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Author Manuscript Published OnlineFirst on August 3, 2015; DOI: 10.1158/1078-0432.CCR-15-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Preclinical Efficacy and Safety Assessment of an Antibody Drug Conjugate Targeting the c-RET Proto-oncogene for Breast Carcinoma

Minh Nguyen1*, Shuichi Miyakawa1, Junichi Kato1, Toshiyuki Mori2*, Toshimitsu Arai2, Mark Armanini1*, Karen Gelmon3, Rinat Yerushalmi3*, Samuel Leung3*, Dongxia Gao3*, Gregory Landes1*, Mary Haak-Frendscho1*, Kathleen Elias1, Andrew D. Simmons1*

1Takeda California, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA; 2Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan; 3British Columbia Cancer Agency, Vancouver, BC, Canada

*Current affiliations: Andrew D. Simmons, Clovis Oncology, Inc.; Minh Nguyen, Clovis Oncology, Inc.; Toshiyuki Mori, Whiz Partners, Inc.; Mark Armanini, Teva Pharmaceutical Industries, Ltd.; Rinat Yerushalmi, Rabin Medical Center; Samuel Leung and Dongxia Gao, Genetic Pathology Evaluation Center; Gregory Landes, DNA Bridges, Inc.; Mary Haak-Frendscho, Igenica, Inc.

Minh Nguyen: [email protected] Shuichi Miyakawa: [email protected] Junichi Kato: [email protected] Toshiyuki Mori: [email protected] Toshimitsu Arai: [email protected] Mark Armanini: [email protected] Karen Gelmon: [email protected] Rinat Yerushalmi: [email protected] Samuel Leung: [email protected] Dongxia Gao: [email protected] Gregory Landes: [email protected] Mary Haak-Frendscho: [email protected] Kathleen Elias: [email protected] Andrew D. Simmons: [email protected]

Type of manuscript: Preclinical Therapeutics Running Title: Pharmacologic Effects of RET Antibody Drug Conjugates Key words: breast cancer, RET, antibody drug conjugate, safety, expression

Funding/Financial Support: Financial support for the conduct of this study was provided by Takeda Pharmaceuticals International Co. Financial support for medical editorial assistance was provided by Millennium: The Takeda Oncology Company.

Corresponding author: Andrew D. Simmons, PhD, Associate Director, Translational Medicine, Clovis Oncology, Inc, 499 Illinois Street, Suite 230, San Francisco, CA 94158; Phone: 415-409- 5452; Fax: 415-552-3427; [email protected]

Disclosure: M. Nguyen, S. Miyakawa, J. Kato, M. Armanini, G. Landes, M. Haak-Frendscho, K. Elias, and A.D. Simmons, were employees of Takeda California, Inc. when the research was performed; T. Mori, and T. Arai were employees of Takeda Pharmaceutical Company Ltd. when the research was performed. K. Gelmon, R. Yerushalmi, S. Leung, and D. Gao report no potential conflict of interest.

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2015; DOI: 10.1158/1078-0432.CCR-15-0468 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Nguyen et al Page 2 Pharmacologic Effects of RET Antibody Drug Conjugates

ABSTRACT

Purpose: The RET proto-oncogene has been implicated in breast cancer, and the studies herein describe the preclinical and safety assessment of an anti-RET antibody drug conjugate

(ADC) being developed for the treatment of breast cancer.

Experimental Design: RET protein expression was analyzed in breast tumor samples using tissue microarrays. The fully human anti-RET antibody (Y078) was conjugated to the DM1 and

DM4 derivatives of the potent cytotoxic agent maytansine using thioether and disulfide linkers, respectively. The resulting compounds, designated Y078-DM1 and Y078-DM4, were evaluated for antitumor activity using human breast cancer cell lines and established tumor xenograft models. A single-dose, 28-day, safety study of Y078-DM1 was performed in cynomolgus monkeys.

Results: By immunohistochemistry, RET expression was detected in 57% of tumors (1596 of

2800 tumor sections) and was most common in HER2-positive and basal breast cancer subtypes. Potent in vitro cytotoxicity was achieved in human breast cancer cell lines that have expression levels comparable with those observed in breast cancer tissue samples. Dose- response studies in xenograft models demonstrated antitumor activity with both weekly and every-3-weeks dosing regimens. In cynomolgus monkeys, a single injection of Y078-DM1 demonstrated dose-dependent, reversible drug-mediated alterations in blood chemistry with evidence of on-target neuropathy.

Conclusion: RET is broadly expressed in breast cancer specimens and thus represents a potential therapeutic target; Y078-DM1 and Y078-DM4 demonstrated antitumor activity in preclinical models. Optimization of the dosing schedule or an alternate cytotoxic agent with a different mechanism of action may reduce the potential risk of neuropathy.

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TRANSLATIONAL RELEVANCE

This report demonstrates that RET is broadly expressed and is a potential therapeutic target in breast cancer. We also describe the pharmacological effects of the antibody drug conjugates (ADCs) Y078-DM1 and Y078-DM4, which demonstrated potent in vitro and in vivo activity in RET-expressing tumor xenograft models. Y078-DM1 was evaluated in a single-dose toxicity study in cynomolgus monkeys and demonstrated drug-mediated alterations in blood chemistry and evidence of dose-dependent neuropathy. Taken together, these data highlight the therapeutic potential, as well as key safety concerns, associated with repeated dosing of

ADCs targeting RET.

Nguyen et al Page 4 Pharmacologic Effects of RET Antibody Drug Conjugates

INTRODUCTION

Antibody drug conjugates (ADCs) are composed of a potent cytotoxic drug chemically attached to a tumor-specific antibody. Their goal is to improve the therapeutic index by specifically targeting and delivering the cytotoxic drug to malignant cells expressing a tumor- specific antigen while reducing non-target tissue and systemic exposure to the cytotoxic drug.

Over the last several years, ADC platforms have evolved, yielding significant technologic improvements through increased drug potency, enhanced and conditional linker stability, linker conjugation methods, target selection, and more recently, antibody engineering (1). Two of the most notable successes that highlight this progress are brentuximab vedotin (Adcetris®, Seattle

Genetics) and trastuzumab emtansine (T-DM1; Kadcyla®, Roche), which have shown significant clinical activity in refractory Hodgkin’s lymphoma, systemic anaplastic large cell lymphoma, and human epidermal growth factor receptor-2–positive (HER2+) advanced breast cancer, respectively (2, 3). Despite these advances, identifying suitable ADC targets remains a significant challenge.

The RET proto-oncogene encodes a cell surface receptor composed of 3 domains: a large N-terminal extracellular domain containing 4 cadherin-like repeats and a cysteine-rich region, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. It is expressed on neural crest-derived and urogenital cells and is essential for normal neuronal and enteric development (4). With the exception of the peripheral enteric, sympathetic, and sensory neurons, as well as the thymus and testis, there is little RET protein detected in adult tissues (5).

Signaling occurs when RET binds to the glial-derived neurotrophic factor (GDNF) family of ligands and corresponding GDNF family α-receptors (GFRα), resulting in RET dimerization and subsequent autophosphorylation of critical tyrosine residues on the intracellular domain of RET

(6, 7). RET activation can trigger multiple signaling cascades involved in regulation of proliferation, differentiation, survival, migration, and chemotaxis. Germline and somatic oncogenic activating mutations in RET play a key driver role in cancer (8), and the multikinase

Nguyen et al Page 5 Pharmacologic Effects of RET Antibody Drug Conjugates

RET inhibitors vandetanib and cabozantinib were recently approved for medullary thyroid carcinoma (9-12). Furthermore, treatment of RET fusion-positive non-small cell lung cancer patients with cabozantinib resulted in confirmed partial responses in 2 patients and prolonged stable disease in a third patient, providing early clinical validation for RET inhibition in this genetically defined population (13).

Although oncogenic mutations of RET have not been observed in breast cancer, several groups have reported preferential RET overexpression or rearrangements in estrogen receptor- positive (ER+) breast cancer cell lines and tumors (14). Increased RET expression has been associated with decreased metastasis-free survival (HR=2.12; P=0.0476) and overall survival

(HR=1.95; P=0.0438) in patients with breast cancer (15), and RET inhibition decreased growth and metastasis of ER+ breast cancer cells (15, 16). Increased expression of RET has also been observed in primary tumors from patients who have failed tamoxifen therapy, suggesting a role for RET in endocrine resistance (17). Although the mechanism of this resistance is not well understood, RET expression is upregulated in response to estrogen treatment, and RET activation by GDNF results in increased ERα phosphorylation and activation of known ERα target genes (18). These data suggest that RET signaling may play an important role in a subset of breast cancers by enabling an alternative proliferation pathway that circumvents targets of standard-of-care treatments and thus would be an attractive candidate for therapeutic intervention.

As one approach to explicitly target RET in breast cancer, we explored the preclinical activity of a fully human anti-RET antibody (Y078) conjugated to maytansinoid analogs that induce mitotic arrest by suppressing microtubule formation. Two linker-cytotoxin combinations were used for these studies: Y078-DM1 contains the noncleavable SMCC-DM1 linker-cytotoxic payload (identical to that used in trastuzumab emtansine) (19), and Y078-DM4 contains the sterically hindered disulfide-containing SPBD-DM4 linker-cytotoxic payload (20). Herein, we explored the expression of RET in clinical breast cancer specimens as well as the feasibility of

Nguyen et al Page 6 Pharmacologic Effects of RET Antibody Drug Conjugates using Y078-DM1 and Y078-DM4 for the treatment of breast cancer by performing in vitro and in vivo pharmacology and safety studies.

MATERIALS AND METHODS

Cell lines

MCF-7, CHO-K1, P3U1, FM3A, TT, and PC-3 cells (ATCC, Manassas, VA); Free-Style

293F cells (Invitrogen, Carlsbad, CA) were maintained as recommended by the supplier. RET- expressing CHO (RET-CHO) and RET-expressing FM3A cells (RET-FM3A) were prepared by transfecting CHO-K1 and FM3A with pEE12.4-RET and pEF1-RET, respectively, using Gene

Pulser (Bio-Rad, Hercules, CA). The 293F cells were purchased directly from Invitrogen and used within 6 months of purchase. Invitrogen has tested 293F cells for contamination of bacteria, yeast, mycoplasma and virus, and characterized cells by isozyme and karyotype analysis. MCF-

7, CHO-K1, P3U1, FM3A, TT, and PC-3 cell lines were obtained from ATCC, and used within 6 months of purchase. ATCC authenticates cell lines routinely by short tandem repeat profiling, monitoring cellular morphology, karyotyping, and cytochrome C oxidase I. All cell lines at were passaged only a few times to establish master cell bank after thawing of initial frozen stocks to reduce total number of passages, from which working cell banks are used and authenticated by cellular morphology. Please see Supplementary Methods for details of plasmid construction.

RET-CHO cells were selected with glutamine synthetase (GS) supplement and 33.3 μM MSX

(MP Biomedicals, Solon, OH), while RET-FM3A cells were selected in 1 mg/mL Geneticin

(Invitrogen). A lentiviral vector was produced using packaging plasmids (System Biosciences,

Mountain View, CA) with the lentivector plasmid encoding RET (Geneocopia, Rockville, MD) in

293F cells, and then used to transduce MCF-7 cells to generate MCF-7/L-RET cells. The Y078 antibody production cell line was generated by cloning Y078 coding sequences into pConPlus

(Lonza Biologics, Basel, Switzerland) and stably transfecting GS-CHO.

Nguyen et al Page 7 Pharmacologic Effects of RET Antibody Drug Conjugates

Generation and selection of anti-RET antibody Y078

Please see Supplementary Methods for details of protein production and purification. KM mice (Kyowa Hakko Kirin, Tokyo, Japan) were immunized 10 times over the course of ~100 days with 20 μg RET-Fc or 1×107 RET-FM3A cells by intraperitoneal administrations.

Splenocytes were collected and fused to P3U1 cells using a CEEF-50B Electrofusion System

(CytoPulse, Holliston, MA). Fused cells were cultured in 96-well plates with Daigo-T medium

(Dainippon Pharmaceuticals, Osaka, Japan) containing HAT supplement for 10 days.

Hybridoma supernatants were screened by direct ELISA using 96-well plates coated with 50 ng histidine-tagged RET, and the bound hybridoma-derived antibody was detected with goat anti- human immunoglobulin G (IgG) horseradish peroxidase (Bethyl Labs, Montgomery, TX) at a

1:3000 dilution. Y078 was selected as the lead antibody based on its binding and internalization profile as well as the ability to block GDNF-induced pERK upregulation in MCF-7 cells (data not shown).

Antibody-maytansinoid conjugates

The Y078 antibody and an isotype control antibody (ISO), a chimeric anti-soybean trypsin inhibitor with human IgG1 constant domains (ImmunoGen, Inc., Waltham, MA), were both modified with either succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate

(SMCC) for DM1 conjugation or N-succinimidyl 4-(2-pyridyldithio) butanoate (SPDB) for DM4 conjugation as previously described (21, 22). The conjugates Y078-DM1, Y078-DM4, and ISO-

DM4 contained an average of 3.3 toxin molecules per antibody. The conjugate ISO-DM1 contained 4.2 cytotoxic drug molecules per antibody.

Immunohistochemistry

Sections were stained on the Ventana Discovery System (Ventana, Tucson, AZ) using

DAB Map Detection Kit and heat-induced antigen retrieval (HIER) with Standard Cell

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Conditioning 1 (Ventana). For a detailed description of the staining protocols, see

Supplementary Methods.

Immunofluorescence and cell cytotoxicity assays

To assess internalization by fluorescent microscopy, RET-CHO cells were incubated with 20 μg/mL Y078 anti-RET for 30 minutes at 4°C or 37°C before washing in 1% (w/v)

BSA/PBS and incubating with 20 μg/mL anti-human IgG AF488 (Invitrogen) for 30 minutes at

4°C. Cells were washed in PBS, and if necessary, AF488 signal was quenched with rabbit anti-

AF488 (Invitrogen) before fixing in 4% paraformaldehyde. Images were taken on the

ImageXpress (Molecular Devices, Sunnyvale, CA) at 20× magnification. To assess cytotoxicity, cells were seeded the day before at 3,000 cells per well in 96-well plates in 100 μL media.

Solutions of ADCs and controls were serially diluted in 100 μL media before adding to cells and tested in triplicates per plate. Cells were cultured for 6 to 7 days before cell viability was assessed using Cell Titer Glo (Promega, Madison, WI) on an Envision 2102 Multilabel Reader

(Perkin Elmer, Waltham, MA). Half-maximal inhibitory concentrations (IC50) were calculated by nonlinear regression analysis using a sigmoid dose response curve with Prism software

(GraphPad, La Jolla, CA).

In vivo tumor xenograft models

Female 6-week-old NCR nu/nu mice (Taconic, Oxnard, CA) were implanted subcutaneously with 17β-estradiol pellet (0.36 mg, 90 days release; Innovative Research of

America, Sarasota, FL) to facilitate tumor growth. Three days later, tumor xenografts were initiated by subcutaneously injecting MCF-7 or MCF-7/L-RET cells (5×106 suspended 1:1 in

Matrigel Matrix; BD Biosciences, Bedford, MA) into the flank of the right hind leg. Two perpendicular tumor diameters (a and b) were measured 2 times a week to follow tumor

Nguyen et al Page 9 Pharmacologic Effects of RET Antibody Drug Conjugates progression, and tumor volumes were calculated (volume = ab2/2). When tumors reached

200 mm3 ~3 weeks after implantation, mice were randomized and treated with controls or ADCs by intraperitoneal injections. All studies were conducted in accordance with the Guide for the

Care and Use of Laboratory Animals.

Cynomolgus monkey toxicity study

The safety of Y078-DM1 was evaluated using 2 male and 2 female cynomolgus monkeys of Chinese origin per group (Charles River, Reno, NV). Animals received a single dose on day 1 of vehicle, 30 mg/kg ISO-DM1, 30 mg/kg unconjugated Y078, or Y078-DM1 at 3,

10, or 30 mg/kg. All compounds were administered by IV infusion over 30 minutes. Animals were observed for clinical signs of morbidity or mortality. Analyses of clinical chemistry, hematology, and coagulation were made with blood collected 1 week before dosing to serve as baseline and on days 2, 7, 15, 22, and 28. Animals were euthanized on day 29, and gross pathologic and histopathologic evaluation was performed. Neurologic examination (behavior, motor function, cranial nerve function, and proprioception) was performed once prestudy, during week 2, and once before necropsy. Electrophysiology was performed once prestudy and once within 1 week of necropsy. Detailed electrophysiology methods are available upon request.

Differences in measures across groups were determined by quantitative analysis of wave form characteristics and direct statistical comparisons of measures at each time point, using standard analysis of variance (ANOVA) multivariate inferential procedures with the α level set at 0.05.

The protocol and any amendments or procedures involving the care or use of animals were reviewed and approved by the Testing Facility Institutional Animal Care and Use Committee before initiation of procedures.

Nguyen et al Page 10 Pharmacologic Effects of RET Antibody Drug Conjugates

RESULTS

RET expression in breast cancer

A comprehensive study to evaluate RET expression was performed at the British

Columbia Cancer Agency (BCCA) with a high density immunohistochemistry tissue microarray

(TMA) of >4,000 clinically annotated primary fresh-frozen breast cancer specimens from

Vancouver Hospital ER Laboratory in Canada (23). Intensity of RET staining was evaluated by a pathologist and was scored as negative, weak cytoplasmic and/or membranous staining, or strong cytoplasmic and/or membranous staining. Overall, 1596 of the 2800 evaluable cores

(57%) were scored positive (Table 1). RET immunoreactivity was found to be most common in

HER2+ and basal breast cancer subtypes. Over 80% of such breast cancers were RET-positive, whereas 47% of luminal cancers (ER+ without HER2 immunoreactivity) were RET-positive.

Outcome analysis revealed that RET-positive versus RET-negative breast cancers had poorer survival in both the training and validation sets (Supplementary Figure S1). However, this association was not maintained when classified by subtype. Because RET immunoreactivity correlates with breast cancer subtypes with a known adverse prognosis, it is not surprising that

RET immunoreactivity is a marker for poor prognosis in the entire cohort.

To further characterize RET expression in breast cancer, RET expression in metastatic disease was assessed in commercially available FFPE tissue microarrays containing breast primary samples (n=97) and matched metastatic lymph node tumor samples (n=104) (Figure

1A). RET immunoreactivity was observed in 37 (38%) of the primary samples (n=16 score 1+, n=21 score 2-3+). In metastatic lymph node tissues, 44 samples (42%) were RET-positive

(n=21 score 1+, n=23 score 2-3+). Greater than 50% of the RET-positive primary samples had matching metastatic lymph node tissues that also expressed RET.

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In vitro characterization of Y078-DM1 and Y078-DM4

To assess in vitro activity of the fully human monoclonal antibody Y078, expression of

RET in MCT-7 parental and MCF-7/L-RET (engineered) xenograft tumors was compared with a panel of primary breast tumors (Figure 1B). RET immunoreactivity in the xenografts was comparable with that observed in the primary breast cancer samples, with MCF-7 on the low range and MCF-7/L-RET on the high range, thus validating these 2 tumor models for RET in vitro and in vivo studies. The MCF-7 and MCF-7/L-RET cell lines express ~30K and ~100K receptors on their surface, respectively, as determined by quantitative flow cytometry (data not shown). Other ER+ or HER2+ breast cancer cell lines (EFM19, MDA-MB-134VI, and BT474) expressed RET mRNA; however, surface expression of RET was undetectable in these cells

(data not shown). Similarly, RET expression was not detected in triple-negative cell lines (data not shown).

Since internalization is a prerequisite for ADC efficacy (24), Y078, which binds to the extracellular domain of RET with high affinity (128 pM; data not shown), was used to demonstrate internalization into RET-CHO cells by immunofluorescence (Figure 2A). The antibody was lysine-conjugated to 2 linker-cytotoxin moieties to obtain Y078-DM1 and Y078-

DM4, and evaluated in cytotoxicity assays using MCF-7 parental and MCF-7/L-RET engineered cells as targets (Figure 2B). The similarly-conjugated isotype controls ISO-DM1 and ISO-DM4, as well as unconjugated Y078, were included as negative controls, while S-methyl-DM4 was used as a positive control to assess the intrinsic sensitivity of the cell lines to the maytansinoid payload as the free drug. In the MCF-7 cytotoxicity assay, Y078-DM4 was 6-fold more potent than its isotype control (IC50 of 6.2 nM versus 36 nM; Supplementary Table S1). Y078-DM1 and its corresponding isotype control had comparable low potency (>10 nM) in MCF-7 cells, perhaps due to the low number of receptors. In the engineered MCF-7/L-RET cells, both Y078-DM1 and

Y078-DM4 revealed potent activity, with IC50 values of 0.37 nM and 0.15 nM, respectively, suggesting that potency is dependent on receptor density. Unconjugated Y078 had a modest

Nguyen et al Page 12 Pharmacologic Effects of RET Antibody Drug Conjugates inhibitory effect on MCF-7/L-RET but not MCF-7 cells; growth of MCF-7/L-RET cells was reduced by 20% at high concentrations (Figure 2B). Y078-DM1 and Y078-DM4 were also tested in RET-CHO cells and in TT cells; the latter is a human MTC cell line with endogenous RET expression. In RET-CHO cells, both ADCs had similar potency, as observed in MCF-7/L-RET cells. The results in TT cells were similar to those in MCF-7 cells, wherein Y078-DM4 demonstrated potent activity and Y078-DM1 had no measurable effect (data not shown).

Bystander activity of Y078-DM1 and Y078-DM4

The cytotoxicity assay was modified to assess the bystander activity of Y078-DM1 and

Y078-DM4. In this assay, target MCF-7/L-RET cells and RET-negative bystander PC-3 prostate carcinoma were cultured individually or at a 1:1 ratio. Both Y078-DM1 and Y078-DM4 demonstrated potent activity against MCF-7/L-RET cells but had only limited activity against

RET-negative PC-3 cells (Supplementary Figure S2A). In co-culture, however, Y078-DM4 demonstrated killing of both MCF-7/L-RET and RET-negative PC-3 cells.

The cytotoxicity assays were confirmed using immunofluorescence as an orthogonal technique. In Y078-DM1-treated co-cultures, PC-3 cells were clearly visible, and a limited number of MCF-7/L-RET cells remained. This was in striking contrast to Y078-DM4-treated cultures that contained a limited number of both cell types (Supplementary Figure S2B). Isotype controls showed limited cytotoxicity in all cell conditions (data not shown).

In vivo studies to evaluate potency of Y078-DM1 and Y078-DM4

MCF-7 and MCF-7/L-RET tumor xenograft models were used to evaluate Y078-DM1 and Y078-DM4. When tumors reached ~200 mm3 in volume, mice were treated with either

Y078-DM1 or Y078-DM4 or their respective isotype control ADCs. In the MCF-7 tumor study, doses of 3 or 10 mg/kg were administered once weekly for a total of 3 doses with no signs of toxicity (data not shown). An equivalent dose of unconjugated S-methyl-DM4 (assuming a drug-

Nguyen et al Page 13 Pharmacologic Effects of RET Antibody Drug Conjugates to-antibody ratio of 3.4 and 100% cytotoxic drug delivery) led to pronounced (≥20%) weight loss in 5 of 10 mice (data not shown). Both Y078-DM1 and Y078-DM4 demonstrated statistically significant (P<0.001), dose-dependent tumor growth inhibition (TGI) of 56% and 83% at the 3 mg/kg dose, and 97% and 108% at the 10 mg/kg dose, respectively, on day 56 (Figure 3A). No significant weight loss was observed in the Y078-DM1- and Y078-DM4-treated mice (data not shown). Although the isotype controls did not significantly reduce tumor growth, weak antitumor activity was apparent at the 10-mg/kg dose. Unconjugated Y078 was tested in both of these models and had no effect on tumor growth (data not shown).

To better understand the in vivo relationship between receptor expression and efficacy, tumor xenograft studies were also performed in mice with established MCF-7/L-RET tumors.

Doses of Y078-DM1 (1, 3, and 10 mg/kg) or Y078 (1 and 3 mg/kg) were administered weekly for a total of 3 doses (Figure 3B). Treatment with both Y078-DM1 and Y078-DM4 led to statistically significant (P<0.001) reductions in tumor volume on day 35: 142% TGI at 3 mg/kg Y078-DM4,

124% TGI at 10 mg/kg Y078-DM1, and 90% TGI at 3 mg/kg Y078-DM1. No antitumor effect was observed at 1 mg/kg. The isotype controls ISO-DM1 and ISO-DM4 did not significantly reduce tumor growth.

Approximately 14 days after the last dose, residual MCF-7 tumors were excised from mice treated with 3 mg/kg Y078-DM4 or 10 mg/kg Y078-DM1. These samples, as well as larger tumors from the vehicle- and isotype-control–treated mice, were analyzed by immunohistochemistry for the presence of RET. Notably, there was no clear qualitative difference in RET expression in samples from control or Y078 ADC-treated animals.

Pronounced necrotic areas were observed in 86% (n=6/7) of the Y078-DM4-treated tumors, whereas size-matched tumors treated with Y078-DM1 had none (n=0/6) (Figure 3C and 3D).

Pockets of necrosis were also observed in the center of the substantially larger tumors from control-treated mice (data not shown). These data demonstrate that despite the comparable size, there is a clear qualitative difference in tumors treated with Y078-DM1 or Y078-DM4.

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Dosing and scheduling studies with Y078-DM1 and Y078-DM4

To evaluate the activity of Y078-DM1 and Y078-DM4 using the every-3-weeks (q3w) schedule commonly used in the clinic, the non-engineered MCF-7 cell line was selected to provide a more challenging tumor xenograft model. Doses of 10 mg/kg and 15 mg/kg Y078-

DM1, administered q3w, demonstrated comparable TGI (~64%; Figure 4A). Evaluation of dose fractionation on a weekly schedule, at doses of 3.3, 5, 10, or 15 mg/kg yielded TGI of 54%, 77%,

78%, and 95%, respectively, on day 63 (Figure 4A). The observation that the TGI achieved with

10 mg/kg and 15 mg/kg q3w was similar to that with 3.3 mg/kg/week and 5 mg/kg/week, respectively (similar dose intensity), suggests that variations in dose and schedule of Y078-DM1 had little effect on the extent of TGI. In a related study, Y078-DM4 was administered at 3 mg/kg either as a single bolus dose or weekly times 3 doses (q1w×3), every 2 weeks for 2 doses

(q2w×2), or every 3 weeks for 2 doses (q3w×2) to evaluate different dosing regimens (Figure

4B). The best tumor suppression resulted from the most frequent dosing (q1w×3) of Y078-DM4 with 91% TGI as calculated on day 56. By contrast, ISO-DM4 administered q1w×3 had a very minor effect on tumor growth. Notably, Y078-DM4 administered as a single dose had a similar effect on tumor growth (TGI 60%) as the q2w×2 and q3w×2 schedules (TGI 55% and 47%, respectively; Figure 4B). Taken together, these data suggest that antitumor activity is exposure- driven and independent of dosing schedule.

Safety evaluation of Y078-DM1 in cynomolgus monkeys

Based on evidence that Y078 binds to the cynomolgus RET ortholog and to human RET with similar affinity, and tissue cross-reactivity studies confirming comparable staining patterns in monkey and human tissues (data not shown), a safety study was performed in the cynomolgus monkey to assess the potential toxicities of Y078-DM1. Although Y078-DM4 was more potent in vitro and in vivo, the Y078-DM1 conjugate was selected for this experiment

Nguyen et al Page 15 Pharmacologic Effects of RET Antibody Drug Conjugates because the stable thioether linker (SMCC) can only release its maytansinoid payload following uptake and intracellular processing within the lysosomal compartment, and the lysine-linker-

DM1 adduct is the sole intracellular metabolite. Thus the SMCC-DM1 conjugate, which exhibits no bystander cytotoxic activity, should provide a clearer picture of the potential for on-target cytotoxicity (25). An important goal of these studies was to assess the anticipated on-target delivery of the potent microtubule inhibitor to neurons.

Y078-DM1 was well tolerated and no significant adverse events or deaths were recorded.

No significant test article-related differences in clinical observations, gross pathology, body weights, or coagulation parameters were observed up to 10 mg/kg. These results are consistent with RET having a restricted expression profile in adult tissues (5). However, Y078-DM1 and

ISO-DM1 dosed at 30 mg/kg were associated with clinical pathology changes consisting of minimal to moderate increases in enzyme levels (including alkaline phosphatase, aspartate aminotransferase, gamma-glutamyl transferase, and lactate dehydrogenase) between days 2 and 15, protein changes suggestive of an acute-phase response beginning on day 7, a delayed reticulocyte response on day 7 (ISO-DM1 only), and/or decreases in platelet counts on day 7

(Y078-DM1 only) (Supplementary Figure S3). Of these effects, only the enzyme levels did not reverse to baseline levels by day 29. These data suggest that DM1 mediates the coincident alterations in clinical pathology observed in both the isotype control and Y078-DM1 groups, and are similar to the clinical pathology observations in preclinical and clinical studies with T-DM1

(26-28).

Although no treatment-related neurologic examination findings were observed in any of the groups, significant alterations in electrophysiologic responses were evident on day 29 in the monkeys treated with 30 mg/kg Y078-DM1 but not with ISO-DM1. There was a notable reduction in mean measures of conduction, prolonged mean measures of latency, and reduction in the response amplitude (Figure 5A). Physiologically relevant slowing of both sensory and motor conduction velocity was observed in peripheral nerves, as well as prolongation of the

Nguyen et al Page 16 Pharmacologic Effects of RET Antibody Drug Conjugates spinal cord absolute latency (consistent with a slowing of conduction in the activated axons), and delays in the somatosensory evoked potentials recorded from the central nervous system brainstem. Microscopic findings were consistent with a dose-related minimal to mild peripheral neuropathy that extended centrally from distal (peroneal/sural) nerves through the spinal nerves associated with the dorsal root ganglia and into the sensory tracks (dorsal funiculi) of the spinal cord. Histologically defined, Y078-DM1-related peripheral neuropathy occurred at all doses and was characterized by condensed and fragmented axons within intact myelin sheaths in the peroneal, sural, tibial, sciatic, and spinal nerves/nerve roots (associated with the dorsal root ganglia), and by the presence of glial cells within myelin sheaths in the dorsal spinal cord.

Cytoplasmic vacuolation occurred in animals given unconjugated Y078, whereas evidence of dose-dependent vacuolation, neuronal degeneration, and necrosis were observed in groups given treated with Y078-DM1 (Figure 5B).

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DISCUSSION

In this study, we demonstrate that RET is expressed in primary and metastatic breast cancer by profiling >4,000 clinically annotated primary fresh-frozen breast cancer specimens.

Immunohistochemical analysis of the BCCA TMA samples has demonstrated broad expression across all breast cancer subtypes, with the highest prevalence in HER2+ and basal breast cancers. This is in contrast to previous studies that reported preferential expression of RET in

ER+ breast cancer but with relatively little expression in HER2+ and basal breast cancers (17, 18,

29, 30). The explanation for the discordance is not clear; however, it may reflect differences in the methods (in situ hybridization versus immunohistochemistry), protocols, reagents, sample set sizes, or patient treatment history. Observed inconsistencies notwithstanding, overall the data suggest that RET is expressed in human primary and metastatic breast cancer, and the high percentage of RET immunoreactive tumors observed in the HER2+ and basal subtypes

(>80%) suggests that targeting RET may be a viable strategy for treating these aggressive subtypes.

ADCs have been demonstrated to function in a multi-step process that includes target antigen binding, internalization, trafficking, cytotoxic drug release, and drug action. After activation, RET is internalized from the plasma membrane to early endosomes through clathrin- coated pits (31). Efficient internalization of naked and conjugated Y078 was confirmed in MCF-

7/L-RET cells by immunofluorescence, and in vitro cytotoxicity assays were used to functionally assess the internalization and trafficking of Y078-DM1 and Y078-DM4 as well as the release and activity of the cytotoxic payload. The SPDB-DM4 linker-cytotoxic drug contains a “cleavable” linker that incorporates a sterically hindered disulfide bond between the linker and DM4. In contrast, the SMCC-DM1 linker-cytotoxic payload contains a “noncleavable” linker with a covalent thioether bond between the linker and DM1 (20, 32). Assessment of HER2-targeted and CanAg-targeted ADCs with similar or identical linkers has suggested that both ADCs are degraded in the lysosome, and that the major active maytansinoid catabolites from SMCC-DM1

Nguyen et al Page 18 Pharmacologic Effects of RET Antibody Drug Conjugates and SPBD-DM4 are lysine-Nε-SMCC-DM1 and S-methyl-DM4, respectively (20, 32). Whereas the lysine-Nε-SMCC-DM1 is charged and membrane-impermeable, S-methyl-DM4 is lipophilic and can reenter target or neighboring cells to induce bystander activity (25); tumors with heterogeneous expression of RET, therefore, may respond differently to Y078-DM1 versus

Y078-DM4. Both of these compounds demonstrated potent cytotoxicity in MCF-7/L-RET cells, whereas only Y078-DM4 demonstrated bystander activity and cytotoxicity in MCF-7 cells. These data suggest an in vitro threshold where the lower receptor number MCF-7 cells may not accumulate enough of the active lysine-Nε-SMCC-DM1 metabolite to result in cytotoxicity under the conditions of the assay; however, other explanations cannot be excluded, such as altered trafficking or differences in payload potency between S-methyl-DM4 and lysine-Nε-SMCC-DM1.

In contrast, both ADCs demonstrated potent activity in vivo using MCF-7 tumor xenografts, suggesting that accumulation and/or improved pharmacokinetics may contribute to the potency of the thioether-containing conjugate. Additional studies in primary and engineered cell lines would be helpful in refining this threshold and elaborating biomarker and patient stratification strategies.

Dose, scheduling, and time-dose fractionation tumor xenograft studies were performed to explore ADC activity in the MCF-7 xenograft model. Although reagents were not available to measure ADC levels, previous reports suggest that the half-life of SMCC-DM1 conjugated

ADCs (typically measured by ELISA and equivalent to ≥1 drug/antibody) is ~5 days (33). In addition, anti-therapeutic antibody formation was observed in 5.3% of patients after repeated dosing of T-DM1 (34). Comparable antitumor responses were observed with lower doses of

Y078-DM1 administered weekly or higher doses administered q3w, suggesting that antitumor activity was related to exposure. Similarly, a relationship between antitumor activity and exposure was observed with T-DM1, but tumor response was independent of dosing schedule

(33). Consistent with these data, more frequent administration and presumably higher exposure of Y078-DM4 dosed at 3 mg/kg also increased potency. The optimal clinical benefit, however, is

Nguyen et al Page 19 Pharmacologic Effects of RET Antibody Drug Conjugates likely to be a dose and schedule that maintains the drug exposure above the minimally efficacious dose while limiting systemic exposure and coincident toxicity. Although no formal safety assessment was performed in rodent models, weekly dosing with 10 mg/kg or 15 mg/kg of either Y078-DM1 or Y078-DM4 did not result in substantial body weight loss. Taken together,

RET targeted conjugates demonstrate potent activity in tumor models with clinically relevant levels of RET, and highlight the potential of RET as an ADC target.

Tumor xenograft studies have clearly demonstrated increased cytotoxicity with Y078-

DM4 versus Y078-DM1, not only by dose response studies but also by evidence of necrosis in the residual tumor masses. This difference might be a potential advantage for Y078-DM4 in that the bystander cytotoxicity may damage tissues involved in supporting the tumor growth, such as tumor neovasculature and stromal cells, which would result in enhanced activity in tumors with heterogeneous RET expression. An example where this may be beneficial would be the case of brentuximab vedotin, which demonstrates potent activity in Hodgkin’s lymphoma that is composed of CD30-positive malignant Reed-Sternberg tumor cells surrounded by a complex mixed inflammatory filtrate (35, 36). However, bystander activity could also be a drawback if vital cells in surrounding tissues are affected. Because of its anticipated lack of bystander cytotoxic activity, Y078-DM1 was selected for safety evaluation in cynomolgus monkeys where the goal was to evaluate the potential for on-target toxicity of the ADC. Additional studies are required to determine which conjugate has the widest therapeutic index.

RET is expressed in the adult peripheral nervous system; therefore, the targeted delivery of a potent microtubule-interfering drug to cells expressing RET has the potential to elicit dose- limiting neural toxicity. This would be particularly concerning in heavily-treated cancer patients who often have preexisting, treatment-related, grade 1-4 peripheral neuropathy (37, 38). Given the essential role of microtubules in maintaining neuronal cell structure and viability, it is also not surprising that peripheral neuropathy is observed with some ADCs that deliver highly potent microtubule-destabilizing cytotoxic drugs (3, 39). In addition, there is a precedent for an ADC

Nguyen et al Page 20 Pharmacologic Effects of RET Antibody Drug Conjugates with a target in the peripheral nervous system. IMGN901 targets neural cell adhesion molecule

(CD56) and is currently being assessed in a phase II trial in small-cell lung cancer in combination with etoposide/carboplatin (40). Although peripheral neuropathy was the most common treatment-related adverse event observed with IMGN901 in phase I clinical trials, grade 3 peripheral neuropathy was seen only in patients treated with doses of ≥90 mg/m2, emerging in most cases past the third treatment cycle, and manageable with dose modification

(41). The cynomolgus monkey study revealed that Y078-DM1, and to a lesser degree unconjugated Y078, was associated with dose-dependent on-target neuropathy. Although the effects were minimal or mild at low doses, there was a clear deficit observed with the ADC at the 30 mg/kg dose. Depending on the dose-activity relationship for ADC-driven on-target toxicity, this observation could translate to a clinically significant toxic polyneuropathy similar to that induced by many anti-neoplastic drugs. ADCs are commonly administered q3w in the clinic, and it is not clear if the minimal or mild effects observed at low doses would worsen with repeated dosing and lead to clinically relevant peripheral neuropathy. Cytoplasmic vacuoles were noted in dorsal root ganglia neurons following ADC administration, but the significance of these vacuoles has been debated without clear consensus (42, 43). Given the potent anti-tumor activity observed, these preclinical data support refining the therapeutic index and clinical utility of RET- targeted ADCs using alternative and repeat dose schedules, evaluating alternative cytotoxic payloads with different mechanisms of action, or utilizing companion diagnostics to select for patients with high RET expression to ensure a favorable risk-benefit profile.

Nguyen et al Page 21 Pharmacologic Effects of RET Antibody Drug Conjugates

ACKNOWLEDGEMENTS

We are grateful to members of Takeda Pharmaceuticals International Co. for helpful support, comments, and discussions. We thank Petter Veiby and Mary Carsillo for feedback regarding the pharmacology and safety studies, and Patrick Vincent, Mathias Schmidt, and

Steven McKinney for critical review. We gratefully acknowledge John Lambert, Chris Perry,

Robert Lutz, Hans Erickson, Erica Hong, and Kate Lai for generating ADCs and for helpful discussions. We are grateful to Joe Arezzo for performing, analyzing, and interpreting electrophysiologic studies.

FUNDING

Financial support for the conduct of this study was provided by Takeda Pharmaceuticals

International Co. Financial support for medical editorial assistance was provided by Millennium:

The Takeda Oncology Company. We thank William Sinkins, PhD, ProEd Communications, Inc.®, for his medical editorial assistance with this manuscript.

Nguyen et al Page 22 Pharmacologic Effects of RET Antibody Drug Conjugates

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TABLES

Table 1. RET expression in breast cancer samples measured by IHC

Markers used to define breast cancer subtypes RET expression by IHC by IHC

Subtype HER2+ ER+ or Ki67 CK5/6+ or 0, 1 or 2, PgR+ ≥14% EGFR+ n (%) n (%)

Luminal A – + – NA 689 (52.6) 621 (47.4)

Luminal B – + + NA 394 (52.3) 360 (47.7)

Luminal/HER2 + + NA NA 52 (25.5) 152 (74.5)

HER2 + – NA NA 22 (9.6) 206 (90.4)

Basal – – NA + 47 (15.5) 257 (84.5) CK5/6, cytokeratin 5/6; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor type 2; IHC, immunohistochemistry; NA, not applicable (marker was not used to define corresponding subtype); PgR, progesterone receptor

Nguyen et al Page 28 Pharmacologic Effects of RET Antibody Drug Conjugates

FIGURE LEGENDS

Figure 1. RET immunoreactivity is broadly observed in primary and metastatic breast cancer specimens. (A) RET expression in primary and metastatic disease was assessed in a commercially available FFPE tissue microarray containing primary breast tumor specimens

(n=97) and matched metastatic lymph node tumor samples (n=104). Representative microscopic pictures with RET immunoreactivity in 4 matched primary and metastatic lymph node tumor specimens are shown. (B) RET staining of breast tumor samples in comparison to tumor xenografts derived from MCF-7 parental and MCF-7/L-RET cell lines. Primary breast cancer specimens BC02, BC05, and BC10 were obtained from screening arrays of BCCA samples and evaluated for RET expression by immunohistochemistry. RET immunoreactivity in the MCF-7 and MCF-7/L-RET cell lines brackets the spectrum of RET expression in the primary tumor samples.

Figure 2. Anti-RET Y078 antibody internalizes and Y078 ADCs elicit potent in vitro cytotoxicity.

(A) Internalization was assessed by incubating Y078 and RET-CHO cells for 30 minutes at either 4oC or 37oC, followed by detection of Y078 using anti-human IgG conjugated to AF488 in the presence and absence of anti-AF488 to block surface staining. Representative fluorescent images are shown; punctuate green intracellular staining shows Y078 internalization. (B) MCF-7 parental (left) and MCF-7/L-RET cells (right) were incubated with serially diluted unconjugated

Y078, isotype ADCs ISO-DM1 and ISO-DM4, or Y078 ADCs Y078-DM1 and Y078-DM4. The concentration of S-methyl-DM4 used was equivalent to an ADC with a drug to antibody ratio of

3.4. Samples were incubated for 6-7 days before viability was assessed using Cell Titer Glo. For all studies, the graphs are representative of 3 experiments carried out in triplicate.

Nguyen et al Page 29 Pharmacologic Effects of RET Antibody Drug Conjugates

Figure 3. RET-targeted Y078 conjugates demonstrate potent in vivo activity in tumor xenograft models. Nude mice bearing established MCF-7 (A) or MCF-7/L-RET (B) xenograft tumors with an average size of ~200 mm3 were treated weekly with vehicle and either Y078-DM1 (left panel) or Y078-DM4 (right panel), and their respective isotype controls ISO-DM1 and ISO-DM4, at the doses indicated. All treatments were administered by intraperitoneal injection and arrows indicate time points for dosing. Tumor growth was measured twice weekly using a caliper, and the lines indicate average tumor volumes for each group ± SEM (n=10). The MCF-7 and MCF-

7/L-RET cell lines have ~30K and ~100K receptors on their surface, respectively, as determined by quantitative flow cytometry (data not shown). (C) Approximately 14 days after the last dose, residual tumors from animals treated with 3 mg/kg Y078-DM4 or 10 mg/kg Y078-DM1, and large tumors from the vehicle- and isotype control-treated mice were excised and analyzed by immunohistochemistry for the presence of RET. Heterogeneous RET staining in the tumors

(n=6-7 for ADC treated tumors) was assessed, and the stacked bars represent the percentage of the tumor that had negative, low, medium, or high levels of RET immunoreactivity. There was no apparent change in RET expression in any of the control or treatment groups. (D)

Representative images of immunohistochemical analysis of RET expression in MCF-7 tumors after treatment with Y078-DM1 (left) and Y078-DM4 (right) at 10 mg/kg and 3 mg/kg, respectively. The vast majority (n=6/7; 86%) of the Y078-DM4-treated tumors had pronounced necrotic areas as compared to size-matched tumors (n=0/6) treated with Y078-DM1. The expression of RET is limited to areas of cell viability and not in areas of necrosis.

Nguyen et al Page 30 Pharmacologic Effects of RET Antibody Drug Conjugates

Figure 4. Observed tumor response to Y078-DM1 and Y078-DM4 administered using different schedules and doses in tumor xenograft models. (A) Nude mice bearing established MCF-7 xenograft tumors with an average size of ~200 mm3 were treated weekly with either a single dose (top panel) or weekly doses (bottom panel) of Y078-DM1. All treatments were administered by intraperitoneal injection and arrows indicate time points for dosing. Tumor growth was measured twice weekly using a caliper, and the lines indicate average tumor volumes for each group ± SEM (n=10). The q3w doses of 10 and 15 mg/kg were chosen to mimic q1w fractional dosing of the same ADC at 3.3 and 5 mg/kg, respectively. (B) Nude mice bearing established MCF-7 xenograft tumors were treated with either a single dose (top panel) or multiple doses (bottom panel) of ISO-DM4 or Y078-DM4. The dosing schedule in indicated in the graph and the day of dosing is indicated by the arrows. Dosing was kept at a constant 3 mg/kg for all administrations. Tumor growth was measured twice weekly using a caliper, and the lines indicate average tumor volumes for each group ± SEM (n=10).

Nguyen et al Page 31 Pharmacologic Effects of RET Antibody Drug Conjugates

Figure 5. Nerve conduction and histologic analysis of Y078-DM1 in monkeys. (A) Cynomolgus monkeys (n=4) were treated with a single dose of 30 mg/kg Y078-DM1 and electrophysiologic assessments were performed before the initial dose (baseline) and on day 29. Animals were anesthetized and placement of the active, reference, and ground electrodes was tailored to each modality and positioned with respect to bony landmarks in each subject. The nerve conduction velocity and amplitude were measured in peroneal and sural nerves to assess potential neurotoxicity from Y078-DM1 administration. The sural sensory nerve response recorded at baseline and at day 29 is shown, and demonstrates a significant slowing of the response onset and peak, as well as a substantial reduction in the response amplitude. (B)

Cynomolgus monkeys (n=4/group) were treated with a single dose of vehicle, 30 mg/kg Y078,

30 mg/kg ISO-DM1, or 30 mg/kg Y078-DM1. Animals were euthanized on day 29 and dorsal root ganglia neurons were collected for pathological analysis. Representative hematoxylin and eosin immunochemistry sections containing vacuolation in Y078- and Y078-DM1-treated animals are shown.

6015 Figure 1 This tagline is for information only; DO NOT PRINT

A Patient A3/F3 Patient A8/F8 Patient G1/H1 Patient E11/F11 Primary Me tastatic

B BC10 MCF-7 BC05 BC02 MCF-7/L-RET

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A 4oC without anti-AF488 4oC with anti-AF488 37oC with anti-AF488

B MCF-7 MCF-7/L-RET 120 120 Y078 unconjugated Y078 unconjugated 100 ISO-DM1 100 ISO-DM1 ili ty ili ty Y078-DM1 Y078-DM1 80 80 ISO-DM4 ISO-DM4 60 Y078-DM4 60 Y078-DM4 Ce ll Viab Ce ll Viab 40 S-methyl-DM4 40 S-methyl-DM4 % % 20 20

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A Y078-DM1 Y078-DM4

1400 Vehicle 1400 Vehicle ISO-DM1 10 mg/kg ISO-DM4 10 mg/kg ISO-DM1 3 mg/kg ISO-DM4 3 mg/kg 1200 1200 Y078-DM1 10 mg/kg Y078-DM4 10 mg/kg Y078-DM1 3 mg/kg Y078-DM4 3 mg/kg 1000 1000 3 3 mm mm e, 800 e, 800

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A B Y078-DM4 Y078-DM1 (All doses 3 mg/kg) 1400 Vehicle 1400 Vehicle 10 mg/kg q3w ISO-DM4 single 1200 15 mg/kg q3w 1200 Y078-DM4 single 3 3 1000 1000

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Preclinical Efficacy and Safety Assessment of an Antibody Drug Conjugate Targeting the c-RET Proto-oncogene for Breast Carcinoma

Minh Nguyen, Shuichi Miyakawa, Junichi Kato, et al.

Clin Cancer Res Published OnlineFirst August 3, 2015.

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