ImmunoPET Imaging of Endogenous and Transfected Prolactin

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ImmunoPET imaging of endogenous and transfected prolactin receptor tumor xenografts Sarah M. Cheal, Shutian Ruan, Darren R Veach, Valerie A. Longo, Blesida J. Punzalan, Jiong Wu, Edward K. Fung, Marcus P. Kelly, Jessica R. Kirshner, Jason T. Giurleo, George Ehrlich, Amy Q. Han, Gavin Thurston, William C. Olson, Pat B. Zanzonico, Steven M. Larson, and Jorge A. Carrasquillo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01133 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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Molecular Pharmaceutics

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ImmunoPET imaging of endogenous and transfected prolactin receptor tumor xenografts

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Sarah M. Cheal1,2,3, Shutian Ruan1,2,3, Darren R. Veach1,2, Valerie A. Longo4, Blesida J.

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Punzalan1,2, Jiong Wu1, Edward K. Fung1,5, Marcus P. Kelly6, Jessica R. Kirshner6, Jason T.

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Giurleo6, George Ehrlich6 , Amy Q. Han6, Gavin Thurston6, William C. Olson6, Pat B.

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Zanzonico2,4,5, Steven M. Larson1,2,3,7, Jorge A. Carrasquillo1,2,3,7*

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Immunotherapy, New York, NY

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Small-Animal Imaging Core Facility, MSK, New York, NY

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Department of Medical Physics, MSK, New York, NY

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Regeneron Pharmaceuticals, Inc., Tarrytown, NY

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Department of Radiology, Weill Cornell Medical Center, New York, NY

Department of Radiology, Memorial Sloan Kettering Cancer Center (MSK), New York, NY Molecular Pharmacology Program, MSK, New York, NY

Center for Targeted Radioimmunotherapy and Diagnosis, Ludwig Center for Cancer

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*Corresponding Author: Jorge A. Carrasquillo, MD Memorial Sloan Kettering Cancer Center 1275 York Avenue New York, NY 10065 Telephone: 212-639-2459 Fax: 212-717-3263 E-mail: [email protected]

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Running title: Antibody PET imaging of PRLR

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ABSTRACT

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Objective: Antibodies labeled with positron-emitting isotopes have been used for tumor

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detection, predicting which patients may respond to tumor antigen-directed therapy, and

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assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is

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overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated

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REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent.

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Methods: REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or

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labeled with I-131/I-124. In vitro determination of half-maximal inhibitory concentration (IC50)

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of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by

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examining the effect of increasing amounts of these on uptake of trace-labeled I-131 REGN2878.

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REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution

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studies were performed in mice bearing tumor xenografts with various expression levels of

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PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and

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T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89

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REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in

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tumor xenografts with and without prior injection of 2 mg of non-radioactive REGN2878.

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Results: Competition binding assay of DFO-REGN2878 at ratios of 3.53 to 5.77 DFO per

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antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM

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for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution

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studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed

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times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ±

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33.8 %IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed low uptake (7.0 ± 2.3


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%IA/mL) of control Zr-89 REGN1932 and very low uptake and rapid clearance of I-124

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REGN2878 (1.4 ± 0.6 %IA/mL).

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Conclusions: Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor

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xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a

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very rapid in vivo turnover half-life of ~14 minutes from the cell membrane. Despite relatively

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modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final

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retention of the Zr-89 REGN2878 antibody, with uptake that appeared to be related to PRLR

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expression. This reagent has the potential to be used in clinical trials targeting PRLR.

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Key words: ImmunoPET, Prolactin Receptor, Zr-89, I-124



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INTRODUCTION

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The prolactin receptor (PRLR) is a transmembrane receptor belonging to the class-1 cytokine

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receptor superfamily; its main ligand is prolactin and its normal function is related to

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reproductive biology.1 PRLR is expressed in a subset of breast and prostate cancers and is

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implicated in the pathogenesis.2-4 A phase I immunotherapy study demonstrated the safety of

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naked antibody adminstration although no anti-tumor response was observed.5 We have

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validated the expression of PRLR in human breast cancers and produced fully human anti-PRLR

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monoclonal antibodies including REGN2878, the antibody used in this study.6 Our related

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studies demonstrated the rapid internalization of PRLR and anti-PRLR antibodies and showed

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that the rapid lysosomal degradation of PRLR antibody drug conjugates (ADC) leads to potent in

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vitro cytotoxicity despite PRLR having only moderate cell surface expression (~30,000 PRLR

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per cell).7 Preclinical pharmacology studies of REGN2878 ADC demonstrated significant anti-

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tumor activity in a number of breast cancer xenograft models.6 Furthermore, PRLR is also

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expressed in ovarian cancer and others have recently taken advantage of a gadolinium magnetic

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resonance imaging ligand that binds to PRLR and internalizes and visualizes tumor xenografts8.

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Thus, PRLR is a potential therapeutic and imaging target in PRLR-bearing tumors.Several

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immunoPET antibodies have been used in pre-clinical and clinical trials for tumor imaging,

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theranostics, and assessment of tumor response.9-12 Although positron-emitting radionuclides

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such as Cu-64 and Ga-68 have been used for immunoPET,13-14 their short half-lives—12.7 hours

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(h) and 1.1 h, respectively—are suboptimal for the slow tumor-localizing kinetics of intact

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immunoglobulins and thus there has been great interest in using longer-lived positron emitters

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such as Zr-89 and I-124, with half-lives of 78.4 h and 100.4 h, respectively. For example, Zr-89

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trastuzumab has been used as a biomarker to evaluate intra-patient heterogeneity of trastuzumab

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uptake in HER2-positive breast cancer, and to correlate uptake with treatment response in

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patients treated with the trastuzumab emtansine (T-DM1) antibody-drug conjugate. In this

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setting, combining Zr-89 trastuzumab and FDG PET predicted response to treatment with T-

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DM1.15 In addition, early changes in Zr-89 trastuzumab uptake in patients with breast cancer

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have correlated with CT changes in size of individual lesions in patients treated with an HSP90

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inhibitor.16 Further, a recent report has suggested that patients with prior biopsy-proven, HER2-

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negative disease may be identified as HER2-positive by Zr-89 trastuzumab imaging and selected

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for a therapeutic intervention that they would not otherwise receive.17

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REGN2878 is a fully human monoclonal antibody to PRLR that has demonstrated therapeutic

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potential as an ADC.6 The rationale for developing a radiolabeled REGN2878 is to potentially

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use this reagent for patient selection, assessment of PRLR engagement during therapy,

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heterogeneity of PRLR expression and prediction of tumor response in patients undergoing

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PRLR directed therapy. As a first step in developing a radiolabeled antibody for imaging or

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theranostic purposes, preclinical studies are required to determine the feasibility of labeling,

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biodistribution, and pharmacokinetics. In this report, we investigated the ability of Zr-89

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REGN2878 to target PRLR-expressing tumor xenografts and characterized its biodistribution in

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tumor xenograft models. REGN2878 binds to human PRLR but not to the mouse.6 We also

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determined the relationship of antigen density with tumor uptake, assessed specificity of uptake,

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and compared Zr-89 REGN2878 targeting to radioiodinated REGN2878 to obtain data on the

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internalization rate in vivo.

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EXPERIMENTAL SECTION

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Antibody Reagents

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REGN2878 is an IgG1 monoclonal antibody that binds to PRLR and was provided at 50.8

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mg/mL.6 The isotype-control antibody REGN1932 is a human IgG1 antibody that recognizes the

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cat allergen Fel d 1, and was provided at 50.9 mg/mL. A 27.7-kD human PRLR ectodomain

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protein with carboxy-terminal myc and histidine tags (hPRLR) was used in antigen binding

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assays. All antibodies and PRLR ectodomain proteins were provided by Regeneron

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Pharmacetuicals, Inc. (Tarrytown, NY).

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Conjugation

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The antibodies were conjugated with isothiocyanate-desferrioxamine B (SCN-DFO,

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Macrocyclics, Dallas, TX) as previously described.18 The DFO chelate-to-antibody conjugation

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ratios were determined by isotope titration as previously described.19 The antibody concentration

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was determined by ultraviolet (UV) spectroscopy (absorbance at 280 nm).

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Radioimmunoassays

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To assess the effect of conjugation with DFO on REGN2878, an in vitro competition assay was

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performed to determine the half maximal inhibitory concentration (IC50) with each of the three

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DFO to REGN2878 conjugation ratios of 3.53, 4.23, and 5.77 to 1, and the original non-

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conjugated REGN2878 as a control, as well as REGN1932 non-PRLR binding antibody. The

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competition assay was performed using I-131 REGN2878 at a specific activity (SA) of 454

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MBq/mg as the tracer. To determine the IC50, 3 ng of I-131 REGN2878 was mixed with

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increasing concentrations of either: non-radioactive REGN2878, the various DFO-REGN2878


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conjugates, or I-127 REGN2878 (labeled using stable I-127, as described below), or REGN1932

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in concentrations ranging from 1x10-6 to 1 µM per tube using 5 x 105 MCF-7/PRLR cells in a

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final volume of 0.5 mL. After a 60-min incubation period at room temperature (RT), the cells

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were centrifuged and washed with PBS and the cell pellet was counted in a gamma counter to

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determine the bound fraction of I-131 REGN2878. The data were then fit using one-site-fit log

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IC50 (Prism version 6.00 for Windows, GraphPad Software, La Jolla, CA, USA).

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Immunoreactivity was determined using increasing numbers of MCF-7/PRLR cells (from 0.5 to

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5 million). Approximately 2 ng of antibody was mixed with the cell in a final volume of 0.5 mL

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for 60 min at RT. The cell-bound activity was counted after centrifugation and discarding the

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supernatant. Data was analyzed as using the Lindmo method.20 In addition, the

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immunoreactivity/specificity of binding was confirmed using an in vitro binding assay with

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excess amounts of hPRLR, followed by size-exclusion high-pressure liquid chromatography (SE

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HPLC; column: TSKgel 3000SW 7.5 mm X 30 cm, 10 µm (TOSOH Bioscience); running

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buffer: 100 mM citrate, 100 mM NaCl pH 6.71; flow rate: 1.2 mL/min; UV detection: 280 nm;

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HPLC system: Shimadzu LC-20AB Prominence Liquid Chromatograph with SPD-20A UV/VIS

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Detector and Bioscan Flow-Count Radiodetector). The SE HPLC traces of non-radioactive

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REGN2878, radiolabeled REGN2878, and Zr-89 REGN1932 were compared to UV or

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radioactivity tracings after these antibodies were mixed together with excess hPRLR to generate

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high-molecular weight antibody-PRLR complexes (Fig. 1).

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The affinities of three non-radioactive conjugated reagents with DFO to REGN2878 ratios of

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3.53, 4.23, 5.77 to 1, and the original non-conjugated REGN2878 as a control were measured in



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Surface Plasmon Resonance Biacore experiments performed on a Biacore T200 instrument (GE

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Healthcare, Picastaway, NJ) using a dextran-coated (CM5) chip at 37°C. The running buffer was

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HBS-ET filtered (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA, 0.05% polysorbate 20, pH 7.4).

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The capture surface was prepared by covalently immobilizing goat anti-human Fc antibody (GE

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Healthcare, Picastaway, NJ) to the sensor chip using (1-ethyl-3-[3-

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dimethylaminopropyl]carbodiimide hydrochloride)/N-hydroxysuccinimide (EDC/NHS) coupling

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chemistry. Antibodies were captured through their Fc regions on the anti-human Fc antibody

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immobilized sensor chip and were tested for binding to the monomeric extracellular domain of

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human PRLR with carboxy-terminal myc and a histidine tag. PRLR solutions were prepared at a

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concentration range between 200 nM to 6.25 nM and individually injected over antibody

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(parental REGN2878), DFO conjugate (3 DFO-REGN2878s), and isotype control (REGN1932)

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captured surfaces. All capture surfaces were regenerated with one 15-s pulse of 10 mM glycine–

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HCl, pH 1.5 (GE Healthcare). Kinetic parameters were obtained by globally fitting the data to a

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1:1 binding model using Biacore T200 evaluation software. The equilibrium dissociation

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constant (KD) was calculated by dividing the dissociation rate constant (kd) by the association

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rate constant (ka).

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Zr-89 was provided in-house by the Radiochemistry and Molecular Imaging Probes Core Facility

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using methods previously described with minor modifications.21 I-124 was either provided in-

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house or purchased commercially (IBA Molecular, Richmond, VA). I-131 was purchased from

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Nordion (Ottawa, ON, Canada). DFO-REGN2878 (DFO to REGN2878 ratio of 3.03) and DFO-

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REGN1932 (DFO to REGN1932 ratio of 2.89) were labeled with Zr-89 at specific activities of


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110 to 223 MBq/mg and used for biodistribution and imaging.18 Post-processing radiochemical

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purity (RCP) averaged 99% as determined by instantaneous thin-layer chromatography (ITLC)

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using 5 mM DTPA. In addition, REGN2878 was labeled with either I-124, I-131, or I-127 using

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the iodogen method22. I-124 REGN2878 was prepared at a SA of 140 MBq/mg and was used for

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imaging and biodistribution; RCP was 98% by ITLC with 0.9% NaCl. For biodistribution and

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radioimmunoassay, REGN2878 was labeled with I-131; the SA was 179 to 453 MBq/mg and

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RCP was 99% by ITLC with 0.9% NaCl. Briefly, to prepare I-127 REGN2878, 2 mg of stock

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REGN2878 (13.4 nmol) was mixed with 80 µL of 0.2 M sodium phosphate pH 7.4 in a pre-

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coated iodogen tube (Thermo Scientific Pierce). Next, 29 µL of 0.1 mg/mL NaI (19.1 nmol) in 1

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mM NaOH was added. After 5 minutes at room temperature, the reaction was transferred to a

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new tube containing 50 µL of iodogen-stop buffer [10 mg/mL of tyrosine (saturated), 10%

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glycerol, 0.1% xylene cylanol in PBS] and purified using a PD10 column that was pre-

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equilibrated and eluted with PBS.

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Cell lines

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A total of five PRLR-expressing cell lines were studied. Cell lines were cultured as described in

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supporting data (cell culture conditions) and used for subsequent xenograft generation and/or

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binding studies. PRLR sites per cell ranged from approximately

ImmunoPET Imaging of Endogenous and Transfected Prolactin

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