Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography a C Hallie M

Published OnlineFirst July 7, 2020; DOI: 10.1158/1078-0432.CCR-20-1358

CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING

Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography A C Hallie M. Hintz1, Joseph P. Gallant1, Donald J. Vander Griend2, Ilsa M. Coleman3, Peter S. Nelson3, and Aaron M. LeBeau1

ABSTRACT ◥ Purpose: Metastatic castration-resistant prostate cancer Results: Analysis of patient data documented FAP overexpres- (mCRPC) is a lethal, heterogeneous disease with few therapeutic sion in metastatic disease across tumor subtypes. PET imaging with strategies that signiﬁcantly prolong survival. Innovative therapies [89Zr]Zr-B12 IgG demonstrated high tumor uptake and long-term for mCRPC are needed; however, the development of new retention of the probe in the preclinical models examined. FAP- therapies relies on accurate imaging to assess metastasis and positive stroma tumor uptake of [89Zr]Zr-B12 IgG was 5-fold higher monitor response. Standard imaging modalities for prostate than the isotype control with mean %ID/cc of 34.13 1.99 versus cancer require improvement and there remains a need for 6.12 2.03 (n ¼ 3/group; P ¼ 0.0006) at 72 hours. Ex vivo selective and sensitive imaging probes that can be widely used biodistribution corroborated these results documenting rapid blood in patients with mCRPC. clearance by 24 hours and high tumor uptake of [89Zr]Zr-B12 IgG Experimental Design: We evaluated the transmembrane prote- by 72 hours. ase ﬁbroblast activation protein alpha (FAP) as a targetable cell Conclusions: Our study reveals FAP as a target for imaging surface antigen for mCRPC. Genomic and IHC analyses were the tumor microenvironment of prostate cancer. Validation of performed to investigate FAP expression in prostate cancer. Our [89Zr]Zr-B12 IgG as a selective imaging probe for FAP- FAP-targeted antibody imaging probe, [89Zr]Zr-B12 IgG, was expressing tumors presents a new approach for noninvasive evaluated by PET/CT imaging in preclinical prostate cancer models. PET/CT imaging of mCRPC.

Introduction reactive stroma in prostate cancer. CAFs in the localized prostate tumor microenvironment are known to play an essential role in Malignant cells are surrounded by a complex and supportive tumor tumorigenesis (5–8). Furthermore, FAP-positive CAFs induce epithe- microenvironment that consists of immune cells, extracellular matrix, lial–mesenchymal transition–driven gain of cancer stemness, inva- vasculature, and fibroblasts (1). Cancer-associated fibroblasts (CAF) siveness, and development of distant metastases (9, 10). Recruitment of are the major cell type in the reactive stroma and are known to promote reactive stroma is essential for metastatic tumor survival and tumorigenesis and metastasis (2). Fibroblast activation protein alpha growth (11–13); however, not much has been published on the role (FAP) is a transmembrane serine protease expressed by CAFs in the of FAP or CAFs in prostate cancer metastasis. microenvironment of epithelial tumors (3). Meta-analysis of FAP Prostate cancer that has metastasized to the bone and visceral tissues expression and clinical outcomes in solid cancers indicate that patients is highly lethal with 5-year survival rates remaining at 30%. The with FAP overexpression are at higher risk of tumor invasion, lymph development of new therapeutics for mCRPC is dependent on accurate node metastasis, and decreased overall survival (4). Thus, FAP has imaging modalities for patient staging and evaluating treatment been gaining momentum as the next pan-cancer target for diagnostic response. The current standard for imaging disease burden is bone and therapeutic development given its absence in normal adult tissue, scintigraphy with 99mTc methyl diphosphonate ([99mTc]Tc-MDP). This upregulated expression in activated fibroblasts, and localization to the imaging modality only detects bone lesions and is plagued by poor tumor microenvironment. Although FAP expression has been anno- sensitivity (14, 15). PET imaging agents used in conjunction with either tated in multiple solid cancers, its presence in metastatic castration- CT or MRI have had various degrees of success imaging prostate resistant prostate cancer (mCRPC) is still largely uncharacterized. Few cancer (16). The commonly used PET radiotracer studies have been performed to accurately define and examine the 18 18 [ F]fluorodeoxyglucose ([ F]FDG) is of little utility (17, 18) while [18F]sodium fluoride (Na[18F]F) has documented improved sensitivity

1 over bone scans, but it cannot detect visceral lesions (15, 19). Department of Pharmacology, University of Minnesota Medical School, Min- 18 ﬂ neapolis, Minnesota. 2Department of Pathology and Surgery, University of [ F] uciclovine was recently approved by the FDA for detecting Illinois at Chicago, Chicago, Illinois. 3Division of Human Biology and Clinical biochemical recurrence; however, its utility in mCRPC has not been Research, Fred Hutchinson Cancer Research Center, Seattle, Washington. investigated (20, 21). Prostate-speciﬁcmembraneantigen(PSMA) 89 68 H.M. Hintz and J.P. Gallant contributed equally to this article. targeted probes such as, [ Zr]Zr-J591 and [ Ga]Ga-PSMA-11, have met with success in the clinic, but loss of PSMA expression is known to Corresponding Author: Aaron M. LeBeau, University of Minnesota, 312 Church – Street SE, Minneapolis, MN 55455-0217. Phone: 612-301-7231; Fax: 612-625- occur in neuroendocrine, androgen receptor negative prostate cancer, 8408; E-mail: [email protected] rendering the probes ineffective (22). Therefore, standard imaging modalities for prostate cancer leave room for improvement and warrant Clin Cancer Res 2020;XX:XX–XX the discovery and validation of new antigens in prostate cancer. doi: 10.1158/1078-0432.CCR-20-1358 The localization of FAP to the tumor microenvironment and its 2020 American Association for Cancer Research. association with aggressive phenotypes make it a compelling imaging

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Antibody production Translational Relevance For antibody expression, the heavy chain (342 bp) and light chain Targeted imaging probes for measuring disease burden and (324 bp) variable domains of the B12 sequence were cloned separately therapeutic response are dependent on the detection of unique into pFUSEss human IgG1 expression vectors (pFUSEss-CHIg-hG1 antigens expressed by the tumor. Men who fail standard-of-care and pFUSE2ss-CLIg-hk, Invitrogen) and cotransfected into HEK293T therapies and progress to metastatic castration-resistant prostate cells. The serum was collected after 72 hours, filtered through a cancer (mCRPC) are left with few therapeutic options. The current 0.45-mm filter, and purified using a 5 mL HiTrap Protein A HP imaging standards for prostate cancer are limited, which hinders column (GE Healthcare). The column was equilibrated with the development of new and urgently needed therapies. Our study 20 mmol/L sodium phosphate at pH 6.8. The serum was loaded onto shows fibroblast activation protein alpha (FAP) is a relevant target the column, washed with equilibration buffer for 10 column volumes, for the PET/CT imaging of mCRPC. FAP is emerging as the next and bound protein was eluted with 0.1 mol/L citric acid at pH 3.5. pan-cancer target given its upregulated expression in cancer- Eluted IgG was collected, concentrated using a 50-kDa centrifugal filter associated fibroblasts and localization to the tumor microenviron- (Millipore), and buffer exchanged into PBS using a Sephadex G-25 ment. Our FAP-targeted, radiolabeled probe was validated in PD-10 desalting column (GE Healthcare). The purity of the final B12 preclinical imaging studies and demonstrated to be highly selective IgG was analyzed by reduced and nonreduced SDS-PAGE, and protein for FAP-expressing tumors. This study suggests [89Zr]Zr-B12 IgG concentration was measured on the basis of absorbance at 280 nm is an ideal candidate for noninvasive PET/CT imaging in mCRPC, using a NanoDrop One UV – Vis Spectrophotometer (Thermo Fisher and more importantly, it represents a platform technology for the Scientific). development of theranostics targeting FAP. Genomic analysis RNA sequencing (RNA-seq) data were extracted from previously published studies of individuals with primary prostate cancer (24) or and therapeutic target for solid tumors including prostate cancer. mCRPC bone and soft tissue tumor biopsies (25). Capture of clinical Previously, we discovered and characterized a mAb targeting the samples was part of standard-of-care approaches for mCRPC that extracellular domain of FAP, termed B12 IgG. Here, we describe the included treatment with abiraterone acetate or enzalutamide followed development of [89Zr]Zr-B12 IgG, a FAP-targeted probe for the by taxane. RNA-seq fragments per kilobase million (FPKM) values detection of prostate cancer by PET/CT imaging. Genomic and IHC were obtained using the previously described methods (26). Micro- analysis revealed overexpression of FAP in metastatic disease com- array data were extracted from previously published studies of a set of pared with primary prostate tumors. In addition, FAP expression was metastatic tumors from men with CRPC (27). The dataset is available found to be uniform across metastatic disease regardless of genomic in the Gene Expression Omnibus under accession GSE77930. subtype, patient treatment history, and location of the metastatic lesion. We demonstrate that [89Zr]Zr-B12 IgG can be used for the IHC noninvasive imaging of FAP-expressing cells in preclinical models of Human studies were conducted in accordance with the Belmont prostate cancer. High tumor uptake and retention of [89Zr]Zr-B12 IgG Report. Samples for IHC analysis were obtained from the Prostate was observed compared with tumors administered the [89Zr]Zr- Cancer Biorepository Network and the University of Minnesota isotype control (IC IgG), suggesting potential for further development Masonic Cancer Center (Minneapolis, MN) using a University of of B12 IgG as a platform technology for radioimmunotherapy or Minnesota Human Subjects Division–approved IRB protocol for antibody–drug conjugates. In addition, [89Zr]Zr-B12 IgG detected tissue acquisition (IRB#1604M86269) and with written patient con- FAP-expressing stromal cells in a relevant prostate tumor microen- sent. IHC was performed on formalin-fixed paraffin-embedded tissue vironment model. Overall, our findings suggest that targeting FAP sections using (1:100) mouse anti-FAP (Abcam, 227703 clone SP325). could result in a next-generation targeted imaging paradigm and, thus, Unstained sections (4 mm) were deparaffinized and rehydrated using positively affect the development of much needed therapeutics for standard methods. For antigen retrieval, slides were incubated in mCRPC. 6.0 pH buffer (Reveal Decloaking reagent, Biocare Medical) in a steamer for 30 minutes at 95Cto98C, followed by a 20-minute cooldown period. A serum-free blocking solution (Sniper, Biocare Materials and Methods Medical) was placed on sections for 30 minutes. Blocking solution was Cell culture removed and slides were incubated in primary antibody diluted in 10% DU145 (#HTB-81) and HEK293T (#CRL-11268) cell lines were blocking solution/90% TBST. The antibody was used according to the purchased from the ATCC and were maintained according to ATCC manufacturer's protocol. guidelines. hPrCSC-44 cells were obtained from Dr. W. Nathaniel Brennen (Johns Hopkins Medicine, Baltimore, MD) and maintained Quantitative real-time PCR in RoosterNourish-MSC media (RoosterBio). CWR-R1-luciferase RNA was prepared from tissue using a RNeasy Kit (Qiagen) cells were obtained from Dr. Scott Dehm (University of Minnesota, and synthesized to cDNA using the High-Capacity RNA-to-cDNA Minneapolis, MN) and maintained with 10 mmol/L enzalutamide. Kit (Applied Biosystems). TaqMan qRT-PCR was performed using CWR-R1 cells were lentivirally transduced to express FAP as described the TaqMan Universal PCR Master Mix (Applied Biosystems) previously (23). FAP-expressing cells were continuously supplemen- and TaqMan Gene Expression Assay Probes: human FAP ted with 3 mg/mL puromycin to ensure stable levels of FAP expression. Hs00990807_m1 and 18s ribosomal 1 Hs03928985_g1. 18s ribosomal All cell experiments were performed within 4 months of thawing cell 1 was used as a normalization control. All qPCRs were performed lines from frozen cell stocks. Cell lines were authenticated using short- on a StepOnePlus Real-Time PCR system instrument (Applied tandem repeat analysis and routinely monitored for mycoplasma Biosystems). Each sample had three technical replicates, and each contamination prior to our studies. reaction was performed in three biological replicates. The data were

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DDC C ¼ t analyzed using the comparative t method (fold change 2 )as HPLC analysis with a radioactive detector was not performed on any of described previously. All data are presented as mean þ SEM. the radiolabeled products.

Western blot analysis Biodistribution Cell lysates were prepared from tissue using 1 laemmli buffer. Acute in vivo biodistribution studies were conducted to evaluate the Twenty micrograms of total protein per sample was denatured with 5% uptake of [89Zr]Zr-B12 IgG in mice bearing subcutaneous CWR- b-mercaptoethanol and separated on a 4% to 12% Bis-Tris Plus precast R1FAP tumors. Mice were randomized before the study and admin- gel (NW04122, Invitrogen) with SDS-PAGE and then transferred at istered [89Zr]Zr-B12 IgG (1.15 MBq, 1–2 mg mAb, in 100 mL of PBS) 20 V onto a nitrocellulose transfer membrane (Thermo Fisher Scien- via tail vein injection. Mice (n ¼ 3 mice for each tissue per time point) tific) with an iBlot 2 Dry Blotting System (Thermo Fisher Scientific) for were euthanized at 24, 72, and 144 hours postinjection. Organs 7 minutes. Membranes were blocked in 5% milk in TBS, 1% Tween 20, (including the tumor) were excised and counted on an automatic and probed with murine anti-human FAP mAb (1:500, sc-100528, gamma-counter (Hidex). The total number of counts [counts per Santa Cruz Biotechnology) overnight at 4C, washed, and then minute (cpm)] of each organ was compared with a standard syringe of incubated in rabbit anti-murine mAb conjugated to peroxidase known activity and mass. Count data were background- and decay- (1:1,000, sc-516102, Santa Cruz Biotechnology) for 1 hour at room corrected, and the percentage injected dose per gram (%ID/g) for each temperature. Equal protein loading was confirmed using murine anti- tissue sample was calculated by normalization to the total amount of human a-tubulin (1:10,000, sc-23948, Santa Cruz Biotechnology), and activity injected into each mouse. anti-murine conjugated to peroxidase (1:10,000). Binding was detected using the SuperSignal West Pico PLUS Chemiluminescent Substrate mPET/CT imaging (Thermo Fisher Scientific) and blots were imaged with a MyECL PET imaging experiments were conducted on an Inveon mPET/CT imager (Thermo Fisher Scientific) and Image Studio 2.0 software Scanner (Siemens Medical Solutions). Mice (n ¼ 3 for experimental (Li-Cor). and control groups) were administered [89Zr]Zr-B12 IgG or [89Zr]Zr- IC formulations (4.3 MBq, 25–30 mg of mAb, in 100 mL of PBS) via tail Animal models vein injection. Mice were anesthetized by inhalation of 2% isoflurane All animal studies were approved by the University of Minnesota and PET images were recorded at 24-hour time points out to 144 hours Institutional Animal Care and Use Committee. All animal studies were postinjection. PET list-mode data were acquired for 30 minutes using a nu performed in 3- to 4-week-old athymic nude-Foxn1 mice (Envigo). gamma ray energy window of 350–650 keV and a coincidence timing For subcutaneous tumor implantation, cells suspended in a 1:1 mixture window of 3.438 ns. CT acquisition was performed for 5 minutes at 80 of PBS and Matrigel (Corning) were injected into the shoulders of the kVp, 500 mA, 384 ms per step, and 340 steps covering 220 degrees. CT mice using a 26-gauge needle. For the engineered cell line model, images were reconstructed using a Hu scaled Feldkamp algorithm animals (n ¼ 3 for experimental and control groups) received sub- resulting in 192 192 matrix and PET utilized Ordered Subset cutaneous injections of CWR-R1FAP cells (1 106 cells in 100 mL). For Expectation Maximization (OSEM-3D) with 18 subsets and 2 itera- the CAF model, animals (n ¼ 3 for experimental and control groups) tions resulting in a 128 128 matrix. Two-dimensional (2D) images received subcutaneous injections of a 2:1 mixture of hPrCSC-44 and were prepared in Inveon Research Workplace and quantified using DU145 cells (2 106 hPrCSC-44 cells and 1 106 DU145 cells in AMIDE. An empirically determined system calibration factor was used 100 mL). Tumor volumes were measured twice weekly with calipers to convert voxel count rates to activity concentrations and the resulting and tumors were allowed to grow to a size of 100 to 300 mm3 before image data were normalized to the administered activity to parame- nuclear imaging and biodistribution experiments. For intratibial terize images in terms of %ID/cc. Drawn ellipsoid ROIs (n ¼ 3 for tumor implantation, luciferase-expressing CWR-R1FAP cells sus- experimental and control groups per time point) were used to deter- pended in PBS (2.5 105 cells in 10 mL) were injected into the tibia mine the mean %ID/cc in various tumors. Three-dimensional (3D) of one leg of each mouse (n ¼ 3 for experimental and control groups). reconstructions were generated using AMIRA. Tumors were allowed to grow for 5 weeks before bioluminescent imaging (BLI) and nuclear imaging experiment. For BLI, the mice were Statistical analysis injected intraperitoneally with D-luciferin (150 mg/kg). Images were All statistical analyses were performed using Prism GraphPad acquired 10 minutes after the injection of D-luciferin using an IVIS software. Patient microarray data were analyzed using a one-way Spectrum (PerkinElmer). ANOVA and corrected for multiple comparisons using Sidak hypothesis testing. Animal studies were performed with n ¼ 3 for experi- Bioconjugation and radiochemistry mental and control groups and the statistical significance of signal For nuclear imaging studies, the B12 IgG and IC IgG were conju- intensities were determined using a two-way ANOVA and corrected gated to p-SCN-Bn-Deferoxamine (DFO, Macrocyclic) as described for multiple comparisons using Sidak hypothesis testing. Quantitative previously (28). Zirconium-89 [89Zr] was purchased from the Uni- RT-PCR results were determined by two-tailed Student t test. Results versity of Wisconsin Medical Physics Department (Madison, WI). are depicted as mean þ SEM. The symbols used to represent [89Zr]Zr-oxalate (277 MBq) in 1.0 mol/L oxalic acid (600 mL) was the P values were as follows; ns, P > 0.05; , P ≤ 0.05; , P ≤ 0.01; P ≤ P ≤ adjusted to pH 7.5 with 1.0 mol/L Na2CO3. To radiolabel the B12 IgG , 0.001; , 0.0001. and IC IgG, the DFO-B12 IgG conjugate (272 mL, 2.2 mg/mL, 600 mg mAb) or DFO-IC IgG conjugate (600 mg mAb) in 0.5 mol/L HEPES (pH 7.5) was added to the neutralized [89Zr]Zr-oxalate solution and Results incubated at room temperature with rotation for 1 hour. The labeled FAP is overexpressed in metastatic prostate cancer product was purified using a size-exclusion PD-10 column preequili- Publicly available RNA-seq datasets were analyzed (24, 25) to brated with PBS buffer and evaluated via radioactive TLC for purity quantify FAP expression between hormone-na€ve primary prostate using standard methods as described previously (29). Size-exclusion tumors and metastatic disease (Fig. 1A). Data analysis revealed a

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Figure 1. FAP is overexpressed in metastatic prostate cancer compared with primary tumors. A and B, RNA-seq analysis of FAP expression in primary tumor versus metastatic disease. Multiple raw FASTQ RNA-seq datasets were obtained via DbGAP and aligned and transcripts were quantified via uniform pipeline, enabling cross- experimental comparisons. Data are reported as FPKM. A, mRNA FAP expression was significantly increased in metastatic disease (n ¼ 51) compared with primary tumors (n ¼ 25). , P ≤ 0.001. B, Similar levels of FAP expression were observed across primary tumor grades, treatment history, and metastatic sites. C, Representative images of IHC staining for FAP in primary prostate cancer tumors. Magnification, 40. D, Representative images of IHC staining for FAP in normal prostate and several metastatic lesions. Magnification, 40.

significant increase in FAP mRNA expression in metastatic disease across all metastatic subtypes regardless of AR status or NE differen- compared with primary (P ¼ 0.0003). Next, the dataset was stratified tiation (Fig. 2B). The gene expression profiles of several CAF markers based on grade, treatment regimen, and location of metastasis to see if were also documented to have no significant difference among tumor any further mRNA expression patterns could be defined (Fig. 1B). genotypes. One exception being the AR /NE S100A4 population, þ FAP expression was similar in primary tumor samples regardless of which was found to be significantly higher than AR /NE tumors grade (Gleason score 6–9). In the metastatic tumor subtypes, treatment (P ¼ 0.001). Together, these analyses suggest that FAP expression is a with second generation antiandrogens (enzalutamide or abiraterone characteristic of mCRPC regardless of genetic subtype, treatment acetate) or chemotherapy (taxane) did not have a significant effect on regimen, or location of metastasis. FAP expression. Finally, we observed no difference in FAP mRNA expression among locations of metastasis (lymph node, bone, liver, PET/CT imaging of FAP in an engineered model other). To corroborate the FAP expression patterns found by RNA Previously, we demonstrated that IRDye-800CW–conjugated sequencing, IHC analysis was performed on two tissue microarrays B12 IgG could potently and selectively detect FAP-expressing cells (TMA). We first analyzed a primary prostate adenocarcinoma TMA. by near infrared optical imaging (23). These results suggested No discernable staining for FAP was observed in any of the primary further development of B12 IgG as a PET imaging probe labeled 89 ¼ prostate tumor cores even across different Gleason scores (Fig. 1C). In with the long-lived positron-emitting radioisotope [ Zr] (t1/2 a mCRPC TMA, we observed medium to strong IHC staining in 3.7 days). We first evaluated [89Zr]Zr-B12 IgG for the targeted metastatic lesions compared with normal prostate (Fig. 1D). imaging of FAP-positive cells in mice bearing subcutaneous CWR- Further genetic analysis was performed to assess whether FAP R1FAP xenografts. We previously engineered a human FAP- overexpression was associated with specific metastatic prostate cancer expressing prostate cancer cell line, CWR-R1FAP,tousefor gene signatures. We investigated FAP expression in the context of proof-of-concept studies (23). Mice were injected with [89Zr]Zr- androgen receptor (AR) status and neuroendocrine (NE) differenti- B12 IgG via tail vein and imaged by mPET/CT at 24, 48, 72, 96, and ation using microarray data of 171 mCRPC tumors from 63 rapid 144 hours postinjection (Fig. 3A). Analysis of 2D and 3D PET autopsy subjects at the University of Washington (Fig. 2A; refs. 27, 30). images revealed the distribution of [89Zr]Zr-B12 IgG. Initial imag- Quantitative analysis revealed that FAP gene expression was similar ing probe uptake at 24 hours was high in the FAP-positive tumors

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FAP ACTA2 S100A4 PDGFRA PDGFRB AR KLK3 TMPRSS2 NKX3-1 FOLH1

− − − − AR+ NE AR+ NE+ AR NE AR NE+ Low High

B FAP ACTA2 S100A4 6 ns o 6 6 ns ns *** 4 4 ns 4 ns ns 2 ns 2 ns 2 0 0 0 mean centered ratio mean centered rati 2 -2 -2 -2 2 mean centered ratio 2 g Log

-4 Log -4 -4

− + − + − + − + Lo − + − + − − + NE + NE - NE - NE + NE + NE NE - NE + NE + NE NE − NE AR AR AR AR AR AR AR AR AR AR AR AR PDGFRA PDGFRB 10 5 ns ns ns ns ns 5 ns 0

0 -5 mean centered ratio mean centered ratio 2 2 -5 -10 Log Log − + − + − + − + − − + NE + NE NE − NE + NE + NE NE − NE AR AR AR AR AR AR AR AR

Figure 2. FAP expression is uniform across metastatic prostate cancer genomic subtypes. A, FAP expression was evaluated in metastatic prostate cancer tumors that displayed gene signatures signifying AR status and NE differentiation. B, FAP expression in mCRPC patient samples was similar across all genetic subtypes. Expression of several CAF markers is also shown (ACTA2, S100A4, PDGFRA, and PDGFRB). Data are represented as log2 mean centered ratio. , P ≤ 0.001. For A and B: ARþ NE (n ¼ 134); ARþ NEþ (n ¼ 7); AR NE (n ¼ 10); AR NEþ (n ¼ 20). and liver. Over the time course, retention of [89Zr]Zr-B12 IgG signal the tumors and secondary organs over time (Fig. 3C). [89Zr] in the tumor remained high, while liver signal diminished. By uptake in the main clearance organs (liver and kidney) and the 144 hours postinjection, probe uptake in the FAP-positive tumors spleen, where IgGs are known to localize nonspecifically, was had cleared. Additional mice bearing subcutaneous CWR-R1FAP observed as anticipated. Distribution of [89Zr]Zr-B12 IgG was xenografts were injected with the nonspecific[89Zr]Zr-IC IgG via similar in all other organs at each time point, except for the tail vein and imaged at 24, 48, and 72 hours postinjection (Fig. 3A). tumors. At 24 hours, [89Zr] uptake by FAP-positive tumors was Although some uptake was observed in the FAP-positive tumors, detected; however, the mean %ID/g value of [89Zr] uptake was the signal was much lower than [89Zr]Zr-B12 IgG administered 3-fold higher at 72 hours (P < 0.0001). Uptake in the FAP- tumors. Furthermore, the probe rapidly cleared from the tumors positive tumors remained significantly higher at 144 hours and almost no signal was visible by 72 hours, suggesting that uptake compared with 24 hours (P < 0.0001). These data further reflect was due to the enhanced permeability and retention effect rather the trends observed in the 2D and 3D imaging data. than selective localization. The ability of [89Zr]Zr-B12 IgG to image FAP-expressing cells in a Quantitative analysis of [89Zr] uptake in the FAP-positive tumors complex microenvironment was investigated in mice bearing intrati- was in concordance with the 2D and 3D imaging data (Fig. 3B). At bial CWR-R1FAP xenografts. Following intratibial injection of lucif- 24 hours, the [89Zr] uptake by tumors in animals administered with erase-expressing CWR-R1FAP cells, BLI was performed weekly to [89Zr]Zr-B12 IgG was 2-fold higher than [89Zr]Zr-IC IgG with %ID/cc monitor tumor growth. Approximately 5 weeks postinjection, meta- values averaging 38.32 3.58 versus 15.69 0.33 (P ¼ 0.003). static lesions in or around the bone had developed (Fig. 4A). Mice were The difference in tumor uptake of [89Zr]Zr-B12 IgG compared to injected with [89Zr]Zr-B12 IgG or [89Zr]Zr-IC IgG via tail vein and [89Zr]Zr-IC IgG increased to 3-fold by 72 hours. %ID/cc values imaged 24 hours postinjection (Fig. 4A). In the [89Zr]Zr-B12 IgG averaged 30.24 2.48 versus 9.83 0.73 (P ¼ 0.0013). An ex vivo administered animals, high probe uptake was observed in the FAP- biodistribution study was performed to evaluate probe localization to positive leg tumor while no uptake was visible in the healthy leg. As

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A [89Zr]Zr-B12 IgG [89Zr]Zr-IC IgG 24 h 48 h 72 h 144 h 24 h 48 h 72 h 50 50 %ID/cc %ID/cc 2D PET/CT 0 0 9.3 9.3 x 10 x 10 -1 -1 3D PET/CT

1.6 1.6

B C 30 24 h 72 h 144 h 50 ** B12 IgG 25 ** **** 40 IC IgG ** 20 **** 30 15 %ID/g

%ID/cc 20 10 10 5

0 0 24 48 72 96 120 144

Blood Heart Lung Liver Bone umor Time postinjection (h) Spleen Kidney Muscle T Intestine

Figure 3. [89Zr]Zr-B12 IgG can detect FAP-positive prostate cancer tumors by PET/CT imaging. A, Representative PET/CT images of mice bearing subcutaneous CWR-R1FAP xenografts (white arrowhead). Mice (n ¼ 3 for experimental and control groups) received 3.7 MBq (25–50 mg, 7.3 mg/MBq) of [89Zr]Zr-B12 IgG or 3.7 MBq (25–50 mg, 8.7 mg/MBq) [89Zr]Zr-IC IgG via tail vein and were imaged at the indicated time points. B, Quantitative analysis of subcutaneous xenografts from mice in Fig. 3A (n ¼ 3 for experimental and control groups per time point) revealed signiﬁcantly higher [89Zr] uptake in the tumors of [89Zr]Zr-B12 IgG administered animals compared with the [89Zr]Zr-IC IgG at 24, 48, and 72 hours postinjection. Values, mean SEM. , P ≤ 0.01. C, Ex vivo biodistribution of [89Zr]Zr-B12 IgG in tissues of mice bearing subcutaneous CWR-R1FAP xenografts. Mice (n ¼ 3 mice per tissue per time point) were injected with 1.15 MBq (5.7 mg, 6.6 mg/MBq) of [89Zr]Zr-B12 IgG via tail vein prior to sacriﬁce at the indicated time points. Values, mean SEM. , P ≤ 0.0001.

shown in the 2D and 3D PET images, [89Zr]Zr-IC IgG signal in the cells (2:1 ratio; 2 106 :1 106 cells; Fig. 5B). Mice bearing FAP-positive leg tumor was considerably lower than [89Zr]Zr-B12 IgG subcutaneous hPrCSC-44/DU145 xenografts were injected with at 24 hours postinjection (Fig. 4A). Insignificant nonspecific locali- [89Zr]Zr-B12 IgG or [89Zr]Zr-IC IgG and imaged serially at 24, 48, zation was also observed in the leg and spine of [89Zr]Zr-IC IgG 72, 96, 120, and 144 hours postinjection (Fig. 5C). Analysis of the 2D administered animals. Quantification of [89Zr] uptake in the FAP- and 3D imaging data revealed high uptake in the FAP-positive stroma positive tumors reiterated these results. [89Zr] uptake in the animals tumors and liver at 24 hours in the animals administered [89Zr]Zr-B12 administered with [89Zr]Zr-B12 IgG was almost 5-fold higher than IgG. Retention of the imaging probe in the tumor and concordant loss with [89Zr]Zr-IC IgG with %ID/cc values averaging 28.57 3.23 of signal in the liver was also observed over time. By 144 hours versus 5.85 0.58 (P ¼ 0.0014). postinjection, the [89Zr]Zr-B12 IgG signal was low but still detectable in the 2D PET images. In contrast, the animals administered PET/CT imaging of FAP-expressing stroma [89Zr]Zr-IC IgG had extremely high uptake in the liver compared with To develop a xenograft model that recapitulated the tumor micro- the low uptake by the FAP-positive stroma tumors. Furthermore, environment where FAP expression is localized to the stroma, and not [89Zr]Zr-IC IgG signal in the tumor had cleared by 72 hours. Quan- cancer cells, we used the immortalized human prostate cancer stroma tification of [89Zr] uptake in the FAP-positive stroma tumors was cell line, hPrCSC-44, in combination with the FAP-null prostate significantly higher in the animals administered [89Zr]Zr-B12 IgG cancer cell line DU145. hPrCSC-44 cells are CAFs that highly express compared with [89Zr]Zr-IC IgG at 24, 48, 72, and 144 hours postin- FAP as demonstrated by flow cytometry with a PE-conjugated com- jection (Fig. 5D). Notably, tumor uptake of [89Zr]Zr-B12 IgG was 4- to mercial anti-human FAP mAb (Fig. 5A). The mouse xenograft model 5-fold higher than [89Zr]Zr-IC IgG from 24 to 72 hours. Mean %ID/cc was developed by subcutaneous injection of hPrCSC-44 and DU145 were 15.39 0.93 (B12 IgG) versus 11.93 2.38 (IC IgG) at 24 hours

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A BLI 2D PET/CT 3D PET/CT B

1.9 50 40 9.3 **

%ID/cc 30 x10 9 c

20 TH Zr]Zr-B12 IgG %ID/c 89

[ 0 0.27 Tumor Healthy x 10 10 -1 1.9 50 0 %ID/cc

x10 B12 IgG IC IgG 9

Zr]Zr-IC IgG TH 89

[ 1.6 0.27 0 Tumor Healthy

Figure 4. [89Zr]Zr-B12 IgG can detect FAP-positive metastatic prostate cancer tumors by PET/CT imaging. A, Representative PET/CT images of mice bearing intratibial CWR- R1FAP xenografts. Intratibial tumors were identiﬁed by BLI. Mice (n ¼ 3 for experimental and control groups) received 3.8 MBq (25-50 mg, 10.6 mg/MBq) [89Zr]Zr-B12 IgG or 4.2 MBq (25-50 mg, 14.3 mg/MBq) [89Zr]Zr-IC IgG via tail vein and were imaged at 24 hours postinjection. The tumor-bearing leg [Tumor (T)] was compared with the healthy leg [Healthy (H)] in each animal. B, Quantitative analysis of intratibial xenografts from the mice in A (n ¼ 2 for experimental and control groups) revealed signiﬁcantly higher [89Zr] uptake in tumors treated with [89Zr]Zr-B12 IgG compared with the [89Zr]Zr-IC IgG. Values, mean þ SEM. , P ≤ 0.01.

(P ¼ 0.0007) and 34.13 1.99 (B12 IgG) versus 6.12 2.03 (IC IgG) at over 50% of the total tumor mass, or more, in cases of desmoplastic 72 hours (P ¼ 0.0006). reaction. Therefore, targeting the tumor microenvironment could To validate our FAP-positive tumor stroma model, tumors and increase imaging sensitivity and have greater utility compared with livers from subcutaneous hPrCSC-44/DU145 xenografts administered current agents. Our group has previously shown that non-specific PET [89Zr]Zr-B12 IgG were excised and frozen. FAP mRNA levels were radiotracers have limited function in preclinical prostate cancer analyzed from the frozen tissue samples and FAP-negative xenograft models (29). CAFs are the major cell population in the tumor stroma tissues by quantitative RT-PCR. Human FAP mRNA was detected in and are critical to tumor growth beyond 1 to 2 mm, indicating that the hPrCSC-44/DU145 xenografts, but not the FAP-negative control CAF-targeted imaging could have improved sensitivity for small when normalized to mouse liver tissue (P ¼ 0.0006; Fig. 5E). Next, tumors (32). In addition, reactive stroma formation occurs in both Western blot analysis using a commercial anti-human FAP mAb was bone and visceral lesions; thus, imaging CAFs could expand tumor performed to investigate FAP protein expression in the tissue samples. visualization for all metastatic sites. FAP is an attractive imaging target Human FAP was detected in all three hPrCSC-44/DU145 xenografts because of its upregulated expression in CAFs and localization to the (Fig. 5F). Our tumor stroma model presents a more relevant tool for tumor microenvironment of epithelial tumors. Therefore, FAP- the evaluation of diagnostic and therapeutic agents for prostate cancer. targeted imaging could be used for heterogeneous tumor populations Overall, these data validate [89Zr]Zr-B12 IgG as a probe for the targeted as a pan-cancer diagnostic and as a predictive biomarker for evaluating imaging of FAP-expressing stroma within the prostate tumor FAP-targeted therapies. microenvironment. In this study, we reveal FAP as a candidate for the targeted imaging and treatment of mCRPC. Genomic and IHC analyses demonstrate FAP expression in metastatic disease across genomic subtypes, treat- Discussion ment regimens, and metastatic sites. Our FAP-targeted nuclear imag- Nuclear imaging has proven invaluable at accelerating the devel- ing probe, [89Zr]Zr-B12 IgG, demonstrated excellent selectivity and opment of diagnostics and therapeutics for cancer. New therapies are sensitivity toward FAP-expressing cells in several preclinical models of needed for mCRPC and the development of these agents is dependent prostate cancer. This was documented by high tumor uptake at on accurate imaging modalities to stage disease and evaluate patient 24 hours postinjection and long-term retention of [89Zr]Zr-B12 IgG response to therapy. Standard imaging modalities for prostate cancer signal compared with the [89Zr]Zr-IC IgG (Figs. 3A, 4A, and 5C). The possess various flaws and need improvement to drive therapeutic ex vivo biodistribution study highlighted the promising pharmacoki- discovery. Current radiotracers lack selectivity for cancer tissue versus netics of [89Zr]Zr-B12 IgG (Fig. 3C). We observed blood clearance by surrounding healthy tissue; have low sensitivity for small tumors and 24 hours and [89Zr]Zr-B12 IgG uptake in the tumor was higher than visceral metastatic lesions; and patients may be required to undergo any other organ by 72 hours. [89Zr]Zr-B12 IgG also showed utility in multiple scans to assess all potential metastatic sites. These limitations the intratibial and prostate tumor stroma xenograft models further, urge the discovery and validation of new antigens in prostate cancer for suggesting its high sensitivity in advanced models (Figs. 4 and 5). In targeted nuclear imaging and the subsequent development of inno- addition, we introduce a clinically relevant model of the prostate tumor vative therapies. Antigens that can function as highly selective and microenvironment to help advance the development of FAP-targeted sensitive imaging targets are key to the development of next- diagnostic and therapeutic agents for mCRPC. FAP is highly expressed generation probes and the advancement of personalized medicine. by CAFs in the tumor stroma, but not cancer cells, and models that Imaging the tumor microenvironment with targeted probes is a key reflect this will be critical for continued studies of FAP and prostate strategy to overcome current limitations given the essential role of the cancer and could improve translation of targeted probes into the clinic. microenvironment in disease progression. In addition, stromal cells This model could be further improved with the development of a have greater genomic stability than malignant cells, which promotes humanized mouse to evaluate [89Zr]Zr-B12 IgG imaging in more retention of imaging targets (2, 31). The tumor stroma can make up advanced prostate tumor microenvironments.

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A B Unstained hPrCSC-44 cells Anti- FAP mAb 300

200 Count

2:1 hPrCSC-44 5 wks 100 DU145 cells :DU145 sc injection

0 0 103 104 105 PE-A :: FAP-PC

C [89Zr]Zr-B12 IgG [89Zr]Zr-IC IgG 24 h 48 h 72 h 144 h 24 h48 h 72 h 144 h 50 50 %ID/cc %ID/cc

0 2D PET/CT 0

14.8 14.8 x 10 x 10 -1 -1 3D PET/CT

2.8 2.8

D EFFAP αTubulin 60 30 *** *** B12 IgG 25 90 kDa IC IgG ** 50 kDa 40 *** 20

15 fold change %ID/cc ** A 20 10 mRN 5 0 0 24 48 72 96 120 144 hPrCSC-44/DU145 Negative Time postinjection (h)

Figure 5. [89Zr]Zr-B12 IgG can detect FAP-positive tumor stroma by PET/CT imaging. A, Validation of membrane bound FAP expression in the hPrCSC-44 cell line by ﬂow cytometry. hPrCSC-44 cells were stained using a commercial PE conjugated mAb (red line) and compared with an unstained control (blue line). B, Schematic of FAP- positive tumor stroma model. hPrCSC-44 and DU145 cells (2:1 ratio; 2 106 :1 106 cells) were implanted subcutaneously in mice. Xenografts grew for 5 weeks before PET/CT imaging. C, Representative PET/CT images of mice bearing hPrCSC-44/DU145 subcutaneous xenografts (white arrowhead). Mice (n ¼ 3 for experimental and control) received 4.6 MBq (25–50 mg, 6.7 mg/MBq) [89Zr]Zr-B12 IgG or 4.7 MBq (25-50 mg, 6.7 mg/MBq) [89Zr]Zr-IC IgG via tail vein and were imaged at the indicated time points. D, Quantitative analysis of subcutaneous xenografts from the mice in C (n ¼ 3 for experimental and control groups per time point) revealed signiﬁcantly higher [89Zr] uptake in the tumors of [89Zr]Zr-B12 IgG administered animals compared with the [89Zr]Zr-IC IgG at 24, 48, 72, and 144 hours postinjection. Values, mean SEM. , P ≤ 0.01; , P ≤ 0.001. E, mRNA levels of human FAP in hPrCSC-44/DU145 (n ¼ 3 technical and biological replicates) and FAP-negative (n ¼ 3 technical and biological replicates) xenograft tissues were analyzed using quantitative RT-PCR. mRNA fold change is normalized to mouse liver tissue. , P ≤ 0.001. F, Representative Western blot detecting human FAP protein in hPrCSC-44/DU145 xenograft tissue using a commercial mAb.

Other FAP-targeted approaches have been developed for theranos- ESC11 and ESC14 were used for SPECT/CT imaging in xenograft tic use in oncology. The ﬁrst-generation anti-FAP mAb, F19, and its models and tested for therapeutic efﬁcacy in a single radioimmu- humanized version, sibrotuzumab, were labeled with 131I and used for notherapy study (35). No data have been published from patient trials; SPECT imaging of patients with metastatic cancer (33, 34). Both were thus, translational potential cannot be assessed. DOTA coupled, FAP- shown to have slow clearance and no reported internalization, which targeted enzyme inhibitors (FAPI) were labeled with 68Ga for PET/CT limits their function for imaging or therapy. Furthermore, the human- imaging and 90Y for radioimmunotherapy in patients with metastatic ized antibody caused an immune response in patients and sibrotu- cancer (36–38). A limited study was done in patients with mCRPC that zumab was withdrawn from clinical trials. The 177Lu-labeled mAbs lacked PSMA expression, but more work is needed to evaluate the

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Imaging FAP in Prostate Cancer

radiotracers utility throughout metastatic disease. In addition, FAPIs therapy to target the tumor microenvironment and advance have short biological half-lives, a key determinant for isotope selection, therapeutic strategies to improve patient outcomes. which limits their theranostic applications. A mAb is better suited for therapeutic development given the higher therapeutic benefit from Disclosure of Potential Conflicts of Interest prolonged exposure to cytotoxic agents. A FAP-targeted peptide, FAP- H.M. Hintz is a co-inventor on a pending patent (16/778,977) regarding the use of 2286, is also being developed for peptide-targeted radionuclide therapy B12 IgG and its variants to target Fibroblast Activation Protein Alpha for imaging and by Clovis Oncology, but so far, no data have been published (39). therapy, which will be owned by the University of Minnesota. P.S. Nelson reports personal fees from Janssen, Bristol-Myers Squibb, and Astellas outside the submitted Although these studies demonstrate the utility of FAP-targeted imag- work. A.M. LeBeau is a co-inventor on a pending patent (16/778,977) regarding the ing, they focus on applications in breast, colorectal, and lung cancers. use of B12 IgG and its variants to target Fibroblast Activation Protein Alpha for To date, almost no work has been published on FAP-targeted imaging imaging and therapy, which will be owned by the University of Minnesota. No in prostate cancer. Although PSMA-targeted imaging probes have potential conflicts of interest were disclosed by the other authors. been used for prostate cancer, variable expression in metastatic tumors and difficulty detecting visceral lesions limits their utility (40). There Authors’ Contributions remains a need for a selective and sensitive imaging probe that can be H.M. Hintz: Conceptualization, data curation, formal analysis, methodology, widely used in patients with mCRPC. FAP-targeted imaging probes writing-original draft, writing-review and editing. J.P. Gallant: Conceptualization, software, formal analysis, investigation, writing-original draft, writing-review and could overcome current limitations given the genomic stability of the editing. D.J. Vander Griend: Data curation, formal analysis. I.M. Coleman: Data tumor microenvironment, the essential role of CAFs in tumor pro- curation, formal analysis, writing-original draft. P.S. Nelson: Data curation, formal gression, and the localized overexpression of FAP in the prostate analysis, supervision, writing-original draft. A.M. LeBeau: Conceptualization, tumor stroma. resources, supervision, validation, investigation, methodology, writing-original Together, our data support [89Zr]Zr-B12 IgG as an ideal candi- draft, project administration, writing-review and editing. date for the noninvasive PET/CT imaging of mCRPC by targeting FAP. In addition to the antibody's favorable pharmacokinetics and Acknowledgments binding affinity, B12 IgG is internalized by FAP-expressing cells as This work was supported by NIH/NCI R01 CA237272 (to A.M. LeBeau), NIH/ NCI R01 CA233562 (to A.M. LeBeau), a 2018 Prostate Cancer Foundation Challenge indicated by the high probe uptake, tumor retention, and previous Award (to A.M. LeBeau), a 2013 Prostate Cancer Foundation Young Investigator in vitro characterization studies (25). Antibody internalization is a Award (to A.M. LeBeau), and NIH/NCI T32 CA009138 (to H.M. Hintz). The authors key property that permits the retention of imaging signal and acknowledge the University Imaging Centers at the University of Minnesota (Min- efficacy of potential immunoconjugate therapies. Thus, these prop- neapolis, MN) for their technical advice and support of various imaging equipment. In erties warrant further development of B12 IgG for translation into particular, the authors thank Jason Mitchell for his PET/CT imaging assistance. We the clinic as a theranostic platform technology. Antibody–chelator thank the University of Minnesota's Research Animal Resources staff for assisting with research animal maintenance and include a special thanks to Jason Austin for his conjugation chemistry facilitates the exchange of nuclides that can help with injections and Yingming Li for her expertise with the IT model. The authors be used for either diagnostic or therapeutic purposes. Therefore, are grateful to Colleen Forster and the rest of the Biorepository and Laboratory B12 IgG can be evaluated as a radioimmunotherapy agent. Clinical Services Division at the University of Minnesota (Minneapolis, MN) for their studies with 177Lu- or 225Ac-PSMA-targeted mAbs are currently histology and pathology support. ongoing and suggest that development of other radioimmunothera- pies could be effective against mCRPC (41, 42). B12 IgG could also The costs of publication of this article were defrayed in part by the payment of page advertisement be used to create a library of antibody–drug conjugates for targeted charges. This article must therefore be hereby marked in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. delivery of cytotoxic agents. Finally, combination therapies that target both CAFs and cancer cells are shown to have a synergistic Received April 15, 2020; revised May 27, 2020; accepted July 2, 2020; published first effect (43, 44). B12 IgG may be used as part of a combination July 7, 2020.

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Imaging Fibroblast Activation Protein Alpha Improves Diagnosis of Metastatic Prostate Cancer with Positron Emission Tomography

Hallie M. Hintz, Joseph P. Gallant, Donald J. Vander Griend, et al.

Clin Cancer Res Published OnlineFirst July 7, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-20-1358

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