Detecting Functional and Accessible Folate Receptor Expression in

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Detecting functional and accessible folate receptor expression in cancer and polycystic kidneys Haiyan Chu, Jonathan M. Shillingford, Joseph A. Reddy, Elaine Westrick, Melissa Nelson, Emilia Z. Wang, Nikki Parker, Albert E. Felten, Jeremy Vaughn, Le-cun Xu, Yingjuan Lu, Iontcho R. Vlahov, and Christopher P. Leamon Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00624 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on August 4, 2019

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Molecular Pharmaceutics

Detecting functional and accessible folate receptor expression in cancer and polycystic kidneys Haiyan Chu, Jonathan M. Shillingford*, Joseph A. Reddy, Elaine Westrick, Melissa Nelson, Emilia Z. Wang, Nikki Parker, Albert E. Felten, Jeremy Vaughn, Le-Cun Xu, Yingjuan Lu, Iontcho R. Vlahov and Christopher P. Leamon# Endocyte, Inc., 3000 Kent Ave., Suite A1-100, West Lafayette, IN 47906, USA *Current

#To

address: Division of Nephrology, Department of Internal Medicine, 1560B MSRB 2, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA

whom correspondence should be addressed: Dr. Christopher P. Leamon 3000 Kent Ave. Suite A1-100 West Lafayette, IN 47906 Phone: (765)463-7175 FAX: (765)463-9271 Email: [email protected]

Running Title: Folate receptor functional IHC Key Words: Folate receptor, targeted drug delivery, immunohistochemistry, cancer, polycystic kidney disease.

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ABSTRACT Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are: i) Folate receptor alpha (FR) protein is expressed in the diseased tissues, and ii) FR in those tissues is accessible and functionally competent to bind systemically-administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FR binding, a multi-lysine containing linker that can crosslink to FR in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FR-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FR protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FR expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.


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1. INTRODUCTION Folate receptor alpha (FR) is a glycophosphatidylinositol-anchored cell surface protein which binds the essential vitamin, folate, and transports this molecule (and congeners thereof) into cells to facilitate 1-carbon metabolism and nucleotide biosynthesis.1 Whereas most normal tissues express low to no FR on their apical membranes or epithelial cells, this protein is highly expressed on the cell surface of various malignancies as well as the cystic membranes of polycystic kidney disease 2-6. For example, epithelial ovarian carcinomas, triple negative breast cancer, and non-small cell lung cancer have been found to have high membrane expression of FRα.7-11 Furthermore, higher levels of FRα in ovarian and breast cancers has been strongly associated with more advanced stage of disease and much poorer disease-free survival.7, 10, 12-13 FRα has attracted widespread interest as a target for drug delivery in cancer therapy due to its expression in various types of cancers and its increased level at more advanced cancer stage. Imaging agents and therapeutics can be targeted to FRα-expressing cancers by coupling high affinity ligands or antibodies to molecular “payloads”. Following intravenous infusion, such coupled drugs (or conjugates) can be distributed to FRα-expressing tumor cells through circulation. IMGN853, a FR-based antibody-drug conjugate (ADC) of DM4, has shown promising anti-tumor activity against platinum-resistant ovarian cancers.14-15 Similarly, EC145, a folate-based small molecule drug conjugate (SMDC) of the microtubule destabilizer, DAVLBH, has also shown clinical activity against ovarian and non-small cell lung cancers.16-18 With the continued development of FRα-targeted therapeutics, a reliable tissue-based method is desired for the selection of those patients with FRα-expressing tumors. Immunohistochemistry (IHC) of formalin-fixed and paraffin-embedded (FFPE) tissue is admittedly a globally applied 3 ACS Paragon Plus Environment


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method for routine tissue collection, processing and storage; and, an anti-FRα IHC method has been described and used for selecting patients in anti-FRα based ADC studies.15 With preserved tissue morphology, IHC analysis can facilitate excellent identification of FRα at the cellular level and it can identify the extent of heterogeneity of expression in diseased tissues. However, IHC does have limitations in selecting patients for targeted therapy. For example, IHC analysis of the FRα antigen in diseased tissues fails to address the protein’s in vivo accessibility and binding ability. Systemically administered radiolabeled anti-FRα antibodies have been explored for in vivo tumor imaging, but these agents couldn’t achieve the needed contrast between FRα-positive tumors and FRα-negative normal tissues, probably because these large molecular weight agents have prolonged circulation time and slow/inefficient penetration into solid tumors.19-20 In contrast, small molecule folate conjugates of metal chelators do show rapid in-vivo clearance and better tissue penetration, thus displaying promising real time imaging capabilities for FR-positive tumors. One agent,

99mTc-EC20

(etarfolatide), has been shown to be a safe and effective tool in

imaging FR-positive tumors in both animal models and over 1000 patients.16,

21-23

While

additional folate-based small molecular imaging agents are under development for improved sensitivity, such imaging agents are limited in that they cannot reveal tissue heterogeneity of FR expression at the cellular level. Herein we describe a new method that combines the excellent in vivo capability of folate-based imaging with the analytical capability (at the cellular level) of IHC to assess the accessibility and functionality of FR expression within tissues. To achieve this, a folate-conjugated small molecule reporter conjugate (SMRC) is desired whereby following intravenous injection it can subsequently


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bind to accessible and functional FR in vivo in tissues of interest such as tumors, polycystic kidneys and even normal organs. Following tissue biopsy, FFPE processing and IHC, the cellular localization and relative abundance of functional and accessible FR can be evaluated. A major barrier hindering this application is that formaldehyde fixation denatures FR which allows the once-bound folate-SMRC to dissociate and wash away from the processed tissue.24 To circumvent this limitation, we have designed the formaldehyde-activated crosslinking SMRC, herein referred to as EC2220. We report on the design and chemical synthesis of EC2220, its biological properties both in vitro and in vivo, as well as its application in determining the accessibility of functional tissue-derived FR.

2. MATERIALS AND METHODS 2.1. Materials. Pteroic acid (Pte) and N10-trifluoroacetyl-Pte were prepared according to Xu et al.25 Peptide synthesis reagents were purchased from NovaBiochem (La Jolla, CA) and Bachem (San Carlos, CA). EC17 and EC0923 were synthesized at Endocyte and as previously described.2627

All other common reagents were purchased from Sigma (St. Louis. MO) or other major

suppliers.

2.2. Cell lines. All cell lines were of human origin and purchased from ATCC except for the IGROV-1 cell line which was a gift from Professor Philip S. Low of Purdue University. KB cells (ATCC CCL-17) are a cell line containing markers of HeLa cervical cancer origin. IGROV1 and OV90 (ATCC CRL-11732) are ovarian carcinoma cell lines. MDA-MB-231 (ATCC HTB26) are triple-negative breast carcinoma cells, and A549 (ATCC CCL-185) are non-small lung adenocarcinoma cells. 5 ACS Paragon Plus Environment


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2.3. Synthesis and analytical characterization of EC2220. EC2220 is a fluorescein-coupled, folate-containing peptide consisting in sequence of Pte-Glu-Lys-Asp-LysAsp-Lys-Lys-fluorescein. EC2220 synthesis was divided into two parts: peptide synthesis and fluorescein isothiocyanate (FITC) coupling. Synthesis of the folate-containing peptide was performed following a previously described procedure.28 In brief, an acid-sensitive Wang resin loaded with fluorenylmethoxycarbonyl-methyltrityl-L-lysine (Fmoc-Lys(Mtt)-OH) was placed in a peptide synthesis vessel, and benzotriazile-1-ul-oxy-tris-pyrrolidino-phosphiniumhexafluorophosphate (PyBOP)-activated, protected amino acid monomers were sequentially applied. Fmoc protecting groups were removed after each coupling step by using 20% piperidine in dimethyl formamide (DMF). After the seven amino acid coupling steps were completed, the 4-methyltrityl protecting groups on Lys were cleaved with 2% trifuoroacetic acid (TFA) in dichloromethane; FITC (Sigma Aldrich) in 30% DMF was then added for the final coupling. The product was then cleaved from the resin and further purified by high-performance liquid chromatography (HPLC) using an Xterra RP18 30X300 mm 7 µm column (Waters) with a 30 min linear gradient from 1% to 30% of solvent B (solvent A = 50 mM ammonium bicarbonate, pH7.0; solvent B = acetonitrile) at a flow rate of 25 mL/min to yield purified folate-peptide-fluorescein, EC2220. A folatefluorescein conjugate without the peptide linker, EC17, and an unconjugated folate peptide derivative, EC0923, were also synthesized as described before.26-27 The mass of purified EC2220, EC17 and EC0923 was analyzed by electrospray-mass spectrometry, and the excitation and emission spectra of EC17 and EC2220 were measured with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) and an UV-VIS spectrophotometer (Beckman Coulter DU640).


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2.4. FR binding assay. KB cells in logarithmic growth phase were suspended in folatefree RPMI-1640 with 0.5% fetal bovine serum (FBS) and incubated in the dark with various concentrations of EC17 or EC2220 in the absence and presence of a 100-fold excess of a nonFITC folate derivative, EC0923. After incubation for 1 h at 4°C, cells were washed three times with phosphate-buffered saline (PBS), and the fluorescence of cell bound EC17 and EC2220 was analyzed using a Gallios Flow Cytometer (Beckman Coulter). GraphPad Prism 7 was used for graphing and analysis of the equilibrium dissociation constant (Kd).

2.5. Fluorescence microscopy. KB cells were plated on Nunc Lab-Tek II chamber slides (Theromo Fisher Scientific) and cultured in folate-deficient RPMI-1640 medium containing 10% FBS. When cells reached 90% confluence, the culture medium was replaced with cold folate-free RPMI-1640 medium containing 50 nM of either EC2220 or EC17. After incubation in the dark for 1 h at 4°C, cells were rinsed twice with folate-free RPMI-1640 and once with PBS, and images of bound EC17 or EC2220 on cells were acquired using a confocal fluorescence microscope (Nikon 90i). After imaging, 10% neutral buffered formalin solution (NBF) (approximate 4% formaldehyde, Sigma Aldrich) was added to each well for cell fixation. When desired fixation time (10 min or 30 min) was reached, cells on chamber slides were gently rinsed with PBS three times and incubated for 45 min with 5 µg/mL rabbit anti-FITC primary polyclonal antibody (Invitrogen, ANZ0202) in PBS containing 0.5% bovine serum albumin (BSA). After three rinses with the same buffer, 8 µg/mL Alexa Fluor 647 labeled F(ab')2-goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, A21246) was added onto cells and incubated for 45 min. Cells in chamber slide plates were rinsed three times and the fluorescent images of Alexa Fluor 647 and FITC on cells were captured using a confocal fluorescence microscope.



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2.6. Flow cytometry. To assess the correlation between EC2220 retention and FR expression, cells with different FRα levels including FR-high KB cells, FR-medium IGROV, FR-low OV90 cells and FR-negative A549 cells, were gently trypsinized, washed and suspended in FACS buffer (1X PBS, 0.1% BSA) with 50 nM of EC17 or EC2220. After incubation for 1 h on ice, cells were washed three times with FACS buffer, and the fluorescence signal of fluorescein from bound EC2220 or EC17 on cells was analyzed on a Beckman Coulter Gallios flow cytometer (Beckman Coulter DU640). These EC17- or EC2220-bound cells were then fixed for 10 min with 4% formaldehyde in PBS, washed twice more with PBS, and the fluorescence of retained EC17 or EC2220 on these fixed cells was analyzed by a flow cytometer. The same batch of trypsinized KB, IGROV, OV90 and A549 cells was also incubated with PE-conjugated antihuman folate receptors α and β antibody (BioLegend, rat IgG2a No.5/FOLR) at a dilution of 1:20 in FACS buffer for 45 min on ice. Cells were washed and resuspended in PBS and the fluorescence signal of PE on cells was analyzed by a flow cytometer.

2.7. Tissue distribution of 3H-EC2220. For 3H-EC2220 synthesis, 3H-Pte-Glu-LysAsp-Lys-Asp-Lys-Lys was custom-made by Moravek, Inc. (Brea, CA), and the coupling of FITC to the radiolabeled peptide and purification was done at Endocyte to yield the final 3H-EC2220 product. Six to seven-week-old nu/nu mice (BALB/c-derived) were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and maintained on a folate-free laboratory chow (Purina Inc., Largo, FL). KB tumor cells (1 million per mouse) were inoculated subcutaneously 4-5 weeks before experiment. When tumors reached 500 mm3, mice were injected intravenously with 100 nmol/0.5 mCi/kg of 3H-EC2220, euthanized 4 h later, and perfused immediately with 15 mL PBS containing 2 mM EDTA through the left ventricle to remove blood. One group was further perfused with 15 mL PBS, while the other group was further perfused with 15 mL NBF to fix 8 ACS Paragon Plus Environment

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organs. The completion of washing or fixation was determined by the pale appearance of the organs. Selected tissues and organs were collected and placed in 20 mL glass scintillation vials. Three mL volume of Soluene-350 was added to the tissue samples and a 1:1 mixture of Soluene350 / isopropanol to the blood samples. The vials were then heated at 55–60 °C for 4 h (tissue samples) or 2 h (blood samples) with slow constant swirling. After cooling the samples to room temperature, 0.2 mL of each sample was aliquoted, and 0.2 mL of 30% hydrogen peroxide was added to aliquoted tissue samples or 0.6 mL to aliquoted blood samples. The sample mixtures were then heated again at 60°C for 30 min to complete decolorization, yielding a pale-yellow color. Fifteen mL of Hionic-Fluor was mixed with each sample, light adapted for at least 1 h and then counted for 3H in a liquid scintillation counter. The final data were expressed as % injected activity per gram wet weight of tissue.

2.8. Preparation of agarose cell blocks with EC2220-bound cells. Cells were incubated with 50 nM EC2220 in folate-free RPMI-1640 medium for 1 h at 4°C in the dark, after which cells were washed three times with PBS to remove unbound EC2220 and then resuspended in a minimal amount of PBS at room temperature. Warm 2% low-melting temperature Seaprep Agarose (Lonza) in PBS was then added to the cell suspension and kept at room temperature for solidification. The solidified agarose cell blocks were immersed in formalin for 24 h prior to paraffin embedding and tissue sectioning.

2.9. Tumor model studies for cellular uptake of EC2220 in vivo. Female nu/nu mice were from Harlan Sprague-Dawley and NOD-scid IL2rγ null (NSG) mice were from Jackson Laboratory (Bar Harbor, ME). Mice were acclimated for at least 1 week before tumor implantation. In one study, each nu/nu mouse was implanted with FR-positive KB cells in the left shoulder and FR-negative A549 cells in the right shoulder. EC17 or EC2220 (2 µmol/kg) was intravenously 9 ACS Paragon Plus Environment


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injected through the tail vein once the tumors had grown to a volume of ~500 mm3. Mice were euthanized 4 h later when tumors and kidneys were collected and fixed with formalin solution. In a different study, FR-positive MDA-MB-231 cells were suspended in folate-deficient RPMI1640 and subcutaneously implanted (2 million per animal) in the shoulder of NSG mice. When tumors reached 1000-1500 mm3, mice were injected intravenously with 2 µmol/kg EC2220 and euthanized 4 h later. Mice were then immediately perfused with PBS through the left ventricle to remove blood. MDA-MB-231 tumors and the selected organs were collected and immersed in formalin solution for further tissue processing and IHC analysis.

2.10. PKD mouse models and treatment conditions. The Pkd1fl/fl:Pax8-rtTA:TetOcre (TET PKD) mouse model has been described previously.29-30 Briefly, systemicallyadministered doxycycline binds to Pax8-rtTA, which is only expressed at high levels in renal tubules31 and subsequently results in kidney-specific deletion of exons 2-4 of Pkd1. These animals were injected intraperitoneally with doxycycline 10 days post birth (P10) to induce PKD, and an obvious PKD phenotype was observed 10 days later (P20) as assessed by enlargement and distension of the belly in affected animals. EC2220 (2 µmol/kg) was intraperitoneally injected into doxycycline-induced PKD mice, and kidneys were collected 4 h later for tissue processing and IHC analysis. The second orthologous PKD model, Pkd1fl/-:NesCre (PKD1fl/-) has also been described previously.32 Briefly, one null allele was introduced into this animal model, and the deletion of exons 2-4 of Pkd1 on the second allele was generated by the inherent germline activity of Nestin-Cre, which is active in a mosaic pattern of expression in multiple tissues including the kidney.33 EC2220 (2 µmol/kg) was intravenously injected into PKD mice, and kidneys were collected 4 h later for tissue processing and IHC analysis.


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2.11. Immunohistochemistry. Formalin-fixed tissues were paraffin-embedded (FFPE) and sectioned at 5 µm using a standard procedure by the Histology lab of Purdue Veterinary School. FFPE tissue sections were deparaffinized, rehydrated, and treated either with 10 mM sodium citrate buffer (pH 6.0) at 91°C for 24 min, or with 1 mM EDTA solution (pH 8.5) at 95°C for 48 min for antigen retrieval. Endogenous peroxidase was blocked using Peroxidazed 1 (Biocare Medical), nonspecific sites were blocked with Background Sniper (Biocare Medical), after which tissue sections were incubated with primary antibodies overnight at 4°C in a humidified chamber. Primary antibodies were rabbit anti-FITC primary antibodies (Invitrogen ANZ0202), rabbit antiFRα primary antibodies (PU-19, rabbit polyclonal IgG which cross-reacts with both human and mouse FRα; Endocyte Inc.), and rabbit isotope control IgG (Jackson ImmunoResearch). After washing away unbound primary antibodies, tissue sections were incubated with universal antirabbit Fab’ immuno-enzyme polymer (N-Histofine Simple Stain Max PO; Nichirei Biosciences Inc.) for 30 min, and bound probes were visualized by using 3,3'-diaminobenzidine (DAKO). Tissue sections were counterstained using CAT hematoxylin (Biocare Medical) and Tacha’s Bluing Solution (Biocare Medical), dehydrated and mounted with Permount (Fisher). PBS solutions with or without 0.05% Tween-20 were used for wash in IHC staining.

3. RESULTS 3.1. Design and synthesis of EC2220. Standard practice has shown that formaldehyde fixation of cells and tissues generally denatures FR which, consequently, allows bound folate ligands to be released and washed away. To effectively track functional and accessible FR expression in vivo, we created a SMRC which could bind to FR with high affinity after intravenous administration and then covalently anchor it to the receptor during tissue fixation 11 ACS Paragon Plus Environment


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prior to IHC detection. Formaldehyde is an electrophilic molecule susceptible to chemical attack by nucleophilic species. Formaldehyde fixation causes covalent crosslinks between protein groups, effectively “gluing” them together into an insoluble meshwork for retention. Because lysine has a nucleophilic amine side chain that can easily react with formaldehyde, incorporating lysine residues within the SMRC enables their crosslinking to proteins in close proximity (i.e. receptors). Therefore, EC2220 was designed to contain three modules: i) a folate ligand that binds to FR with high affinity and specificity, ii) a multi-lysine containing peptide linker that can be crosslinked to proteins in close proximity during the formaldehyde fixation process and retain EC2220 in situ, and iii) a small hapten, fluorescein, which doesn’t exist naturally in human tissue but can be easily detected by IHC using commercially available anti-FITC antibodies (Fig. 1A). EC2220 consists of Pte-Glu-Lys-Asp-Lys-Asp-Lys-Lys-[C(S)NH-fluorescein]-OH, and its structure is shown in Fig. 1B. A related folate-fluorescein conjugate which lacks the multilysine residues, EC17, was also synthesized and used as a non-crosslinking control (Fig. 1C). To evaluate whether the peptide linker in EC2220 would interfere with the fluorescent properties of the attached fluorescein, the excitation and emission spectra of both EC2220 and EC17 were measured, as shown in Fig. 1D. EC2220 displayed an absorbance maximum at about 495 nm and an emission maximum at 520 nm, which overlapped with the EC17 spectral pattern. This result indicates that the multi-lysine containing peptide linker in EC2220 does not interfere with the fluorescent property of the fluorescein module.


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Figure 1. Chemical structures and in vitro properties of EC2220 and EC17. (A) Modular design of folate-based small molecule reporter conjugate, EC2220. (B) Chemical structure of EC2220. (C) Chemical structure of the non-crosslinking EC17 analog. (D) Absorption and emission spectra of EC2220 and EC17. (E) Chemical structure of the non-fluorescein folate compound, EC0923. (F) Binding affinities of EC2220 and EC17 with FRα-positive KB cells in the absence (solid symbols) and presence (open symbols) of 100-fold excess of the competitor, EC0923. All data represent mean ± S.D. (n=3 for ligand binding groups and n=2 for EC0923 competition groups). GraphPad Prism 7 was used for Kd analysis. Kd values for EC2220 and EC17 binding are 0.154 ± 0.0312 nM and 0.201 ± 0.0430 nM, respectively. The difference between these two Kd values is not statistically significant. (ns = not significant)



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3.2. Binding affinity of EC2220 to FRα-positive KB cells. The binding affinity of EC2220 to FRα was tested using KB cells that naturally express FRα. EC2220 and EC17 were incubated with KB cells for 1 h at 4°C in the presence and absence of 100-fold excess EC0923, an unconjugated folate derivative (Fig. 1E). Unbound conjugates were removed by washing, and cellassociated fluorescence of bound EC2220 and EC17 was measured by flow cytometry. As shown in Fig. 1F, EC2220 displayed strong FR binding with an apparent dissociation constant of 0.15 nM, which is similar to the Kd of EC17 for FRα-positive KB cells (0.20 nM) when tested under the same conditions. Our results indicate that the addition of the multi-lysine containing linker in EC2220 did not compromise its binding affinity for FRα. Importantly, with the presence of a 100fold excess of unconjugated folate (i.e. EC0923), neither EC2220 nor EC17 bound to KB cells, thereby demonstrating the specificity of EC2220 and EC17 for FR.

3.3. Formaldehyde can crosslink EC2220 to its binding sites on FRpositive cells. To evaluate whether the lysine-containing peptide linker can help retain EC2220 on FR-positive cells during formaldehyde fixation, as designed, association of EC2220 and EC17 with KB cells before and after formaldehyde fixation were compared. As shown in Fig. 2A, EC2220-treated KB cells displayed strong membrane-localized FITC fluorescence signal before fixation, and the FITC signal remained associated with the cell membrane following a 30 min formaldehyde fixation. On the contrary, while EC17-treated KB cells showed strong membranelocalized FITC signal prior to fixation, the fluorescent signal greatly diminished after these cells were fixed with formaldehyde for 10 min and was virtually eliminated after 30 min. To confirm these results, the same fixed cells were also immunostained with anti-FITC antibodies. As shown in Fig. 2A (bottom panels), EC17-treated KB cells showed no immunofluorescence staining for 14 ACS Paragon Plus Environment

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FITC after the 30 min formaldehyde fixation, whereas EC2220-treated KB cells displayed very strong immunofluorescence staining for FITC. From these results we conclude that formaldehyde fixation anchored EC2220 to its original FR binding sites on the KB cell membrane as mediated by the multi-lysine containing peptide linker, whereas EC17 merely washed away due to the formaldehyde-mediated denaturation of the FR. To explore whether the level of retained EC2220 on formaldehyde-fixed cells is proportional to cell surface FR expression, four types of tumor cells with various FRα expression levels were compared relative to their EC2220 retention signals. As shown in Fig. 2B, KB cells bound the most EC2220 followed by IGROV and OV90 cells. In contrast, the known FRnegative A549 cells28,

34

did not bind EC2220. The net level of cell-associated EC2220

fluorescence was not significantly reduced following fixation, which is consistent with the data presented in Fig. 2A. Complementary anti-FRα immunofluorescence staining among these same cell types revealed that the order of FRα expression (from high to low) was also KB, IGROV, OV90, and A549 respectively (Fig. 2C). In comparing these two data sets, we found that a linear correlation existed between the logarithmic mean fluorescence intensity (log MFI) of cellassociated EC2220 and the log MFI of surface FRα level of these cells, both before and after formaldehyde fixation (Fig. 2D). As expected, EC17 showed an identical pattern of expression to EC2220 prior to formaldehyde fixation, but significantly lower binding resulted following formaldehyde fixation due to EC17’s dissociation from the cells (Figs. 2 B and 2D). Based on these findings, we conclude that EC2220 crosslinks and remains attached to FR in situ during formaldehyde fixation, and the level of retained EC2220 after tissue fixation is proportional to the level of FR expression.



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Figure 2. Comparison of EC2220 and EC17 retention on FR-positive cells before and after formaldehyde fixation. (A) Effect of formaldehyde fixation on EC17 and EC2220 retention in cultured KB cells. Cells were cultured on slide chambers and incubated with EC17 or EC2220 for 30 min on ice, then fixed with 4% formaldehyde. After washing, some of the fixed cells were further stained with rabbit anti-FITC primary polyclonal antibody followed with AF647-labeled anti-rabbit IgG secondary antibody, and the cell-associated fluorescence was imaged using a confocal fluorescence microscope. FITC fluorescence is shown in green, and the fluorescence from AF647 is shown in red. (B) Retention of EC2220 and EC17 on various cell types before and after formaldehyde fixation. Cells were incubated with 50 nM of EC17 or EC2220 for 1 h on ice, washed and then either fixed with 4% formaldehyde first or analyzed directly for cell-associated fluorescence by flow cytometry. (C) FR expression on various cells measured by immunofluorescence staining using an anti-FR antibody and flow cytometry. (D) Correlation of EC2220 binding with FR expression on cells before and after formaldehyde fixation. GraphPad Prism 7 was used for 16 ACS Paragon Plus Environment

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graphing and analysis of the coefficient of determination (R2). All data represent mean ± S.D. (n=3). One-way ANOVA with post-hoc Tukey tests were performed for statistical analysis. (****denotes a p-value < 0.0001, ns = not significant).

3.4. EC2220 has similar tissue distribution as

99mTc-EC20

in animal

models. We have previously studied the tissue distribution of intravenously administered folate conjugates using

99mTc-EC20,

a folate-based radioimaging agent that has been safely and

successfully administered in over 1000 patients across multiple clinical trials.16, 21-23 Mouse studies revealed that folate-conjugated agents primarily accumulate in FR-positive tumor tissues and kidneys.35 To evaluate whether the inclusion of the multi-lysine containing linker and the fluorescein module in EC2220 would change its biodistribution properties, 3H-EC2220 was produced and investigated in nu/nu mice bearing subcutaneous FRα-positive KB tumors. As shown in Fig. 3 FRα-positive tissues, such as KB tumors and kidneys, accumulated the highest uptake of 3H-EC2220. Other organs including liver, spleen, intestine and skin also showed far lower levels of 3H-EC2220 uptake, whereas the remaining examined organs including heart, lung, muscle, stomach, brain and bone showed no uptake. Because 3H-EC2220 has similar tissue distribution as what has been previously reported for 99mTc-EC20,35 our data suggest that the multilysine containing linker and the fluorescein module in EC2220 don’t interfere with FR binding in vivo. We further explored whether tissue-bound 3H-EC2220 could be retained after NBF fixation and, as shown in Fig. 3, organs from NBF-perfused mice had similar 3H-EC2220 retention as those perfused with PBS. These data suggest that like cells in culture, formaldehyde fixation does not dissociate 3H-EC2220 from its in vivo FR binding sites within tissues.



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Figure 3. Biodistribution of EC2220 in mice bearing FR-positive KB tumors. KB tumor-bearing nu/nu mice were injected intravenously with 100 nmol/kg of 3H-EC2220 and euthanized 4 h later. Following PBS perfusion to remove blood, mice were further perfused with either PBS (grey bars) or formalin solution (white bars), and 3H-EC2220 uptake by organs was assessed and quantified. Bar graphs represent mean ± S.D. (n=3). A t-test was used for comparison. (*denotes a p-value < 0.05, ns = not significant).

3.5. EC2220 remains bound to the FR in situ during FFPE tissue processing and IHC. FFPE tissue processing and IHC analysis require repeated exposure of tissues to organic solvents as well as aqueous solutions, either of which may dissociate small molecules from tissues. To elucidate whether the bound EC2220 in FR-positive tissues can stay bound during tissue processing, FFPE agarose blocks of EC2220-treated KB cells were prepared and sectioned for IHC staining using a standard procedure. As shown in Fig. 4A, IHC staining for FITC without an antigen retrieval step displayed very strong EC2220 retention on KB cells, indicating that formaldehyde-crosslinked EC2220 can be retained in situ during FFPE tissue processing. Because the antigen retrieval step is used to partially reverse formaldehyde crosslinks and recover/expose over-crosslinked proteins for IHC detection, two antigen retrieval conditions


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were compared for their effects on EC2220 retention. As shown in Fig. 4A, the mild antigen retrieval with 10 mM sodium citrate (pH 6.0) for 24 min at 91°C led to less EC2220 staining on KB cells as compared to those not treated with antigen retrieval, and the extended antigen retrieval treatment with 1 mM EDTA solution (pH 8.5) for 48 min at 95°C was found to dissociate almost all of the EC2220 molecules from the remaining FR-positive KB cells. Because antigen retrieval reverses formaldehyde crosslinks and reduces EC2220 retention on formaldehyde-fixed tissue sections, we conclude that it is indeed the formaldehyde-mediated crosslinking that “glues” the EC2220 small molecule to its original FR binding sites within tissues. We also assessed whether the IHC staining intensity for EC2220 correlates to the level of functional FR on tissue. For this purpose, FR-high KB cells, FR-low OV90 cells, and FRnegative A549 cells were incubated with EC2220, FFPE cell block sections were prepared, and EC2220 bound to these cells was visualized by anti-FITC IHC staining without the disruptive antigen retrieval step. As shown in Fig. 4B, FR-high KB cells exhibited more intense anti-FITC staining than FR-low OV90 cells, and A549 cells lacking FR expression showed no anti-FITC staining. These data confirm that our EC2220-based IHC assay can be used as a semi-quantitative method for analyzing functional FR level in tissues.



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Figure 4. Retention of EC2220 after tissue processing and IHC staining. (A) Effect of the antigen retrieval condition on EC2220 retention with FR-positive KB cells. EC2220-treated KB cells were embedded in agarose and FFPE blocks were prepared. Sections were treated with three different antigen retrieval conditions, as indicated, and retained EC2220 was analyzed by IHC staining for FITC. (B) IHC analysis of EC2220 bound to cells expressing various levels of FR. A549, OV90 and KB cells, representing FR-negative, FR-low, and FRhigh cells, respectively, were incubated with 50 nM EC2220 for 1 h on ice. After washing, agarose blocks were prepared, FFPE-processed and sectioned, and cell-bound EC2220 was analyzed by IHC staining for FITC without an antigen retrieval step.

3.6. EC2220-based IHC assay for in-vivo assessment of FR. Our EC2220based functional IHC assay was evaluated to determine if functional and accessible FR was detectable in animal models. Here, nu/nu mice were implanted with FR-positive KB tumor on the left shoulder and FR-negative A549 tumor on the right shoulder, and EC2220 was intravenously administered through the tail vein when tumors reached 500 mm3. Mice were


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euthanized 4 h later and both tumors and kidneys were collected, fixed with formalin, embedded in paraffin and sectioned using standard histological procedures. EC2220 in tissues was examined by anti-FITC IHC staining without any antigen retrieval step. As shown in Fig. 5A, very strong staining for EC2220 was observed in FR-positive KB tumor cells in contrast to completely negative staining in FR-negative A549 tumor cells, indicating that EC2220 specifically targeted FR-positive cells in vivo. In addition, mouse kidney tissues also showed strong IHC staining for EC2220. Based on observed tissue histology, we could identify that proximal tubules had very strong IHC staining for EC2220, while both distal tubules and glomeruli showed little EC2220 uptake (consistent with literature findings of FR expression;36-37). It is worthwhile to mention that the staining for EC2220 was found in both the cell membrane and cytoplasm which implicates the delivery of folate-conjugates (e.g. EC2220) into the cell cytoplasm through FR-mediated endocytosis in vivo. As a control lacking the multi-lysine containing peptide linker, EC17 was also administered to tumor-bearing mice through tail veins, and its presence within tumors and kidneys was examined, and shown in Fig. 5A. Although EC17 was detectable in FR-positive KB tumor cells and renal proximal tubule cells, the staining intensity was significantly lower as compared to that for EC2220. Because both EC2220 and EC17 have similar binding affinity for FR (Fig. 1E), the prominent tissue retention of EC2220 further confirms that the multi-lysine containing linker in EC2220 helps to anchor the small molecule in situ for IHC analysis, as designed (Fig. 5B).



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Figure 5. Validation of EC2220-based IHC assay in visualizing accessible and functional FR in vivo. (A) Balb/c-derived nu/nu mice bearing FR-positive KB tumor in the left shoulder and FRnegative A549 tumor in the right shoulder were intravenously injected with EC2220 or EC17. Mice were euthanized 4 h later, both tumors and kidneys were collected and their FFPE sections were prepared using standard histological procedures. EC2220 and EC17 uptake was visualized by anti-FITC IHC staining using a standard IHC procedure without an antigen retrieval step. (B) Schematic representation of formaldehyde crosslinking and EC2220 retention on FR-positive tumor cells versus no crosslinking with EC17.

3.7. Assessment of in vivo FR accessibility using EC2220-based IHC assay. Although FRα is most highly expressed on cancer cells and kidney proximal tubule cells, it is also reportedly expressed on some normal tissues.2, 4-6 To assess whether FRα on normal tissues is accessible by intravenously administered folate-conjugates, mice bearing FRα-positive MDA-MB-231 tumors received i.v. EC2220, and select organs were collected 4 h later for analysis of both FRα expression and EC2220 uptake. As shown in Fig. 6, both MDA-MB-231 tumor cells and proximal tubular cells of the kidney showed high FR expression and high uptake of EC2220 as assessed by anti-FITC IHC. Some organs like liver, brain and spleen showed very low to 22 ACS Paragon Plus Environment

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negative staining for FRα as well as little to no cellular uptake of EC2220 (Supplemental Fig. 1). Interestingly, although epithelial cells of lung bronchioles and columnar epithelial cells of the small intestine showed small regions of strong immune-staining for FRα, these particular epithelial cells didn’t show any EC2220 uptake. This divergence implies that intravenously administered folate conjugates are not accessible to all FRα-expressing cells in vivo (see Discussion).

Figure 6. Accessibility of FR in normal tissues to intravenously administered EC2220. NSG mice were implanted subcutaneously with FR-positive MDA-MB-231 tumors in the shoulder. When tumors reached 1000-1500 mm3, mice received an intravenous injection of 2 µmol/kg EC2220 and were euthanized 4 h later. After perfusion with PBS to remove blood, tumors and selected organs were collected and FFPE sections prepared. EC2220 uptake was visualized by anti-FITC IHC staining without an antigen retrieval step. FRα expression in serial sections was 23 ACS Paragon Plus Environment


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detected by anti-FR IHC staining using a mild antigen retrieval treatment (10 mM sodium citrate (pH 6.0) for 24 min at 91°C). Tissue sections for rabbit IgG control were not treated with antigen retrieval step.

3.8. Evaluation of accessible and functional FR in mouse PKD models using EC2220-based IHC assay. Folate-conjugated rapamycin has shown promise in slowing the progression of polycystic kidney disease (PKD) without significant side effects in mouse models.3 In search of a suitable orthologous PKD animal model for preclinical evaluation of newer folate-based SMDCs, our EC2220-based IHC assay was used to evaluate whether cystic renal cells from two different PKD animal models express functional FR. For this purpose, Pkd1fl/-:Nestin-Cre (PKD1fl/-)mice were intravenously administered with EC2220 and their cystic kidneys were collected 4 h later for IHC analysis. As shown in Fig. 7, strong IHC staining for both FRα and EC2220 was observed in both normal kidney as well as in small cysts (dilated, cuboidal but not yet flattened renal cells) in early stage PKD. Although weak staining for EC2220 and FRα could be found in cells lining some of the larger renal cysts, most late-stage PKD cysts with flattened lining cells didn’t show EC2220 staining (i.e. uptake). We also evaluated the uptake of EC2220 by another orthologous PKD mouse model, Pkd1fl/fl:Pax8-rtTA:tetO-Cre (TET PKD). Kidneys from this model were also collected 4 h after intraperitoneal injection of EC2220. As shown in Fig. 7, renal cysts in the TET PKD mouse model displayed strong IHC staining for both FRα and EC2220, suggesting that FRα protein on renal cysts of this orthologous PKD model is functional and accessible to intraperitoneally injected folate-SMDCs. These data further support our previous study that utilized EC2220, referred to as FC-reporter, in the bpk and Pkd1fl/-:NestinCre models of PKD.32 24 ACS Paragon Plus Environment

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Figure 7. Evaluation of functional and accessible FR in renal cysts of orthologous PKD animal models. PKD mouse models were injected with 2 µmol/kg EC2220, their kidneys were collected 4 h later and then processed for IHC analysis. EC2220 uptake was visualized by anti-FITC IHC staining without an antigen retrieval step. FRα expression was detected in serial sections by anti-FR IHC staining with a mild antigen retrieval treatment (10 mM sodium citrate, pH 6.0, for 24 min at 91°C). TET PKD: Pkd1fl/fl:Pax8-rtTA:tetO-Cre mouse; PKD1fl/-: Pkd1fl/-:Nestin-Cre mouse.

4. DISCUSSION In this study, we designed the folate-based SMRC, EC2220, and explored its application in assessing functional and accessible FR in vivo. EC2220 is composed of three modules: a folate ligand for FR binding, a multi-lysine containing linker for formaldehyde crosslinking, and fluorescein for hapten-based IHC detection. Our study demonstrates how the unique design of EC2220 makes it an ideal molecule for assessing the functionality of FR, both in vitro and in vivo. First, EC2220 showed high binding affinity and specificity for FR-positive cells, and the level of bound and retained EC2220 on cells was proportional to the FR level, both before and after formaldehyde fixation. Second, EC2220 preferentially accumulated in FR-positive tumors 25 ACS Paragon Plus Environment


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and kidneys of animal models, and it showed similar patterns of tissue distribution as that for 99mTc-EC20,

a folate-conjugate successfully used for in vivo imaging of FR-expressing tumors

in mice and humans. Third, FR-expressing cells retained EC2220 in situ during FFPE processing and anti-FITC IHC staining, and the staining intensity was also proportional to the respective level of FR expression on the various cell lines tested. Based on these findings, the accessibility of functional FR in vivo can be examined by first administering EC2220, collecting tissues-ofinterest by either surgery or biopsy, and then “visualizing” EC2220 in respective FFPE tissue sections using anti-FITC IHC staining techniques. Importantly, this EC2220-based IHC assay has been tested using various cultured tumor cells, tumor xenograft animal models and PKD models; the sensitivity and specificity of this assay is apparent. Folate has been conjugated to various therapeutics used in cancer therapy, including radionuclides, chemotherapeutic agents, immunotherapy agents, liposomal drugs, gene transfer vectors and protein toxins.38 Because some normal epithelial cells may have some measurable level of FR expression, there has been concern about whether these FR-expressing normal cells would get attacked by FR-targeted agents. By using our EC2220-based IHC assay, we found that although some epithelial cells, like that of lungs and the small intestine, had small pockets of FRα expression, those cells didn’t show any uptake of intravenously administered EC2220 (Fig. 6). Our finding reveals that these FRα-expressing normal epithelial cells likely have limited or no access to systemically-administered EC2220. This result is concordant with previous animal studies, as well as clinical trials, which showed little or no toxicity to FR-positive normal tissues, such as lungs and intestines, following intravenous administration of folate-SMDCs.17-18, 28, 39-46 Compared to anti-FRα IHC analysis, our EC2220-based IHC assay provides a more accurate depiction of the


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in vivo fate of folate-SMDCs, and it may more precisely predict the risk for on-target-off-tumor toxicity. Folate-based SMDCs have shown mixed outcomes on targeted therapies in both animal models and clinical trials. Because targeted therapy is only effective in a subset of patients, a sensitive method that can predict a patient’s response to therapy could improve the likelihood for success in a clinical trial. Various folate-conjugated small molecule radioimaging agents, such as 99mTc-EC20

and

111In-DPTA-folate,

have successfully identified FR-positive cancer in

patients.16, 21-23, 47 Yet, some patients with positive tumor imaging didn’t respond well to FRtargeted SMDCs. Among many probable reasons, heterogenic expression of FR within a tumor is perhaps the most concerning. For example, a tumor with 10% FR-expressing cells and 90% negative cells may be declared FR positive by in vivo imaging; but, that tumor would probably not respond well to FR-targeted SMDCs because of limited targeted drug accumulation. As demonstrated in this study, our EC2220-based IHC assay has cellular-level resolution in addressing the “functional” heterogeneity of cancer (and PKD), and this novel method could possibly offer a more accurate prediction of patient response to folate-SMDCs. The EC2220-based IHC assay is also a powerful tool in selecting proper animal models for preclinical evaluation. PKD is discussed here as an example. Autosomal dominant PKD is a genetic disease associated with uncontrolled growth of renal cysts that leads to end-stage renal disease in 50% of patients. With a better understanding of the abnormal pathology in PKD, inhibitors to these abnormalities have been found to slow the disease progression in animal models 48-53.

However, these inhibitors were not specifically targeted to renal cysts, and their systemic side

effects and impact on non-diseased organs limit their therapeutic applications, especially when chronic long-term treatment (i.e. decades) is required. Because high FR expression was found in 27 ACS Paragon Plus Environment


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the cystic renal cells of human PKD patients3, folate-conjugated PKD inhibitors seem to be a promising solution. Therefore, selection of a suitable animal model is critical in obtaining relevant and meaningful information from preclinical studies. Using our EC2220-based IHC assay, renal cysts were found to be functional and accessible. Furthermore, cyst lining cells in the TET PKD mouse model showed much stronger EC2220 uptake compared to those in PKD1fl/- mouse model, suggesting that the TET PKD model may be the preferred model for the evaluation of folateSMDCs; admittedly, additional studies are necessary to support this viewpoint. Interestingly, although large cysts in PKD1fl/- model exhibited little uptake of EC2220, the lining cells of small cysts still maintained strong EC2220 uptake. It is possible that the cyst lining cells may lose functional FR expression on their epithelial surface during disease progression as they differentiate. If autosomal dominant PKD patients show the same trend during disease progression, treatment with folate-conjugated, kidney-targeted inhibitors that impact the renal cystic phenotype and are used in an early line of therapy (e.g. prior to significant renal impairment) could potentially yield a better overall clinical outcome. EC17, the non-crosslinking folate-fluorescein conjugate, has been previously confirmed to “light up” FR-positive tumors during fluorescence-guided surgical applications.54-55 As demonstrated in this current study, EC2220 has similar capability as EC17 in tracking FR both in vitro and in vivo. Addition of a multi-lysine containing linker in between the folate ligand and the fluorescein module enables its cell and tissue retention during FFPE tissue processing and IHC staining without apparent alteration to its bioactivity and biodistribution. Besides FR, many other protein targets such as carbonic anhydrase IX (CAIX), somatostatin receptor 2, epidermal growth factor receptor, human epidermal growth factor receptor 2, C-X-C chemokine receptor type 4 and fibroblast activation protein, have also been extensively studied for small ligand-based drug 28 ACS Paragon Plus Environment

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delivery. A similar strategy may also be applied for in vivo assessment of other small ligands for their functional binding with corresponding receptors and membrane-expressed proteins that have high expression in tumor cells. Using CAIX as an example, to assess in vivo accessibility of functional CAIX on cancer tissues, a SMRC composed of a CAIX binding ligand, a lysinecontaining peptide linker, and fluorescein as a reporter for IHC staining would be a useful tool in evaluating accessible and functional CAIX in animal models (and potentially in clinical patients). It is also worth mentioning that, instead of fluorescein, other artificial small haptens, such as rhodamine and dinitrophenyl, may also be used as the reporter module in SMRC design. In fact, the folate conjugate with a near-infrared dye, OTL38, has been shown successful in FR-positive tumor imaging56 and is being tested in a Phase III clinical trial. Folate-lissamine conjugates with different spacers have been examined to compare the influence of hydrophilicity and charged groups in the spacer on the biodistribution and interaction with target cells in PKD animal model.57 If lysine-containing linkers were incorporated, these conjugates could potentially be used for functional FR immunostaining and extend the detection limit onto the cellular level. Admittedly, the impact of a lysine-containing linker and a small hapten to other small molecular ligands may yield different results compared to folate. Thus, evaluation of the bioactivity and biodistribution of SMRCs would be necessary before applying SMRC-based IHC assay to in vivo assessment of potential target for drug delivery.

ASSOCIATED CONTENT Attached supplemental material Figure S1 shows IHC staining for FRα and EC2220 to evaluate the accessibility of FRα in mouse brain, spleen and liver tissues to intravenously administered EC2220.



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AUTHOR CONTRIBUTIONS The manuscript was written by H.C. and C.P.L. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGEMENTS We thank Drs. Terry Watnick and Patricia Outeda-Garcia (Johns Hopkins University) for providing PKD tissues from the Pkd1fl/fl:Pax8-rtTA:TetO-Cre (TET PKD) mouse model injected with EC2220. We also thank Drs. Thomas Weimbs and Kevin Kipp (University of California at Santa Barbara) for providing PKD tissue from the orthologous Pkd1fl/-:NesCre (PKD1fl/-) mouse model injected with EC2220.

ABBREVIATIONS FR, Folate receptor alpha; SMDC, small molecule drug conjugate; SMRC, small molecule reporter conjugate; PKD, polycystic kidney disease; FFPE, formalin fixed and paraffin embedded; IHC, immunohistochemistry; NBF, neutral buffered formalin.


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Detecting functional and accessible folate receptor expression in cancer and polycystic kidneys Haiyan Chu, Jonathan M. Shillingford*, Joseph A. Reddy, Elaine Westrick, Melissa Nelson, Emilia Z. Wang, Nikki Parker, Albert E. Felten, Jeremy Vaughn, Le-Cun Xu, Yingjuan Lu, Iontcho R. Vlahov and Christopher P. Leamon#

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Detecting Functional and Accessible Folate Receptor Expression in

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