N87 or N87-TM cells (7.5 x 106 cells per injection, with 50% Matrigel).

Mice were randomized into study groups when tumors reached ~0.3 g (~250 mm3). ADCs or vehicle

were administered intravenously in saline on day 0 and repeated for a total of four doses, four days apart

(q4d x 4). Tumors were measured with calipers weekly and tumor mass calculated as volume = (width x

width x length)/2.

Immunohistochemistry

Five micrometer thick formalin fixed, paraffin embedded tissue sections were stained

immunohistochemically. Detailed sample preparations and staining procedures are provided in the

supplementary methods.




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RESULTS

Generation and Characterization of T-DM1 Resistant Cells

To identify potential mechanisms that may drive clinical resistance to T-DM1 ADC, we

generated T-DM1 resistant versions of the HER2-expressing cancer cell lines N87, BT474 and HCC1954.

We utilized a cyclical dosing schema of a 3-day exposure to high dose T-DM1 ADC followed by a

washout and recovery period to mimic patient exposure and pharmacokinetics when they receive bolus-

chemotherapy treatment cycles (Figure 1A).

N87 cells were inherently sensitive to T-DM1 (IC50 = 1.6 nmol/L payload concentration; 62 ng/ml

antibody concentration) (Figure 1B). Two populations of N87 cells were exposed to the treatment cycles

and, after approximately four months of cyclical dosing, these two populations (henceforth named N87-

TM-1 and N87-TM-2) became refractory to the ADC by 109-fold compared with parental N87 cells

(Figure 1B, Table 1). Interestingly, minimal cross-resistance (1.8x) to the corresponding unconjugated

maytansinoid free drug, DM1-SMe, was observed (Supplementary Figure S1, Table 1) which suggests the

alterations in N87-TM cells that lead to T-DM1 resistance are specific to the whole ADC biomolecule and

not solely to the payload.

Previous reports of T-DM1 resistance in other cell models have implicated ATP-binding cassette

(ABC) transporter protein overexpression (e.g. ABCB1/MDR1, ABCC1/MRP1) or target antigen down-

regulation as key drivers of T-DM1 resistance (13,14). However, proteomics and immunoblots

demonstrate that N87-TM cells do not overexpress MDR1 (Figure 1C) or MRP1 (Figure 1D) and retain

similar levels of HER2 as compared to parental N87 cells (Figure 1E). Despite retaining high levels of

HER2, N87-TM cells display ~50% reduced binding to the unconjugated antibody of T-DM1 (i.e.

trastuzumab) as measured by flow cytometry (Figure S2). In contrast, T-DM1 resistant populations of

BT474 (BT474-TM) and HCC1954 (HCC1954-TM), selected in a similar manner as N87-TM, show

significantly decreased HER2 expression (Supplementary Figures S3A & S3B), suggesting that reduced

antigen is the primary mode of resistance in these two cell models. Hence, we focused additional

characterization on the N87-TM model.




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Taking advantage of the modular nature of ADCs, we hypothesized that altering key components

of the biomolecule would result in an agent able to overcome T-DM1 resistance in N87-TM cells. N87-

TM cells displayed cross-resistance to several trastuzumab-based ADCs (T-ADCs) (Figure 1F and Table

1) which were conjugated to auristatins via a non-cleavable maleimide linker. Remarkably, N87-TM

were sensitive to T-ADCs conjugated to auristatins utilizing a protease-cleavable linker sequence (ValCit-

PAB) in an antigen-dependent manner (Figure 1G, Supplementary Figure S4 and Table 1, Supplementary

Table S1). The sensitivity of N87-TM cells to other trastuzumab-based ADCs suggests that the reduced

level of HER2 in these cells is not a mode of resistance.

To determine if the ADC-resistant N87-TM cells were broadly resistant to other standard-of-care

chemotherapeutics, N87 and N87-TM cells were treated with DNA-damaging agents (e.g., cisplatin,

doxorubuicin), kinase inhibitors (e.g., rapamycin, dacomitinib) and microtubule inhibitors (e.g.,

docetaxel, vinblastine). Importantly, N87-TM cells remained sensitive to these small molecule inhibitors

when compared to the parental N87 cells, discounting general growth defects as the mechanism of

resistance to T-DM1 ADC (Supplementary Figure S4, Table 1).

N87-TM tumors maintain T-DM1 resistance in vivo

A problem that often plagues in vitro drug resistance models is their lack of translatability into the

in vivo setting. In order to determine if the resistance observed in cell culture was recapitulated in vivo,

N87 cells and N87-TM cells were injected into the flanks of female NOD scid gamma (NSG)

immunodeficient mice and treated with T-DM1 ADC (at doses of 6 mg/kg (mpk) and 10 mpk) and a T-

ADC with a cleavable-linked auristatin (T-vc-Aur0101) (at 3 mpk), on a q4d X 4 schedule. The N87

tumors retained high expression of HER2 in vivo and were quite sensitive to all three T-ADC treatment

conditions (Figure 2A). Remarkably, N87-TM tumors also retained high expression of HER2 in vivo and

were refractory to both regimens of T-DM1 ADC, yet tumors regressed with T-vc-Aur0101 (Figure 2B).

Thus, N87-TM tumors retain a similar response profile to T-DM1 and T-vc-Aur0101 ADCs in vitro and

in vivo.




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In order to better understand how N87-TM tumors are resistant to T-DM1 and sensitive to T-vc-

Aur0101, we first interrogated the tumoral distribution of the ADCs. N87-TM tumors, from the

previously described efficacy study, were collected 24 hours following the last ADC dose, and ADC

distribution was evaluated through immunohistochemistry staining with an anti-human IgG antibody.

N87-TM tumors treated with 6 mpk T-DM1 ADC displayed almost complete tumor coverage of IgG

binding, whereas those treated with 3 mpk T-vc-Aur0101 only had ADC binding at the most vessel-

proximal regions of the tumor (Supplementary Figure S5). Thus, tumoral ADC distribution is unlikely to

explain the efficacy response of these ADCs in N87-TM tumors.

Next, we evaluated a biomarker of downstream response to an anti-microtubule targeted agent to

determine how T-DM1 and T-vc-Aur0101 ADCs differentially impacted N87 and N87-TM tumors.

Phosphorylation of histone H3 (pHH3) is a marker of mitotic cells and can be used to evaluate the activity

of anti-mitotic agents. To determine if N87-TM tumors had reduced pHH3 staining following T-DM1

ADC treatment as compared to N87 tumors, mice were administered a single-dose of T-DM1 ADC (6

mpk) and tumors were collected 24 hours later. Co-immunostaining revealed ADC and pHH3 double

positive cells in the N87 tumors (Figure 2C, top left panel), whereas the majority of the ADC-bound N87-

TM tumor cells were pHH3 negative (Figure 2C, bottom left panel). These data are consistent with the

efficacy data showing N87, but not N87-TM, tumors are sensitive to T-DM1 ADC. In a similar study

conducted with T-vc-Aur0101 ADC (3 mpk), N87 and N87-TM tumors had ADC and pHH3 double

positive cells (Figure 2C, top and bottom right panels, respectively). Intriguingly, both T-vc-Aur0101

ADC treated tumors display many pHH3 positive cells without ADC-bound to them, a property known as

bystander effect whereby antigen negative tumor cells neighboring antigen positive tumor cells are

susceptible to membrane permeable payloads. Thus, it is clear from this in vivo biomarker study that T-

ADCs utilizing a cleavable linker that releases a membrane permeable cytotoxic payload can overcome T-

DM1 resistance in N87-TM tumors.

The active released species from a catabolized T-DM1 molecule is the amino acid capped linker

payload, Lysine-MCC-DM1 (16,17). The structural and ionic nature of Lysine-MCC-DM1 in the tumor




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microenvironment renders the molecule membrane impermeable and is likely why T-DM1 does not show

strong bystander effect (18). To determine if amino-acid capping of the released ADC species dictates

differential ADC cytotoxic response in N87-TM as compared to N87 cells, we prepared a series of ADCs

utilizing the auristatin analog (Aur8261) conjugated to the same targeting antibody (trastuzumab) via

different linkers (19). As shown above, N87-TM cells are cross-resistant to a T-ADC with a non-

cleavable-linked payload with a released species of cysteine-mc-Aur8261. However, N87-TM cells are

sensitive to a T-ADC with Aur8261 attached via a cleavable linker where the released species is the

unmodified, membrane-permeable auristatin compound. N87-TM cells were cross-resistant to a T-ADC

with a cleavable linker but with a released species of cysteine-mc-Aur8261 (Supplementary Figure S6).

Thus, T-ADCs whose active catabolite is an amino-capped linker payload are effective in killing N87, but

not N87-TM, cells. Taken together, these data show that N87-TM tumors maintain T-DM1 resistance in

vivo and T-ADCs with cleavable linkers and permeable payloads overcome T-DM1 resistance in vitro and

in vivo.

The role of CAV1 in ADC biology and resistance

To further characterize the mechanism of T-DM1 resistance in N87-TM cells in an unbiased

approach, we performed a proteomic evaluation of membrane fractions to profile proteins differentially

expressed in the N87-TM cells as compared to the parental N87 cells. Significant changes in expression

level of 523 proteins between the cell lines were observed (Figure 3A). Immunoblot analysis validated a

number of the proteomic hits (Figure 3B). Of interest was the over-expression of caveolin-1 (CAV1) and

polymerase I and transcript release factor (PTRF) in N87-TM cells. As CAV1 and PTRF are essential

protein constituents of caveolae (20), we hypothesized that N87-TM cells are enriched for caveolar

endocytic compartments. Caveolae-mediated endocytosis is thought to be induced by Src-mediated

phosphyloration of CAV1 at Tyr 14 (21). Indeed, N87-TM cells have enhanced phospho-Src and

phospho-CAV1 as compared to N87 cells (Figure 3C). Immunofluorescence for CAV1 in N87 and N87-




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TM cells demonstrates that N87-TM cells contain many more intracellular CAV1+ puncta than N87 cells

(Figure 3D).

We next hypothesized that N87-TM cells can internalize T-DM1 into these caveolar endocytic

compartments. We treated N87 and N87-TM cells with T-DM1 for 24 hours and co-immunostained for

anti-human IgG and CAV1 to interrogate the co-localization of the ADC with CAV1+ puncta. Indeed, T-

DM1 ADC co-localized with CAV1 in N87-TM, but not N87, cells (Figure 3E). To determine if this

positive correlation between T-DM1 ADC and CAV1 occurred at the early phases of ADC endocytosis,

we analyzed trastuzumab and CAV1 co-localization at multiple early time points using imaging

cytometry. N87-TM, but not N87, cells showed enhanced trastuzumab and CAV1 co-localization over

time (Figure 3F). Since the internalization of trastuzumab was into differential compartments in N87 and

N87-TM cells, we next sought to determine if N87-TM cells displayed impaired lysosomal trafficking of

trastuzumab because T-DM1 is catabolized in the lysosome. Lysosomal co-localization of trastuzumab

was decreased in N87-TM as compared to N87 cells (Figure 3F).

Caveolar endocytic compartments are phenotypically distinct from the endo-lysosomal pathway

(22). For example, the lumina of caveolar endocytic compartments have a neutral pH, whereas the

lumina of endo-lysosomal compartments are increasingly acidic during pathway maturation (23). To

determine if T-ADCs were internalized into compartments with different pH, we generated a T-ADC

conjugated to two fluorescent moieties: (1) a pH-insensitive AlexaFluor-modified auristatin non-

cleavable linker payload and (2) a pH-sensitive dye, pHRodo, whereby the fluorescence signal is

proportional to the acidity of the micro-environment. We performed live-cell imaging with this

fluorescent T-ADC monitoring ADC internalization into pHRodo-positive areas within the cell. As

expected, T-ADC showed increasingly higher co-localization with low pH compartments through the

time course in N87 cells (Supplementary Figure S7A, black line). However, the T-ADC did not traffic

into low pH compartments in N87-TM cells during the same time course (Supplementary Figure S7A, red

lines). Using the pH-insensitive AlexaFluor signal intensity as a normalization factor, the distribution of




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T-ADC into low pH compartments was decreased in N87-TM cells as compared to N87 cells

(Supplementary Figure S7B).

Caveolae-mediated ADC internalization is likely to be the predominant mechanism by which T-

ADCs enter N87-TM, but not N87, cells. To determine if we could induce caveolae-mediated ADC

internalization in N87 cells, we transfected CAV1-GFP into N87 cells and performed live-cell imaging.

CAV1-GFP introduction resulted in the formation of similar intracellular CAV1+ puncta as N87-TM cells

(Supplementary Figure S8). Interestingly, T-ADC containing an AlexaFluor-modified auristatin non-

cleavable linker payload internalized into the induced caveolar endocytic compartments in N87 cells

(Supplementary Figure S8).

These data suggest T-ADC internalization in N87-TM cells is mediated via caveolae endocytosis.

We next hypothesized that this caveolae-mediated endocytosis drives resistance to T-DM1 in N87-TM

cells. To determine if CAV1 knockdown re-sensitized N87-TM cells to T-DM1, we performed

doxycycline (DOX)-controlled shRNA-mediated CAV1 knockdown in N87-TM cells. Following DOX

withdrawal, CAV1 targeted shRNAs were expressed and resulted in reduced CAV1 protein levels as

compared to a non-targeted control sequence (Supplementary Figure S9). However, CAV1 knockdown

was not sufficient to re-sensitize N87-TM cells to T-DM1 (Supplementary Figure S9). Taken together,

these data suggest that N87-TM cells are enriched for caveolar endocytic compartments capable of

internalizing T-DM1 and differentially providing an alternative route for ADC endocytosis and

trafficking.

Caveolae-mediated T-DM1 endocytosis across HER2+ cell lines

Caveolae-mediated endocytosis has been implicated as a possible mechanism of inherent

insensitivity to ADCs targeting other antigens (e.g. melanotransferrin) (24). We investigated a panel of

HER2+ cell lines to determine their inherent sensitivity to T-DM1 and whether CAV1 protein level

correlated with the magnitude of T-DM1 response. We did not observe a positive correlation between

CAV1 protein level and decreased T-DM1 sensitivity in this panel of HER2+ cell lines (Figure 4A).




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Given that high levels of CAV1 protein alone do not necessarily induce the formation of caveolar

endocytic compartments (25), we next determined the propensity for caveolae-mediated T-DM1

internalization across this cell line panel. Interestingly, some CAV1 high cell lines (e.g. JIMT1, N87-

TM) showed colocalization of T-DM1 and CAV1+ puncta whereas other CAV1 high cell lines (e.g.

SKOV3, HCC1954) did not (Figure 4B; similar data observed with T-vc-Aur0101, Supplementary Figure

S10). Intriguingly, the amount of ADC colocalization with CAV1+ puncta positively correlated with

reduced response to T-DM1 across this cell line panel (Figure 4C). Collectively, these data suggest that

the inherent proclivity of tumor cells to internalize T-DM1 via caveolae-mediated endocytosis may

predict their response to the ADC.




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DISCUSSION

In this study, we generated and characterized multiple in vitro models of T-DM1 resistance

utilizing a drug selection paradigm designed to mimic the clinical dosing regimen of T-DM1. Using this

dosing schema, two models (BT474-TM and HCC1954-TM) acquired T-DM1 resistance via a previously

reported mechanism (i.e. decreased HER2 expression) and another model (N87-TM) acquired T-DM1

resistance likely via a novel mechanism of differential ADC internalization and trafficking. Herein, we

report the first model of acquired T-DM1 resistance that implicates caveolae-mediated endocytosis of T-

DM1 as a novel mechanism of T-DM1 resistance.

Despite using the same approach to generate resistance, these three models and our two

previously reported models of T-DM1 resistance (13) highlight the diversity of molecular mechanisms

that cells may adopt to overcome the challenge of chronic drug exposure. While the background genetics

of each model may dictate the eventual mechanism of resistance to a drug, one might expect a myriad of

changes that may impact a tumor’s sensitivity to T-DM1 given the complexity of its mechanism of action.

For T-DM1 to effectively kill a tumor cell, it must (1) bind to HER2; (2) internalize into the intracellular

portion of the cell; (3) transit through the endosomal maturation pathway with final delivery to the

lysosome; (4) be catabolized within the lysosomal milieu releasing a membrane impermeable payload that

is likely transported out of the lysosome via a lysosomal membrane transporter to interact with tubulin

(the target of DM1). Indeed, there are examples of potential alterations to some of these steps in

experimental models that may ultimately lead to T-DM1 resistance. As previously mentioned, decreased

HER2 expression could prevent sufficient T-DM1 binding but other factors have been shown to influence

trastuzumab binding to HER2, including the accumulation of the truncated p95-HER2 isoform that lacks

a trastuzumab binding site (26). Efficient internalization of T-DM1 may not always be achieved since

trastuzumab-HER2 complexes have been shown to have a rapid rate of plasma membrane recycling once

they are internalized (27). It remains to be seen if a similar phenomenon occurs with T-DM1-HER2

complexes. Lysosomal delivery of T-DM1 is another key event and the results of this study show that

alternative T-DM1 internalization and trafficking parameters can influence lysosomal accumulation of T-




17

DM1. The mechanism of lysosomal escape of lysine-MCC-DM1 (the released species of T-DM1) has

long been debated but the recent description of SLC46A3 as a lysosomal membrane transporter protein

that mediates the lysosome-to-cytoplasm transfer of this metabolite has provided insights into potential

mechanisms of transport (28).

Despite the complexity of these biomolecules, the modular nature of ADCs allows for the

interchanging of various components to assess their influence on the properties of the ADC. The T-DM1-

resistant N87-TM cells were cross-resistant to trastuzumab-ADCs delivering other non-cleavable linked

tubulin inhibitors. By switching the linker to an enzymatically cleavable linker, ADCs carrying other

microtubule-targeting payloads (e.g. ValCit-linked auristatins) were able to overcome T-DM1 resistance

in N87-TM cells. In a similar fashion, cell line models made resistant to CD22- or CD79b-targeting

ADCs employing the vcMMAE linker-payload remained sensitive to the respective ADCs delivering a

different payload (e.g. PNU-159682) (29). Thus, by swapping each module of an ADC with a different

functional group, ADC activity can be modulated. This “component switching” property of ADCs

implies that patients with ADC-refractory tumors may still derive benefit from a similar ADC, targeting

the same antigen, but with altered composition.

This report implicates caveolae-mediated endocytosis of T-DM1 as a potential mechanism of

acquired resistance, yet trastuzumab-ADCs delivering tubulin inhibitors with an enzymatically cleavable

linker overcame T-DM1 resistance. It is important to note that caveolae contain enzymatically-active

cysteine cathepsins capable of processing the ‘ValCit’ linker utilized herein (30). Thus, while the

caveolar microenvironment may not favor the antibody catabolism necessary to release the non-cleavable

linker-payload of T-DM1, it may be suitable to process enzymatically cleavable linkers. Thus, while the

caveolar microenvironment may not favor the antibody catabolism necessary to release the non-cleavable

linker-payload of T-DM1, it may be suitable to process enzymatically cleavable linkers. The impact of

caveolae-mediated endocytosis of other ADCs has been reported as well. Melanotransferrin- and CD133-

targeting ADCs showed potent cytotoxic activity in antigen-positive cell lines where the ADC

preferentially accumulated in lysosomes; however, the conjugates were not active in antigen-positive cell




18

lines in which the ADC colocalized with caveolin-1 (24,31). Both of these ADCs employed the

‘vcMMAF’ linker-payload which contains the same enzymatically cleavable dipeptide linker used here

but releases a different payload (monomethyl auristatin F, MMAF). Due to its carboxylic acid functional

group, MMAF is a charged species at neutral pH; thus, while the linker may be processed in caveolae, the

charged nature of MMAF renders it membrane impermeable and not able to enter into the cytoplasm of

the cell. It is this property that prevents ADCs utilizing MMAF from having substantial bystander effect

(2). Along similar lines, a CD20-targeting ADC utilizing a ‘vcMMAE’ linker was reported to be

internalized via both clathrin- and caveolae-mediated endocytosis; however, the nature of endocytosis did

not impact sensitivity of antigen-positive cell lines to the ADC (32). Unlike MMAF, monomethyl

auristatin E (MMAE) does not exist as a charged molecule at neutral pH and is membrane permeable.

Data herein implicate caveolae-mediated endocytosis in T-DM1 resistance; however, another group has

suggested that caveolae-mediated endocytosis may sensitize cells to T-DM1 (33). While these data

implicate a possible role for CAV1, the inherent differences in sensitivity to free DM1 (18) may explain

the differences in basal sensitivity to T-DM1 seen across the high HER2 cell lines evaluated in that study.

The functional studies in this report implicate a role for caveolae-medicated endocytosis in the

mechanism of resistance to T-DM1 in N87-TM cells. However, CAV1 knockdown was not sufficient to

re-sensitize N87-TM cells to T-DM1. Incomplete CAV1 protein loss may prevent re-sensitization, or as

eluded to previously, the formation of caveolae is not strictly dictated by the level of CAV1 expression,

but rather the interplay of caveolar-membrane coat proteins (caveolin family) interacting with caveolar-

membrane stabilizing scaffold proteins (cavin family) (20). N87-TM cells are enriched for proteins from

both families (i.e. CAV1 and PTRF). Thus, it may be that the driver of caveolae formation in N87-TM

cells is a combination of altered proteins. Indeed, another caveolar coat protein may be compensating for

the loss of CAV1 in the CAV1-knockdown N87-TM cells which, in conjunction with the enhanced

expression of PTRF, allows for the continued formation of caveolae. Along similar lines, CAV1

expression solely did not correlate with T-DM1 sensitivity across a panel of HER2+ cell lines; rather, the

functional aptitude for cells to internalize/traffic T-DM1 into caveolar compartments correlated with T-




19

DM1 sensitivity. The function of caveolae in cancer biology remains largely unknown. However, this

report adds to the growing body of literature that suggests caveolae may dictate sensitivity of cancer cells

to ADCs that utilize certain types of linker-payloads.

It has become increasingly clear that ‘precision medicine’ will only be as effective as our ability

to define patient sub-populations through the use of reliable biomarkers. For many targeted therapies,

such as ADCs, the paramount biomarker is the expression of the target for that agent (e.g., HER2 for T-

DM1). However, given tumoral genetic complexity and heterogeneity, target expression does not solely

predict a patient’s tumor sensitivity to a targeted therapy, particularly in solid tumor indications. For

example, in the EMILIA trial, only 43% of breast cancer patients with high HER2-expressing tumors

responded to T-DM1 (11). Thus, there continues to be an unmet need in our ability to better identify

patients most likely to respond to T-DM1 and other targeted therapies. While alterations in some genes

(e.g. EGFR, HER3, and PTEN expression and PIK3CA mutations) (34) have been found to not correlate

with T-DM1 response, data in this report warrant the investigation of caveolae-mediated endocytosis of

T-DM1 in patient tumors as a potential predictive biomarker for response to T-DM1 and other non-

cleavable linked ADCs.




20

ACKNOWLEDGEMENTS

The authors appreciate the contributions of Pfizer Oncology colleagues Elwira Muszynska,

Nadira Prashad, Kiran Khandke, Manoj Charati, William Hu, My-Hanh Lam, Sylvia Musto, Xiang

Zheng, Anton Xavier, Andreas Giannakou, Mike Cinque, Eric Upeslacis, Anna Maria Barbuti, and

Katherine Yao for technical assistance. The authors also thank Pfizer Medicinal Chemistry colleagues

Jeff Casavant, Zecheng Chen, Matthew Doroski, Gary Filzen, Andreas Maderna, Ludivine Moine,

Alexander Porte, Hud Risley, and Christopher O’Donnell for design and synthesis of the payloads, linker-

payloads and conjugates utilized in this study.




21

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24

TABLES

N87: Parental versus TM-resistant

Compound Linker N87 N87-TM-2 Relative

name type

(IC50,

nmol/L)

(IC50,

nmol/L) resistance

T-MCC_DM1 NC 1.6 174 109x

T_mc_Aur8261 NC 5.3 >1,000 >188x

T_mc_MMAD NC 16 492 31x

T_mc_Aur0101 NC 138 >1,000 >7x

T_vc_Aur8261 C 0.43 0.49 1.1x

T-vc_MMAD C 0.4 0.88 2.2x

T_vc_Aur0101 C 1.1 0.8 0.73x

T_vc_Cys-mc-

Aur8261 C 3.9 136 35x

DM1-Sme -- 19 35 1.8x

Aur-8254 (8261) -- 0.61 0.92 1.5x

MMAD -- 0.14 0.49 3.5x

Aur-0101 -- 1.5 1.9 1.3x

Cys_mc-Aur8261 -- 2,334 4,392 1.9x

Vinblastine -- 3.6 4.8 1.3x

Docetaxel -- 2.9 3.8 1.3x

Doxorubicin -- 38 37 1.0x

5-fluorouracil -- 1,611 3,135 1.9x

Camptothecin -- 21 9.8 0.47x

Gemcitabine -- 7.3 8.4 1.2x

Oxaliplatin -- 623 1,511 2.4x

Temsirolimus -- 2,131 6,706 3.1x

Rapamycin -- 4,682 12,403 2.6x

Dacomitinib -- 7.8 33 4.2x

Pelitinib -- 42 119 2.8x

Neratinib -- 2 11 5.5x

NOTE: IC50 values of ADCs reported here represent payload concentrations.

Data are mean of multiple determinations. These relative resistance values are

represented graphically in Supplementary Figure S4. Relative resistance is the

ratio of the mean IC50 for the TM-resistant cell line versus the parental cell

line. NC = non-cleavable, C = cleavable

Table 1. Resistance profiles of N87 and N87-TM cells to ADCs, standard-of-care

chemotherapeutics and other unconjugated drugs.




25

FIGURE LEGENDS

Figure 1: N87-TM model generation and characterization.

(A) Schematic model of cyclical dosing paradigm used to generate T-DM1 resistant cells. Parental cells

were dosed with T-DM1 for three days, followed by a washout and recovery period. This process was

cycled multiple times until a T-DM1 resistant population emerged.

(B) Parental N87 (closed circle) and two T-DM1 resistant populations (open square and open circle),

selected simultaneously (N87-TM-1 and N87-TM-2), were treated with T-DM1 for 4 days. Cell viability

is plotted against ADC payload concentration.

(C-E) Whole cell lysates from parental and T-DM1-resistant N87 cells were separated by SDS-PAGE

and levels of MDR1 (C), MRP1 (D), and HER2 (E) were determined by immunoblot analyses. Whole

cell lysates from positive and negative control cell lines for MDR1 (KB85 and KBV1) and MRP1 (A549

and H69AR) are shown.

(F) Parental and T-DM1-resistant N87 cells were treated with a trastuzumab-ADC conjugated to a non-

cleavable linker and auristatin payload for 4 days. Cell viability is plotted against ADC payload

concentration.

(G) Parental and T-DM1-resistant N87 cells were treated with a trastuzumab-ADC conjugated to a

cleavable-linked auristatin payload for 4 days. Cell viability is plotted against ADC payload

concentration.

Figure 2: N87-TM cells retain T-DM1 resistance and T-vc-Aur0101 sensitivity in vivo.

(A-B) Parental (A) and T-DM1 resistant (B) N87 cells were implanted into NOD-SCID mice and treated

with ADCs when the average tumor volume of 250 mm3 was achieved. Mice were dosed q4dx4 with

vehicle (black circles), T-DM1 at 6 mg/kg (mpk) (red squares), T-DM1 at 10 mpk (red triangles), T-vc-

Aur0101 at 3 mpk (blue diamonds), or a negative control, isotype ADC with ‘vc-Aur0101’ at 3 mpk

(hashed blue line with downward-pointing triangles). Tumors at staging size for each cell line were

collected, formalin fixed and immunostained for HER2 by immunohistochemistry (insets).




26

(C) N87 and N87-TM tumors were treated with a single dose of T-DM1 at 6 mpk, T-vc-Aur0101 at 3

mpk or a negative control, isotype ADC with ‘vc-Aur0101’ at 3 mpk. After 24 hours, mice were perfused

and tumors formalin fixed and co-immunostained for anti-human IgG (brown color) and phospho-histone

H3 (pHH3) (purple color). Bystander effect is denoted by the prevalence of pHH3+ cells devoid of ADC

binding.

Figure 3: N87-TM cells are enriched for intracellular caveolar compartments and internalize

trastuzumab-ADCs via caveolae-mediated endocytosis.

(A) Membrane fractions from N87 and N87-TM cells were prepared for proteomic profiling. A volcano

plot is shown representing the relative fold change for each protein (x-axis) by the significance level for

each protein. Points in the upper right quadrant of the plot represent proteins enriched in N87-TM cells as

compared to N87 cells.

(B) Whole cell lysates of N87 and N87-TM cells were prepared, separated by SDS-PAGE and

immunoblotted for proteins identified in the proteomic profiling to validate the hits.

(C) Whole cell lysates from N87 and N87-TM cells were separated by SDS-PAGE and levels of total

SRC, phospho-SRC, total CAV1, and phospho-CAV1 were determined by immunoblot analysis.

(D) CAV1+ puncta were identified via immunofluorescence staining of N87 and N87-TM cells.

Average of number of puncta calculated as total number of CAV1+ puncta normalized by number of

nuclei for each field of view.

(E) N87 and N87-TM cells were treated with T-DM1 overnight at 37°C, fixed and co-immunostained

with anti-human IgG (green color) and anti-CAV1 (red color). Image analysis was performed to

determine co-localization between T-DM1 and CAV1 in N87 and N87-TM cells.

(F) N87 and N87-TM cells were incubated with fluorescently-labelled trastuzumab and kinetic co-

localization analysis between T-DM1 and CAV1 and LAMP1 markers were performed via image

cytometry.




27

Figure 4: Caveolae-mediated endocytosis of T-DM1 predicts reduced sensitivity to T-DM1.

(A) A panel of breast and gastric cancer cell lines were evaluated for their relative HER2 and CAV1 at

basal state by immunoblot analysis. CAV1 score was assigned based on basal CAV1 expression from

this experiment. The same panel of cell lines was tested for sensitivity to T-DM1 when treated for 4 days

in a cell viability assay. The correlation of CAV1 score vs. T-DM1 IC50 is represented.

(B) The panel of cell lines were incubated with T-DM1 and analyzed for co-localization to CAV1+

puncta as previously described in Figure 3. Representative images from each cell line show T-DM1

(green color) and CAV1 (red color) immunostaining. White arrows indicate examples of T-DM1 co-

localized with CAV1+ puncta (yellow/orange color).

(C) The T-DM1 and CAV1 co-localization scores from (B) for each cell line were plotted against IC50 of

each cell line to T-DM1. The correlation between T-DM1 and CAV1 colocalization score vs. T-DM1

IC50 is represented.




A

Figure 1

Naïve Cells

“N87”

ON OFF

T-DM1-resistant cells

“N87-TM”

Treatment: T-DM1 ADC @ 10X IC50

ON OFF ON OFF

B

C

N8

7

N8

7-T

M-1

N8

7-T

M-2

IB: HER2

IB: Actin

D

E F

G

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

C o n c (n M )

% S

urv

iva

l

N 87

N 8 7 -T M -1

N 8 7 -T M -2

T -D M 1

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

C o n c (n M )

% S

urv

iva

l

N 87

N 8 7 -T M -1

N 8 7 -T M -2

T -v c -A u r 0 1 0 1

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 5

5 0

7 5

1 0 0

1 2 5

C o n c (n M )

% S

urv

iva

l

N 87

N 8 7 -T M -1

N 8 7 -T M -2

T -m c -A u r 8 2 6 1




IHC: HER2

IHC: HER2

A

B

C T-vc-Aur-0101 T-DM1

N87

N87-TM

IHC: Hu IgG, pHH3

0 2 0 4 0 6 0 8 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

1 8 0 0

2 0 0 0

T im e (D a y s )

Tu

mo

r v

ol

(mm

3)

V e h ic le (P B S )

T -D M 1 @ 6 m p k

T -D M 1 @ 1 0 m p k

T -v c -A u r0 1 0 1 @ 3 m p k

N 8 7

N e g C tl-v c -A u r0 1 0 1 @ 3 m p k

0 2 0 4 0 6 0 8 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

1 8 0 0

2 0 0 0

T im e (D a y s )

Tu

mo

r v

ol

(mm

3)

V e h ic le (P B S )

T -D M 1 @ 6 m p k

T -D M 1 @ 1 0 m p k

T -v c -A u r0 1 0 1 @ 3 m p k

N 8 7 -T M

N e g C tl-v c -A u r0 1 0 1 @ 3 m p k

NegCtl-vc-Aur-0101

Figure 2




A B

C

E

Down in N87-TM

Up in N87-TM

Fold Change (log2)

Sign

ific

ance

Sco

re

IB: IGF2R

IB: LAMP1

IB: Tubulin

N87 N87-TM

IB: CAV1

IB: PTRF

IB: Tubulin

IB: Clathrin

N87 N87-TM

N87

N87-T

M

0

2 0

4 0

6 0

8 0

1 0 0

# o

f C

AV

1 p

un

cta

pe

r c

ell

N87

N87-T

M

0 .0

0 .2

0 .4

0 .6

0 .8

A D C C o lo c a liz a t io n w ith C A V 1 + p u n c ta

a fte r 2 4 h o u r A D C tr e a tm e n t

Ov

erla

p C

orre

lati

on

R

0 1 0 0 2 0 0 3 0 0 4 0 0

0 .2

0 .4

0 .6

0 .8

T im e (m in )

Co

loc

ali

za

tio

n S

co

re

N 87

N 87-T M

C A V 1 C o lo c a liz a t io n w ith T r a s tu z u m a b

0 1 0 0 2 0 0 3 0 0

0 .2

0 .4

0 .6

0 .8

T im e (m in )

Co

loc

ali

za

tio

n S

co

re

N 87

N 87-T M

L y s o s o m e C o lo c a liz a t io n w ith T ra s tu z u m a b

IB: pSRC (Y416)

IB: CAV1

IB: pCAV1 (Y14)

IB: Tubulin

N87 N87-TM

IB: SRC

D

F

Figure 3




A B N87 N87-TM

BT474 HCC1954

SKBR3 SKOV3

JIMT1

C

0 .1 1 1 0 1 0 0 1 0 0 0

0 .0

0 .5

1 .0

1 .5

[T -D M 1 ] (P a y lo a d n M )

AD

C:C

AV

1 C

olo

ca

liz

ati

on

N 8 7

N 8 7 -T M

B T 4 7 4H C C 1 9 5 4

J IM T 1

S K O V 3S K B R 3

R2

= 0 .7 4

p = 0 .0 1

IB: HER2

IB: CAV1

IB: Tubulin

0 .1 1 1 0 1 0 0 1 0 0 0

0

1

2

3

[T -D M 1 ] (P a y lo a d n M )

CA

V1

Sc

ore

(im

mu

no

blo

t)

N 8 7

N 8 7 -T M

B T 4 7 4

H C C 1 9 5 4

J IM T 1S K O V 3

S K B R 3

R2

= 0 .3 0

p = 0 .2 0

Figure 4




Published OnlineFirst October 20, 2017.Mol Cancer Ther Matthew Sung, Xingzhi Tan, Bingwen Lu, et al. resistance to trastuzumab emtansine (T-DM1)Caveolae-mediated endocytosis as a novel mechanism of

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