Journal of Nuclear Medicine, published on June 8, 2020 as doi:10.2967/jnumed.120.249557

ORIGINAL RESEARCH

Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Philipp Diebolder1,2, Cedric Mpoy1, Jalen Scott1, Truc T. Huynh1,3, Ryan Fields2,4, Dirk Spitzer2,4,

Nilantha Bandara1, and Buck E. Rogers1,4*

1Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO,

USA

2Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA

3Department of Chemistry, Washington University, St. Louis, MO, USA

4Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA

B.E.R: [email protected], P: +1-314-362-9787, F: +1-314-362-9790 *Corresponding author

P.D.: [email protected], P: +1-314-362-9787, F: +1-314-362-9790 Postdoctoral Research

Associate

Words: 4,975

Running title: Engineered scFv-Fc Targeting CD44

Funding: Please refer to acknowledgements.

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ABSTRACT CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of (HA) to CD44 activates signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific fragment and evaluated it for imaging CD44-positive cancers utilizing positron emission tomography (PET).

Methods: A human single-chain antibody fragment (scFv) was generated by phage display, employing the extracellular domain (ECD) of recombinant human CD44. Specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell-expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray.

The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG1-Fc

(fragment crystallizable). The scFv-Fc-CD44 was radiolabeled with copper-64 (64Cu) and zirconium-89 (89Zr). The purified reagents were injected into athymic nude mice bearing CD44- positive human tumors (MDA-MB-231, breast cancer, triple negative). Biodistribution studies were performed at different time points after injection of [64Cu]Cu-NOTA-scFv-Fc-CD44 or [89Zr]Zr-

DFO-scFv-Fc-CD44. PET/computerized tomography (CT) imaging was conducted with [89Zr]Zr-

DFO-scFv-Fc-CD44 on days one and seven post-injection and compared to a scFv-Fc control antibody construct targeting A (GPA).

Results: Epitope mapping of the scFv binding site revealed a linear epitope within the ECD of human CD44, capable of blocking binding to native HA. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor- to-blood (TTB) ratios of [89Zr]Zr-DFO-scFv-Fc-CD44 between days five and seven. PET imaging

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confirmed excellent tumor specificity of [89Zr]Zr-DFO-scFv-Fc-CD44 when compared to the control scFv-Fc.

Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64Cu or 89Zr, it demonstrated specific uptake in CD44 expressing MDA-MB-231 tumors. The high tumor uptake (~56%ID/g) warrants clinical investigation of [89Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with

CD44-positive tumors.

Key Words: immunoPET; CD44; scFv-Fc; zirconium-89, copper-64

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INTRODUCTION

CD44 is a heavily glycosylated transmembrane and plays key roles in adhesion to the , , and cytoskeleton remodeling. CD44 is one of the major receptors for the hyaluronic acid (HA), which is an abundant part of the extracellular matrix. The standard form of CD44 (CD44s) is encoded by nine constant that are spliced together with 1-5 forming the ECD (1). Furthermore, CD44 is one of the most studied in cancer and is overexpressed by various solid tumors and hematological malignancies, while different splice variants of CD44 have been identified in squamous cell carcinomas (2). Many cancer-related studies have used monoclonal for targeting extracellular regions on CD44s (exon 1-5) and CD44v6 (exon 10). Both the standard form and splice variants have been associated with tumor progression, and disease progression making CD44 a promising target in cancer therapy (3).

Disruption of the CD44-HA interaction is believed to be the underlying mechanism of

CD44-targeted cancer therapeutics. Bivatuzumab, a humanized directed against the variable region CD44v6 does not appear to block the interaction with HA. Therefore, it was conjugated to the antimicrotuble drug mertansine to induce a direct cytotoxic effect upon internalization. However, subsequent phase I clinical trials were halted due to severe, at times lethal, toxic epidermal necrolysis (4). RG7356, a humanized monoclonal antibody targeting the constant region of the extracellular domain of all CD44 isoforms, binds to an epitope near the HA- binding region and has been shown to have anti-tumor activity as a monotherapy in mouse xenografts that are CD44+ and HA+, but not in xenografts that are CD44+ and HA- (5). Since CD44 is also expressed in human epithelial tissues such as the lung, skin, and mammary gland, it was hypothesized that radiolabeled RG7356 could be used to determine the in vivo expression of

CD44 by PET imaging. This would be critical for the development of CD44-targeted, antibody- based cancer therapies.

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Consequently, RG7356 was radiolabeled with 89Zr and evaluated both preclinically (6) and in cancer patients (7,8). Tumor targeting was confirmed in mice bearing CD44-positive tumor xenografts but was also found in CD44-expressing tissues, such as the spleen and bone marrow of cynomologous monkeys (6). A dose escalation study of RG7356 in patients with CD44+ solid tumors demonstrated excellent tolerability and modest efficacy with disease stabilization in 21% of the patients (8). 89Zr-labeled RG7356 was used in a subset of these patients and showed tumor targeting when doses of >200 mg of RG7356 were given. Further analysis of this data demonstrated how [89Zr]Zr-DFO-RG7356 could be used to quantify dose dependent uptake in normal tissues (7).

In general, antibody fragments with intermediate sizes and clearance rates (i.e., diabodies, minibodies, and scFv-Fcs with molecular weights between 50 to 110 kDa) may be better suited as imaging agents than full length IgGs due to their higher penetration capacity into solid tumors

(9-11). In addition, intermediate sized antibody fragments may allow for better imaging results

(high signal-to-noise ratio) due to a faster clearance rate from blood and other well perfused tissues in comparison to whole antibodies, which usually have longer serum half-lives. Therefore, we sought to develop an anti-CD44 antibody using phage display that could be used for PET imaging of CD44+ cancers.

The aim of our current study was to screen a human scFv phage display library and identify a novel, high affinity antibody fragment to CD44 for diagnostic and potentially therapeutic applications. Our scFv lead was converted into a scFv-Fc (fragment crystallizable) format and radiolabeled with both 64Cu and 89Zr and evaluated in vitro and in vivo using biodistribution and

PET imaging studies. These studies show specific uptake of the scFv-Fc in a CD44+ tumor model.

We believe that, compared to currently explored full length Ab-based detection and therapy concepts, scFv-based formats offer distinct advantages because of flexibility when it comes to downstream customization efforts tailored toward specific therapeutic goals, such as size

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variations for superior tumor uptake, half-life alterations or altered Fc effector functions through

Fc point mutations.

MATERIALS AND METHODS

Production of scFv-Fc Constructs

The scFv-CD44 was isolated from WashU-1 phage display library (Supplemental Fig. 1) by biopanning against the ECD of recombinant human CD44(Q21-E268) (Immune Technologies,

#IT-300-001p) and the control scFv (GPA) as described (12). ScFv-Fcs comprising of a C-terminal human IgG1-Fc (T106-K330) region were cloned in pYD11 vector and produced by transient HEK293-6E cell expression and purified by G as described before (13).

Protein concentrations were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a XCell SureLock Mini-Cell system with NuPAGe 4-12% Bis-

Tris gels (Thermo Fisher Scientific) and Western Blot (WB) using a Trans-Blot SD Semi-Dry

Transfer Cell (Bio-Rad). Fast protein liquid chromatography (FPLC) analysis was done on an

ÄKTA pure protein purification system (GE Healthcare) employing a Superdex 200 Increase

10/300 GL column and 20 mM Tris, 150 mM NaCl, pH 8 as running buffer.

Saturation Binding Studies

The affinities of the antibody fragments were determined from equilibrium binding curves

5 (EC50) generated by flow cytometry as described before (14) by staining of 3x10 /well of MDA-

MB-231 cells. ScFv-CD44 binding was detected by primary murine monoclonal antibody 9E10

(7.5 μg/mL) and secondary goat anti-mouse polyclonal antibody fluorescein isothiocyanate (FITC) conjugate (Jackson ImmunoResearch, #115-095-008). The scFv-Fc-CD44 was detected by a

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rabbit anti-human polyclonal antibody FITC conjugate (Jackson ImmunoResearch, #309-095-

008). To measure the affinities by enzyme-linked immunosorbent assay (ELISA), MaxiSorp plates

(Thermo Fisher Scientific) were coated with CD44(Q21-E268) (3 μg/mL in 2% milk PBS). Serial dilutions (1:2) of the antibody fragments in 2% milk in phosphate buffered saline (MPBS) were incubated in MPBS-blocked plates in triplicates. The scFv-CD44 was detected with primary murine monoclonal antibody 9E10 (7.5 μg/mL) and secondary goat anti-mouse polyclonal antibody peroxidase conjugate (Jackson ImmunoResearch, #115-035-008). The scFv-Fc-CD44 was detected by a goat anti-human polyclonal antibody peroxidase conjugate (Jackson

ImmunoResearch, #109-035-098). TMB substrate solution (Thermo Fisher Scientific) was added to PBS-washed plates and the reaction stopped with 2 M H2SO4 before reading the absorbance at 450 nm in a BioTek ELx800 microplate reader. Background signals were subtracted from the measured absorbance (ELISA) or median fluorescence intensities (flow) and relative affinities calculated by nonlinear regression using GraphPad Prism 8 (GraphPad Software).

Radiolabeling and Characterization

Briefly, scFv-Fcs were conjugated with 2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-

1,4,7-triacetic acid (NOTA) (#B605) or 1-(4-Isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21- tetraoxo-27-(Nacetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (DFO) (#B705) from Macrocyclics in a 1:5 molar ratio in 0.1 M carbonate buffer (pH 9.0) for 1 hour at 37°C. The conjugates were buffer exchanged (NOTA-scFv-Fc; 0.1 M NH4OAc, pH 6.0 and DFO-scFv-Fc; 1

M HEPES, pH 7.2) and frozen until radiolabeling. The number of chelates per scFv-Fc were determined by mass spectrometry as described in the supplemental data. Radiolabeling was performed for 1 hour at 37°C using 200 μg of conjugated scFv-Fc and with 18.5 MBq (500 μCi) of 64Cu or 89Zr.

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Cell Culture

All human lines were purchased from the American Type Culture Collection and cultivated in complete growth medium (DMEM supplemented with 10 mM HEPES and 10% of heat-inactivated FBS) in low passage number in a humidified cell incubator (5% CO2, 37°C).

In Vivo Biodistribution Studies

Animal experiments were conducted in compliance with the Guidelines for Care and Use of Research Animals established by the Washington University Animal Studies Committee using a protocol approved by the committee. The 5–6-week-old female NCI athymic NCr-nu/nu mice

(strain 553) (n=4-5 per group) were purchased from Charles River and housed under pathogen- free conditions in the WUSM animal facility. Mice were anesthetized by the inhalation of 2% isoflurane in 100% oxygen. MDA-MB-231 cells were harvested by trypsinization at 80% confluency, washed twice (PBS), and implanted subcutaneously on the right shoulder with

1.0x107 MDA-MB-231 cells in 100 µl PBS/mouse. Tumors grew for 2 weeks reaching final tumor weights of 123.8±18.9 mg (±SE). In vivo biodistribution studies were conducted in the Washington

University Small Animal Imaging Facility. Isoflurane-anesthetized mice were intravenously injected via the tail vein with 0.37 MBq (10 µCi) of radiolabeled scFv-Fc targeting either human

CD44 or GPA. At the designated time points, mice were sacrificed and tumors and selected organs were collected, weighed, and activity counted with a Beckman Gamma-8000 counter.

Tumor and organ uptake were analyzed and calculated as a percentage of injected dose per gram

(%ID/g). Tumor-to-organ ratios were calculated independently for each mouse based on %ID/g and reported as mean ±SE. [89Zr]Zr-DFO-scFv-Fc-CD44 was also evaluated in mice bearing PC-

3 tumors (n=5 per group) expressing lower levels of CD44 and in non-tumor bearing mice (n=5) to determine the blood half-life as described in the supplemental data.

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Small Animal PET Imaging

Small animal PET/CT imaging studies were conducted at the Washington University

School of Medicine (WUSM) using the MDA-MB-231 mouse model described above. About

2.22 MBq (60 μCi) of the [89Zr]Zr-DFO-scFv-Fc targeting CD44 or GPA was administrated to mice

(n=2 for each construct) via tail vein injection. Mice were anesthetized using 2% isofluorane/oxygen and imaged using an Inveon small animal PET/CT imaging system (Siemens) at day one and seven post-administration. Static images were collected for 30 min and reconstructed with a maximum a posteriori probability algorithm followed by CT co-registration using the Inveon Research Workstation image display software (Siemens). The standard uptake values (SUVs) were calculated by image analysis of muscle and tumor employing the decay- corrected concentrations of injected radioactivity and animal weight. Regions of interest (ROI) were selected from trans-axial PET images at the location corresponding to CT anatomical location of tumors, and the associated activity was measured using Inveon Research Workstation software. The standard uptake value was calculated as the regional activity concentration (Bq/cc) x animal weight (g) / decay-corrected amount of injected dose (Bq).

Statistics

GraphPad Prism 8 was used for statistical analyses. Data are represented as the mean

±SE. Biodistribution data were analyzed by multiple comparison via one-way ANOVA, followed by Tukey’s multiple comparison test (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; nsP≥0.05).

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RESULTS

Antibody Generation

Description of the generation of the human scFv phage display library is provided in

Supplemental Fig. 1. Biopanning against the ECD of recombinant human CD44(Q21-E268) resulted in the isolation of a scFv lead candidate. The yield of the scFv-CD44 after purification was 3.3 mg per liter of culture bacteria with high purity (>98%) (Supplemental Fig. 2) (15). A truncated version of human CD44(Q21-P220) was expressed and purified to demonstrate binding of different recombinant formats and to demonstrate no cross-reactivity toward murine CD44, despite sharing 76% on the amino acid level, or other (Supplemental

Figures 3 and 4).

Epitope Mapping

A peptide microarray identified a single, linear binding epitope of scFv-CD44 comprising the consensus sequence TNPEDIYPS, which is encoded by exon 5 of the CD44

(Supplemental Fig. 5). This motif corresponds to region T163-S171 that is conserved in most human CD44 isoforms but only shares 56% homology with the corresponding mouse homolog

(THQEDIDAS). This membrane-distal region of the ECD is structurally close to a region involved in HA-binding (Supplemental Fig. 6). Blocking studies confirmed that binding of HA to recombinant human CD44 can be efficiently inhibited by the scFv-Fc-CD44 (IC50=2.1 nM) (Supplemental Fig.

7).

Affinity

The affinity of monovalent scFv-CD44 toward recombinant human CD44(Q21-E268) was determined by generating ELISA-based equilibrium binding curves with an EC50 of 26.3±5.3 nM

(Table 1). Specificity to tumor cell-expressed CD44 was tested by flow cytometry using human tumor cell lines with different CD44 expression levels including the breast cancer cell line MDA-

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MB-231 (Supplemental Fig. 8). However, the binding affinity of the monovalent scFv-CD44 toward this cell line was reduced compared to recombinant CD44. The analysis of the saturation binding curve by flow cytometry revealed a reduction of binding affinity by ~20-fold (EC50=512.4±50.5 nM)

(Table 1). To increase the functional affinity we cloned the monovalent scFv-CD44 into a bivalent scFv-Fc-CD44 format comprising a C-terminal human IgG1-Fc (T106-K330) region. Transient expression of the scFv-Fc-CD44 in HEK293-6E suspension culture revealed high yield (45.6 mg/L) and excellent purity (>98%) after only a one step protein G purification procedure, confirmed by SDS-PAGE and WB analysis (Supplemental Fig. 9). FPLC analysis resulted in a main single peak and confirmed the primarily monomeric state of native scFv-Fc-CD44 (94%).

Compared to the monovalent scFv-CD44, the avidity of bivalent scFv-Fc-CD44 to human

CD44(Q21-E268) increased ~40-fold (EC50=0.7±0.1 nM) (Table 1). Importantly, the avidity improved nearly 200-fold on CD44-expressing human MDA-MB-231 breast cancer cells using flow cytometry (EC50: 2.3±0.2 nM) (Table 1).

Radiolabeling and In Vitro Characterization

The scFv-Fc-CD44 was conjugated with either NOTA or DFO resulting in a NOTA:scFv-

Fc-CD44 ratio of 0.30±0.02 and a DFO:scFv-Fc-CD44 ratio of 0.58±0.06 (Supplemental Fig. 10), followed by radiolabeling with 64Cu and 89Zr, respectively (Supplemental Fig. 11) (16-18). The radiochemical purity of the final products was >99.9% for both the [64Cu]Cu-NOTA-scFv-Fc and the [89Zr]Zr-DFO-scFv-Fc (Table 2). Specific activity of [64Cu]Cu-NOTA-scFv-Fc-CD44 was

84.7±11.0 MBq/mg and 80.0±5.5 MBq/mg for [89Zr]Zr-DFO-scFv-Fc with labeling yields of 60% and 75%, respectively (Table 2). Immunoreactive fractions were 88.1±3.7% (64Cu) and 87.9±1.9%

(89Zr) (Table 2, Supplemental Fig. 12A) (19). Binding specificity was confirmed by blocking experiments with native, scFv-Fc-CD44, achieving >97% signal reduction (Table 2, Supplemental

Fig. 12B). The specific half-lives of internalization into MDA-MB-231 cells was determined to be

57.8±34.9 min (64Cu) and 56.4±6.1 min (89Zr), respectively (Table 2, Supplemental Fig. 12C) (20).

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Serum stability showed that both [64Cu]Cu-NOTA-scFv-Fc-CD44 and [89Zr]Zr-DFO-scFv-Fc-

CD44 were > 93% intact over the time course studied (Supplemental Fig. 13).

Biodistribution Studies

Biodistribution was conducted in mice bearing subcutaneous MDA-MB-231 (Fig. 1,

Supplemental Tables 1-4). Tumor uptake of the radiolabeled scFv-Fcs-CD44 increased over time from 3.3±0.3%ID/g ([64Cu]Cu-NOTA-scFv-Fc) after 1 hour up to 55.5±8.6%ID/g ([89Zr]Zr-DFO- scFv-Fc) after day 7 (p<0.0001). One day after injection, the tumor uptake of both radiolabeled scFv-Fcs-CD44 were comparable (64Cu: 22.7%±5.1%ID/g; 89Zr: 33.3±6.4%ID/g, p=0.7979). In contrast, blood detection levels declined over time from 20.7±1.7%ID/g (64Cu at day 1) to

2.3±0.8%ID/g (89Zr at day 7). As a negative control, human GPA-specific scFv-Fc that does not bind to MDA-MB-231 cells was radiolabeled with 89Zr and used (Supplemental Fig. 14).

Biodistribution of the scFv-Fc-GPA control revealed reduced tumor uptake that never exceeded that of 8.1±1.1%ID/g (Supplemental Table 3 and 4). Evaluation of [89Zr]Zr-DFO-scFv-Fc-CD44 and [89Zr]Zr-DFO-scFv-Fc-GPA in mice bearing PC-3 tumors (Supplemental Tables 5 and 6) with a medium level of CD44 expression (Supplemental Fig. 8) showed lower uptake than in the MDA-

MB-231 xenografts at both 1 day (20.7±2.5 vs. 33.3±6.4%ID/g) and 7 days (31.9±3.1 vs.

55.5±8.6%ID/g) demonstrating the difference in CD44 expression. In addition, uptake of [89Zr]Zr-

DFO-scFv-Fc-GPA was 6.0±0.3 and 3.5±0.1%ID/g at 1 and 7 days, respectively, demonstrating the specificity for CD44. To emphasize the specificity of our scFv-Fc-CD44 in targeting CD44, the tumor-to-organ (TTO) ratios for the MDA-MB-231 tumors were calculated (Fig. 2, Supplemental

Tables 7 and 8) based on the biodistribution data and compared to those of the control

(Supplemental Tables 9 and 10). TTB ratios increased over time for the scFv-Fc-CD44 with similar ratios after 1 day for the 64Cu-labeled (2.3±0.6) and 89Zr-labeled scFv-Fc-CD44 (4.5±1.1). The highest TTB ratios were reached after day 5 (39.3±9.4) and after day 7 (35.6±11.0) for 89Zr. The highest tumor-to-muscle (TTM) ratios were observed for [89Zr]Zr-DFO-scFv-Fc-CD44 with

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51.8±7.2 (5 d) and 74.2±5.9 (7 d), respectively. For the scFv-Fc control, the TTB and TTM ratios were low, never exceeding 1.4±0.2 (89Zr: 7 d) and 5.6±1.1 (89Zr: 1 d), respectively. An increase in bone uptake was measured for the [89Zr]Zr-DFO-scFv-Fc-CD44 (11.9±1.1%ID/g on day 7), not uncommon for 89Zr-labeled antibodies. The ratios of 64Cu-labeled and 89Zr-labeled scFv-Fc-CD44 to scFv-Fc-GPA in tumors and normal organs are shown in Figure 3 (Supplemental Tables 11 and 12). As expected, the ratios for the normal organs is ~1-1.5 as the uptake of the scFv-Fc-

CD44 is not expected to be different than the uptake of the scFv-Fc-GPA since the scFv-Fc-CD44 does not bind to murine CD44. However, at time points of 1 day or longer, there is clearly specific uptake in the tumor. The blood half-life of [89Zr]Zr-DFO-scFv-Fc-CD44 was determined to be 77.2 hours (Supplemental Fig. 15).

PET Analysis

Based on the higher TTB ratios, in vivo PET imaging was performed for the [89Zr]Zr-DFO- scFv-Fc-CD44 after day one and seven post-injection and compared to the control (Fig. 4A).

Tumor uptake of the scFv-Fc-CD44 was visible after one day although background signal in other organs could also be detected. In contrast, high and specific tumor uptake could be detected after seven days with almost no remaining background in other organs. For the control, uptake was visible in all highly vascularized tissues, including tumor after one day, likely due to high levels in the blood, presumably caused by Fc receptor recycling and/or an effect known as enhanced permeability and retention. After day seven, however, the control gave only a minor signal for the entire mouse, including a lack of tumor detection. The PET images were further analyzed by calculating the mean and maximum standard uptake values (SUV) (Fig. 4B, Supplemental Table

13). In case of the scFv-Fc-CD44, SUVs of the tumors were increased after day seven (SUVMax:

20.3±2.1) compared to day one (SUVMax: 12.2±0.2). In contrast, much lower uptake in muscle was detected for both day one (SUVMax: 1.3±0.2) and day seven (SUVMax: 0.8±0.2). For the control,

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SUVMax uptake in tumor (1 d: 5.6±0.6; 7 d: 2.9±0.5) and muscle was low (1 d: 1.1±0.1; 7 d:

0.7±0.2).

DISCUSSION

Although CD44 is an attractive target for cancer therapy due to high expression levels on tumor cells, it is also present in normal . Therefore, non-invasive imaging of its expression in patients will be needed for CD44-directed therapies. Biopsies can provide initial information regarding target expression, but not all lesions are accessible for this procedure and the regions obtained may not be representative of the entire tumor mass due to tissue heterogeneities. Furthermore, expression in distant normal tissue cannot be determined using tumor-directed biopsies. For CD44, radiolabeled antibodies can be used to determine tumor and whole body expression. Importantly, non-invasive imaging should be able to increase the therapeutic efficacy of antibodies targeting CD44 by determining appropriate doses for tumor accumulation and limiting normal tissue toxicities. Therefore, the development of an engineered antibody in which in vivo pharmacokinetics can be optimized for non-invasive imaging would be valuable for the development of future CD44-targeted therapeutics.

Here, we report the generation of a human CD44-specific antibody fragment (scFv) utilizing a human scFv-bacteriophage library. In order to improve its pharmacokinetic properties, we generated a bivalent antibody via incorporation into a human Fc format. While this reformatting increased the apparent affinity of the molecule to human CD44 as anticipated, it has been shown for different CD44-specific monoclonal antibodies that pharmacokinetics plays a more important role in tumor uptake than mere affinities (21). Since we did not know a priori the blood half-life of our scFv-Fc, we radiolabeled our reagents with either 64Cu or 89Zr to investigate short and long time points in vivo, respectively. Our data demonstrate that 89Zr is the appropriate choice for this

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particular scFv-Fc since tumor accumulation and TTB ratios were greatest after 5-7 days. In addition, configuration into the scFv-Fc format resulted in a blood half-life of 77.2 hours for [89Zr]Zr- scFv-Fc-CD44 (Supplemental Figure 15) which was shorter than the >125 hours reported for

[89Zr]Zr-DFO-RG7356 (6).

A humanized antibody, RG7356, that targets all isoforms of CD44 has been previously studied because it disrupts the binding of HA to CD44, causing anticancer activity (5). This is distinct from CD44 antibodies that target CD44v6 in which this interaction is not disrupted. Vugts et al., evaluated [89Zr]Zr-DFO-RG7356 in mice bearing MDA-MB-231 xenografts and found that tumor uptake was ~30%ID/g at 3-6 days after injection (6), which is less than our 56%ID/g achieved at 7 days. Similarly, our [89Zr]Zr-scFv-Fc-CD44 had superior TTB ratios at 5-7 days of

~36 compared to [89Zr]Zr-DFO-RG7356 which had a ratio of 8.7 at 6 days (6). It should be noted that neither RG7356 nor our scFv-Fc bind to murine CD44. Evaluation of [89Zr]Zr-DFO-RG7356 in monkeys showed uptake in the spleen, salivary glands, and bone marrow which is believed to be CD44-specific. Subsequent clinical evaluation of [89Zr]Zr-DFO-RG7356 demonstrated tumor targeting and its ability to quantify dose dependent uptake in normal tissues (7,8). These results demonstrate the utility of probing CD44 expression in vivo and that our scFv-Fc appears to be superior to RG7356 in terms of tumor uptake and TTB ratios in mouse models. However, while our mapping studies demonstrate binding of the scFv-Fc to the HA binding domain, we have yet to show in vivo therapeutic efficacy using this construct.

While we have demonstrated that the [89Zr]Zr-scFv-Fc-CD44 has good tumor localization, late time points are needed to achieve high TTB and TTO ratios. Therefore, future studies will evaluate other antibody formats, such as diabodies and minibodies, shown to achieve faster clearance patterns from blood and normal tissues thus leading to higher ratios at earlier time points. This will allow for labeling options using radionuclides that have shorter half-lives such as

64Cu, 68Ga, or 18F and could potentially lead to same-day imaging to determine CD44 expression.

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CONCLUSION

Overall, [89Zr]Zr-DFO-scFv-Fc-CD44 achieved higher TTB and TTO ratios at later time points (i.e., day 5 and 7) compared to a [64Cu]Cu-NOTA-scFv-Fc-CD44 at earlier time points (up to 1 day). We believe that our engineered scFv-Fc is superior to [89Zr]Zr-DFO-RG7356 when compared in the same mouse model and could be valuable for PET imaging in patients with

CD44-positive malignancies. Future studies are required to determine if our novel anti-human

CD44 scFv-Fc exhibits therapeutic efficacy or if other antibody formats, such as diabodies and minibodies provide more favorable activity profiles in vivo.

ACKNOWLEDGMENTS

This work was supported by the Department of Radiation Oncology at WUSM and NIH

R21 CA181022-01 (B.E.R). The authors would like to thank the small animal imaging facility at

WUSM for technical assistance in conducting biodistribution and imaging studies. We would like to acknowledge the isotope production group at Washington University for the production of 64Cu and 89Zr. We would like to thank Dr. Brian Mooney from the University of Missouri (Columbia,

MO) for performing the mass spectrometry analysis to determine chelate to antibody ratios. The authors acknowledge Dr. Abhay Singh for providing assistance with FPLC operations and Dr.

Heping Yan for providing the anti-Myc monoclonal antibody.

Disclosure

The authors report no conflicts of interest in this work.

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KEY POINTS

QUESTION: Can an anti-CD44 scFv be isolated from a human phage display library and used to image CD44 expression in a preclinical mouse model?

PERTINENT FINDINGS: Biodistribution of [89Zr]Zr-DFO-scFv-Fc-CD44 shows high tumor- specific uptake (up to 56%ID/g) in a CD44-expressing xenograft tumor and high TTB (up to 39) and TTM (up to 74) ratios compared to a control [89Zr]Zr-DFO-scFv-Fc-GPA. PET imaging shows good tumor uptake in this model at seven days with low background compared to control.

IMPLICATIONS FOR PATIENT CARE: Development of a novel anti-CD44 antibody in which pharmacokinetics can be optimized in the future will lead to a clinical tool for imaging CD44 expression and thus impacting the development of CD44-targeted therapeutics.

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4. Tijink BM, Buter J, de Bree R, et al. A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus. Clin Cancer Res. 2006;12:6064-6072.

5. Weigand S, Herting F, Maisel D, et al. Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist. Cancer Res. 2012;72:4329-4339.

6. Vugts DJ, Heuveling DA, Stigter-van Walsum M, et al. Preclinical evaluation of 89Zr- labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to

Phase 1 clinical studies. MAbs. 2014;6:567-575.

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7. Jauw YWS, Huisman MC, Nayak TK, et al. Assessment of target-mediated uptake with immuno-PET: analysis of a phase I clinical trial with an anti-CD44 antibody. EJNMMI Res.

2018;8:6.

8. Menke-van der Houven van Oordt CW, Gomez-Roca C, van Herpen C, et al. First-in- human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget. 2016;7:80046-80058.

9. Kenanova V, Olafsen T, Crow DM, et al. Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti- single-chain Fv-Fc antibody fragments. Cancer Res. 2005;65:622-631.

10. Sundaresan G, Yazaki PJ, Shively JE, et al. 124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice. J Nucl Med. 2003;44:1962-1969.

11. Thurber GM, Schmidt MM, Wittrup KD. Antibody tumor penetration: transport opposed by systemic and antigen-mediated clearance. Adv Drug Deliv Rev. 2008;60:1421-1434.

12. Spitzer D, Mitchell LM, Atkinson JP, Hourcade DE. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly. J Immunol. 2007;179:2600-2608.

13. Diebolder P. “LYmph Node Derived Antibody Libraries” (LYNDAL):

19

A concept for recovering human monoclonal antibodies with therapeutic potential University of

Stuttgart; 2014.

14. Diebolder P, Keller A, Haase S, et al. Generation of "LYmph Node Derived Antibody

Libraries" (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

MAbs. 2014;6:130-142.

15. Diebolder P, Krawczyk A. Detailed Protocols for the Selection of Antiviral Human

Antibodies from Combinatorial Immune Phage Display Libraries. Antibody Engineering; 2018.

16. Diebolder P, Vazquez-Pufleau M, Bandara N, et al. Aerosol-synthesized siliceous nanoparticles: impact of morphology and functionalization on biodistribution. Int J Nanomedicine.

2018;13:7375-7393.

17. Kume M, Carey PC, Gaehle G, et al. A semi-automated system for the routine production of copper-64. Appl Radiat Isot. 2012;70:1803-1806.

18. Wooten AL, Madrid E, Schweitzer GD, et al. Routine Production of 89Zr Using an

Automated Module. Applied Sciences. 2013;3:593-613.

19. Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA, Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods. 1984;72:77-89.

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20. Craft JM, De Silva RA, Lears KA, et al. In vitro and in vivo evaluation of a 64Cu-labeled

NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing . Nucl Med Biol. 2012;39:609-616.

21. Glatt DM, Beckford Vera DR, Parrott MC, Luft JC, Benhabbour SR, Mumper RJ. The

Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor

Targeting of Monoclonal Antibodies. Mol Pharm. 2016;13:1894-1903.

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Table 1. Affinity of the human antibody lead as recombinant scFv-CD44 or scFv-Fc-CD44

scFv-CD44 scFv-Fc-CD44

ELISA EC50±SE (nM) 26.3±5.3 0.7±0.1 (n=3) R2 0.927 0.923

Flow cytometry EC50±SE (nM) 512.4±50.5 2.3±0.2 (n=3) R2 0.970 0.985

Binding affinities (EC50) were calculated from the equilibrium-binding curves towards recombinant human CD44(Q21-E268) by ELISA and by flow cytometry on MDA-MB-231 tumor cells.

22

Table 2. Characteristics of the scFv-Fc-CD44 after radiolabeling with 64Cu ([64Cu]Cu-NOTA-scFv- Fc-CD44) or 89Zr ([89Zr]Zr-DFO-scFv-Fc-CD44)

[64Cu]Cu-NOTA- [89Zr]Zr-DFO- scFv-Fc-CD44 scFv-Fc-CD44

ITLC % of ROI±SE 100.00±0.0 99.9±0.0 (n=3)

Specific Activity MBq/mg±SE 84.7±11.0 80.0±5.5 (n=3) (mCi/mg) (2.3±0.3) (2.2±0.1)

Labeling efficiency %±SE 59.6±3.5 75.0±7.1 (n=3)

Immuno-reactivity (64Cu: n=2; %±SE 88.1±3.7 87.9±1.9 89Zr: n=3)

Blocking efficiency (64Cu: n=2; %±SE 99.8±0.0 97.5±0.5 89Zr: n=3)

Internalization: specific t 1/2 min±SE 57.8±34.9 56.4±6.1 (64Cu: n=2; 89Zr: n=3)

23

Figure 1. In vivo biodistribution of the scFv-Fc-CD44. Athymic nude mice bearing MDA-MB-231 tumors were injected intravenously with 0.37 MBq (10 µCi) of either [64Cu]Cu-NOTA-scFv-Fc-

CD44 (grey bars; n=5) or [89Zr]Zr–DFO-scFv-Fc-CD44 (red bars; n=4) and sacrificed after various time points. Data are expressed as mean ±SE of %ID/g.

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Figure 2. Tumor-to-organ ratios of the scFv-Fc-CD44. Ratios were calculated separately for each mouse based on the %ID/g data and expressed as mean ±SE for [64Cu]Cu-NOTA-scFv-Fc-CD44

(n=5) or [89Zr]Zr-DFO-scFv-Fc-CD44 (n=4).

25

Figure 3. Ratios for specific (scFv-Fc-CD44) versus non-specific uptake (scFv-Fc-GPA). Ratios were calculated based on the average %ID/g data from the biodistribution study in mice with MDA-

MB-231 tumor-xenografts.

26

FIGURE 4. Representative PET images of [89Zr]Zr-DFO-scFv-Fc-CD44 and [89Zr]Zr-DFO-scFv-

Fc-GPA (NC: negative control) in athymic nude mice bearing MDA-MB-231 tumors. (A) PET imaging one or seven days after injection of [89Zr]Zr-DFO-scFv-Fc-CD44 (left mice) or [89Zr]Zr-

DFO-scFv-Fc-GPA (right mice). White arrows indicate the position of the MDA-MB-231 tumor xenografts. (B) SUV analysis in tumor and muscle after injection of [89Zr]Zr-DFO-scFv-Fc-CD44

(n=2) or [89Zr]Zr-DFO-scFv-Fc-GPA (n=2).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 1. Summary about WashU-1 phage display library. The human scFv antibody library was generated with a similar strategy used for cloning of LYNDAL (13,14). In brief, human antibody repertoires (IgG and IgM) were amplified by PCR from cDNA of mixed human spleen samples and cloned randomly into phagemid vector pIT2.

This vector encodes for a 16 aa linker (G4S)3T between the VH and VL domain of the scFv as well as adds an additional C-terminal 6xHis and Myc tag. First, the VH and VL repertoires were amplified in a first set of PCRs and completed by SfiI/XhoI (VH) or SalI/NotI (VL) restriction sites in a second set of PCRs. The VH repertories were cloned first by electroporation in E. coli TG1 (Lucigen, #60502), followed by cloning of the VL- kappa or VL-lambda repertories. WashU-1 possess a diversity of 3.3x108 independent scFvs.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 2. Characterization of scFv-CD44. The antibody fragment was isolated from a human phage display library by 3 rounds of biopanning towards recombinant human CD44(Q21-E268) (Immune Technologies, #IT-300-001p). The phagmid vector containing the scFv-CD44 gene with C-terminal 6xHis and Myc tag was transformed into the amber codon non-suppressor E. coli strain Express Iq (NEB). ScFv- CD44 was solubility expressed and purified from the periplasmic fraction by IMAC as described before (15). (A) ScFv-CD44 possessed VH-VL orientation connected by a flexible 16 aa peptide linker. Based on the scFv-CD44 sequence, the theoretical molecular weight (MW) was calculated to be 28.1 kDa with an extinction coefficient (ε) of 38,690 M-1cm-1. (B) Characterization by Coomassie (Coom) stained gradient SDS-PAGE (3 µg/lane) of the reduced scFv-CD44 revealed high purity (>98%) with a single dominant band running at the expected MW. The integrity was analyzed by WB using a primary mouse anti-His monoclonal antibody (mAb) (Santa Cruz Biotechnology, #sc-8036) and a secondary goat anti-mouse polyclonal antibody (pAb) peroxidase conjugate (Jackson ImmunoResearch, #115-035-008) showing no obvious degradation products. (C) Analysis of the oligomeric state of scFv-CD44 (31 µg) by FPLC showed a main peak of monomeric product (60.4% at 16.5 ml) and slight formation of a dimeric fraction (8.9% at 15.4 ml).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 3. Characterization of recombinant human CD44. De novo synthesized DNA encoding for the ECD of human CD44(Q21-P220) was amplified by PCR and cloned into pTT28, a derivative of the pTT vector (National Research Council of Canada). The gene was cloned via restriction enzymes NheI and AgeI to add a C- terminal 8xHis tag. Endo-free plasmid DNA was transiently transfected into HEK293-6E cells and purified after five days expression from supernatant via IMAC purification. (A) Based on the amino acid (aa) sequence, the theoretical MW was calculated to be 23.5 kDa with an extinction coefficient (ε) of 20,775 M-1cm-1. (B) Characterization by Coomassie-stained gradient SDS-PAGE (10 µg/lane) of the reduced protein revealed high purity (>98%) with a broad band spanning from about 35 kDa up to 100 kDa due to the highly glycosylated nature of the mammalian cell produced protein. The integrity was confirmed by WB using a primary mouse anti-His mAb (Santa Cruz Biotechnology, #sc- 8036) and secondary goat anti-mouse pAb peroxidase conjugate (Jackson ImmunoResearch, #115-035-008). (C) Analysis of the oligomeric state of recombinant CD44 proteins (56 µg) by FPLC revealed a broad main peak of monomeric product (84.7% at 14.1 ml).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 4. Specificity and cross-reactivity analysis of the antibody lead towards human and murine CD44, and a panel of irrelevant proteins by ELISA. Proteins were diluted in PBS and coated with 3 μg/ml (milk powder: 20 mg/ml) overnight at 4°C on an ELISA plate (100 μl/well). The milk-blocked plate was incubated with the antibody in different recombinant formats (i.e., scFv, diabody, and scFv-Fc) with 1 μM in 2% MPBS for 2 hours at room temperature (RT). The scFv-CD44 (monovalent) and diabody-CD44 (bivalent) were expressed in periplasm of E.coli and purified via IMAC purification (all >98% pure). The bivalent scFv-Fc-CD44 was expressed by transiently transfected HEK293-6E cells and purified by protein G purification (>98% pure). Both the scFv-CD44 and the diabody-CD44 were detected via their C-terminal Myc tag using a primary mouse

mAb 9E10 (7.5 μg/ml) and secondary goat anti-mouse pAb, F(ab’)2-specific peroxidase conjugate (Jackson ImmunoResearch, #115-035-072). The scFv-Fc-CD44 was detected via a goat anti-human pAb peroxidase conjugate (Jackson ImmunoResearch, #109-035- 098, minimal cross-reactivity to mouse serum) using TMB substrate and 2 M sulfuric acid as stop solution. Absorbance was measured in a microplate reader at 450 nm. Data are represented as the mean of triplicates ±SE.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 5. Epitope mapping of the antibody lead by PEPperMAP technology. The human CD44(Q21-P220) aa sequence (UniProtKB: P16070) was translated into linear overlapping 15 peptides with a 14 aa overlap and elongated with neutral GSGSGSG linkers at the C- and N-terminus. The resulting CD44 peptide microarrays contained 200 different peptides printed in duplicate and were framed by additional hemaglutinin (YPYDVPDYAG, 76 spots) control peptides. The microarray was incubated with scFv-Fc-CD44 in incubation buffer (PBS, pH 7.4 with 0.05% Tween 20 and 10% blocking buffer (Rockland), followed by staining with a secondary goat anti- human IgG (Fc) DyLight680 conjugate. A mouse anti-hemaglutinin mAb (12CA5) DyLight800 conjugate served as control. A LI-COR Odyssey Imaging System was used for the read-out of the fluorescence intensity (arbitrary unit, a.u.) at a scanning intensity of 7/7 (red/green). Quantification of the spot intensities and peptide annotation were done with PepSlide Analyzer. (A) Positive signals (red) with high signal-to-noise ratios were observed for 8 adjacent peptides with well-defined staining of the control peptides (green). The strong response against a single epitope-like spot pattern formed by adjacent peptides suggest a consensus motif TNPEDIYPS as linear epitope of the lead antibody.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 6. Structural representation of human CD44 (left) and scFv-CD44 (right). A high-resolution (1.6 Å) crystal structure (PDB file: 4PZ4) of the ECD of human CD44(Q21-S171) is shown on the left in green. CD44 possess two putative HA binding domains within the distal ECD that are rich on basic amino acids. The major HA bindig

site is situated near the NH2 terminus as highlighted in dark blue (R41, Y42, R78, and Y79). The second HA binding site is located more membrane proximal as shown in bright blue (N100, N101, R150, R154, K158, and R162). The putative linear epitope of the lead antibody is depicted in red (T163-S171). The scFv-CD44 structure was modeled by the ROSIE Rosetta Online Server based on its VH and VL aa sequence and the CDRH3 loop with the highest obtained score. The framework regions of the VH domain is shown in black with the different CDR regions in orange. The framework regions of the VL domain is depicted in grey and the corresponding CDRs in yellow. The CDRs of the variable domains form a pocket enclosing the finger-like epitope of human CD44. The structures were visualized with PyMOL molecular graphics system, version 2.3.0 (Schrödinger).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 7. ScFv-Fc-CD44 efficiently blocks the binding of HA to recombinant human CD44 in concentration dependent manner. One µg/ml of recombinant human CD44(Q21-E268) (Immune Technologies, #IT-300-001p) was coated overnight in PBS at 4 ºC on an ELISA plate (100 µl/well). After blocking (1 hour with 2% BSA in PBS), 0.1 µg/well of HA-biotin (Sigma-Aldrich, #B1557) and different concentration of the scFv-Fc-CD44 was incubated at RT for 1 hour. Detection of the HA- biotin binding to recombinant CD44 was performed by high sensitivity streptavidin-HRP (Thermo Fisher Scientific, #21130) using TMB solution and 2 M sulfuric acid as stop solution. Absorbance was read at 450 nm in a BioTek ELx800 microplate reader. The IC50 for blocking the binding of HA-biotin to human CD44(Q21-E268) by the scFv-Fc-CD44 was 2.1 nM (R2=0.9230). Absorbance data (triplicates, minus background) were

normalized and the IC50 calculated based on nonlinear regression analysis in GraphPad Prism 8 using log(inhibitor) vs. response (three parameters).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 8. ScFv-CD44 specifically binds to human tumor cell lines expressing different levels of CD44. The scFv-CD44 showed a similar concentration- dependent pattern of fluorescence signals in flow cytometry when compared to a CD44- specific mouse mAb clone B-F24 (Abcam, #ab27284) (data not shown). The scFv-CD44 was diluted in flow buffer (2% FBS in PBS) and incubated with 0.3x106 cells/well (2 hours at RT). Detection of scFv-CD44 was performed via the C-terminal Myc tag using murine mAb 9E10 (7.5 μg/ml) and secondary goat anti-mouse pAb fluorescein isothiocyanate conjugate (Jackson ImmunoResearch, #115-095-008). The positive control was detected via the same secondary goat anti-mouse pAb fluorescein isothiocyanate conjugate. Mean fluorescence intensities of cells were analyzed in duplicates in a MACSQuant Analyzer 10 (Miltenyi Biotec) using the MACSQuantify Software. Data are represented as the mean of duplicates ±SE.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 9. Characterization of scFv-Fc-CD44. The scFv-CD44 gene was amplified by PCR with scFv-specific primers to add NarI and EcoRV restriction sites and cloned into vector pYD11 (National Research Council of Canada) adding an intrinsic human IgG1 Fc part at the C-terminus. Plasmid DNA was transiently transfected into HEK293-6E cells and purified after five days of expression from supernatant via IMAC purification. (A) Based on the aa sequence, the theoretical MW was calculated to be 103.7 kDa with an extinction coefficient (ε) of 151,830 M-1cm-1 for the native scFv-Fc-CD44. (B) Characterization by Coomassie-stained gradient SDS-PAGE (5 µg/lane) of the reduced protein revealed high purity (>98 %) with a distinct band for a reduced single-chain (theoretical MW: 51.9 kDa). The integrity was confirmed by WB using a goat anti-human pAb peroxidase conjugate (Jackson ImmunoResearch, #109-035-098). (C) Analysis of the oligomeric state of scFv-Fc-CD44 (45 µg) by FPLC revealed a broad main peak of monomeric product (94.4% at 13.7 ml). (D)

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 10. Analysis of chelated scFv-Fc-CD44 by mass spectrometry. Intact mass of the unconjugated (black curves), or conjugated scFv-Fc (red curves) were analyzed by high-resolution QTOF (Quadrupole Time-of-Flight) LC/MS (Liquid Chromatography/Mass Spectrometry). Samples were concentrated by Amicon Ultra-0.5 centrifugal filters to exchange buffer to 200 mM ammonium acetate buffer (pH 6.9) by centrifugation (10x times) to about 20 µM. Frozen samples were thawed on ice and diluted to 2 pmol/µl in 0.1% formic acid. Two ul of centrifuged supernatants injected via an autosampler and run on a fused silica column packed with Magic C8 (3.5 µm particles, 100Å) linked to a C8 Captrap column (both Michrom Bioresources) by elution with a 30 min gradient of acetonitrile. Data were acquired on a calibrated Agilent 6520 QTOF and examined using the Qualitative Analysis software provided with the instrument. The maximum entropy was used to deconvolute multi-charge-state peaks to intact mass using the protein deconvolution model. The unconjugated scFv-Fc-CD44 (black, top profiles) shows a main peak at about 106.3 kDa and several additional peaks presumably representing different states of glycosylation. NOTA-conjugated (A) or DFO-conjugated (B) profiles (red, middle profiles) show additional peaks for the conjugated products. The zoomed in overlays (bottom profiles) revealed the additional peaks of the conjugated samples with a mean calculated mass of 513.9837±0.7071 for NOTA and 804.7126 ±1.4531 for DFO. Based on the relative peak heights of the conjugated samples, the average number of NOTA or DFO per scFv-Fc-CD44 was 0.30±0.02 and 0.58±0.06, respectively. The ratio of unconjugated to conjugated species was 1/0.42 (scFv-Fc- CD44/NOTA-scFv-Fc-CD44) and 1/0.65/0.21 (scFv-Fc-CD44/DFO-scFv-Fc-CD44/DFO- DFO-scFv-Fc-CD44).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 11. Schematic representation of scFv-Fc-CD44 modified with chelator p-SCN-Bn-NOTA (A) and p-SCN-Bn-DFO (B) and labeled with either 64Cu (A) or 89Zr (B). Frozen aliquots of the scFv-Fc-CD44 (1-2 mg/ml) were dialyzed against 0.1 M carbonate buffer (pH 9.0) and conjugated via its primary amines with chelator (both Macrocyclics, 2 mM in DMSO) p-SCN-Bn-NOTA (#B605) or p-SCN-Bn-DFO (#B705) in a molar ratio of 1 (scFv-Fc) to 5 (chelator) for 1 hour at 37°C while gently rocking at 40 rpm. Buffer-exchanged conjugates NOTA-scFv-Fc-CD44 (0.1 M NH4OAc pH 6.0) and DFO-scFv-Fc-CD44 (1 M HEPES, pH 7.2) were kept frozen until labeling with 64Cu 64 89 89 ( CuCl2 diluted in 0.1 M NH4OAc, pH 6.0) or neutralized (pH 6.8-7.4) Zr solution ( Zr- oxalate diluted in 1 M HEPES, pH 7.2) for 1 hour at 37°C (300 rpm) using about 18.5 MBq (500 μCi) for 200 μg of scFv-Fc. To complex unreacted radiometals, chelex-treated 50 mM DTPA solution (pH 7.0) was added (1/10 of total volume) and incubated for further 5 min at 37°C (300 rpm). Final products were obtained by buffer exchange with PBS (pH 7.2) via Zeba spin columns (Thermo Fisher Scientific). The radiochemical purity of the 64 + - products was confirmed by ITLC as described before (16). Cu (t1/2=12.7 h, β =17%, β 64 =39%, EC=43%, Emax=0.656 MeV) was produced by a (p,n) reaction on enriched Ni on a TR-19 biomedical cyclotron (Advanced Cyclotron Systems) at Washington University Cyclotron Facility and purified with an automated system by standard procedures (17). 89 Zr (t1/2=78.4 h, EC=76.6 %, β+=22.3%, Eave.(β+)=396.9 keV, Rave.(β+)=1.18 mm) was produced via the 89Y(p,n)89Zr transmutation reaction on a CS-15 cyclotron (Cyclotron Corporation) at Washington University Cyclotron Facility and purified with an automated module by standard procedures (18).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 12. (A) Cell binding studies. Representative plot of activity (total/bound) versus 1/[normalized cell concentration] used to calculate the immunoreactive fraction of [64Cu]Cu-NOTA-scFv-Fc-CD44 (black) and [89Zr]Zr-DFO- scFv-Fc-CD44 (red) in MDA-MB-231 cells. (B) Fifteen μg of native scFv-Fc-CD44 was used to specifically block the binding of the radiolabeled scFv-Fcs to 2.5x106 MDA-MB- 231 cells/well. (C) Representative internalization assays of the radiolabeled scFv-Fcs- CD44 into MDA-MB-231 cells. Data are presented as triplicates of mean ±SE. The immunoreactivity towards binding to CD44+ MDA-MB-231 cells was done by Lindmo assay (19). Five thousand cpm (50 μl) were added to MDA-MB-231 cells in increasing concentrations (0.25 to 2.5x106 cells/500 μl PBS) in triplicate for 1 hour at RT. Pelleted cells were washed twice with PBS, and counted for 64Cu or 89Zr activity. The specific binding was calculated as the ratio of bound radioactivity to the total amount of administered activity after background correction. To determine the blocking efficiency, 15 μg of the non-radiolabeled scFv-Fc lead was added to 2.5x106 of MDA-MB-231 cells/well in triplicate and the ratio of blocked to non-blocked activity calculated. Internalization was performed as described before (20) using 5x105 of MDA-MB-231 cells/well. Here, CD44-specific blocking agent (15 μg scFv-Fc-CD44) was added in triplicate, followed by addition of either [64Cu]Cu-NOTA-scFv-Fc-CD44 or [89Zr]Zr-DFO- scFv-Fc-CD44 corresponding to 50,000 to 100,000 cpm. The time-corrected counts per minute (cpm) of the internalized fraction was converted into fmol based on the time- corrected specific activity, the MW of the scFv-Fc-CD44, and the efficacy of the gamma counter, and subsequently normalized to the number of tumor cells.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 13. ScFv-Fc-CD44 retained stable in human serum at 37° when conjugated and radiolabeled with [64Cu]Cu-NOTA (black) or [89Zr]Zr-DFO (red). The radiochemical purity (>99.5%) of the freshly radiolabeled constructs in PBS were first confirmed by ITLC representing the initial time point (0 h). To assess the stability in vitro, 2.7 MBq of the [64Cu]Cu-NOTA-scFv-Fc-CD44 complex (74 μCi, 100 μl) was added to human serum (500 μl) in a microcentrifuge tube in triplicates and incubated at 37° while gently shaking at 300 rpm on an Eppendorf ThermoMixer. To the designated time points (1 h, 2 h, 4 h, 1 d, 2 d, and 3 d), 2 μl samples were withdrawn and evaluated by ITLC. Similarly, 2.0 MBq of the [89Zr]Zr-DFO-scFv-Fc-CD44 complex (54 μCi, 100 μl) was incubated with human serum (500 μl) at 37° in triplicates, and 2 μl samples measured to later time points (1 d, 2 d, 6 d, 7 d, and 8 d) due to the longer half-life of 89Zr compared to 64Cu (78.4 h versus 12.7 h).

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 14. Characterization of control scFv-Fc-GPA. The sequence of the variable domains was obtained from a hybridoma of parental mAb BRIC 256 (International Blood Group Reference Laboratory). The GPA binding site was cloned as

scFv by connecting the VH and VL domain with an (G4S)3 linker, followed by sublconing into vector pYD11 (National Research Council of Canada) via NarI/EcoRV to add an C-

terminal human IgG1 Fc part. Plasmid DNA was transiently transfected into HEK293-6E cells and purified after five days of expression from supernatant via IMAC. (A) Based on the aa sequence, the theoretical MW was calculated to be 105.9 kDa with an extinction coefficient (ε) of 193,770 M-1cm-1 for the native scFv-Fc-GPA. (B) Characterization by Coomassie-stained gradient SDS-PAGE (5 µg/lane) of the reduced protein revealed high purity (>98%) with a distinct band for a reduced single-chain (theoretical MW: 52.95 kDa). The integrity was confirmed by WB using a goat anti-human pAb peroxidase conjugate (Jackson ImmunoResearch, #109-035-098). (C) Analysis of the oligomeric state of scFv- Fc-GPA (42 µg) by FPLC revealed a main peak of monomeric product (68.3% at 13.6 ml) and two smaller peaks corresponding to putative non-covalently linked dimers and trimers. (D) Flow cytometry confirms specific binding of the scFv-Fc-GPA to GPA expressed by human erythrocytes but not to MDA-MB-231 cancer cells. Detection was performed with a rabbit anti-human Fc fluorescein isothiocyanate conjugate (Jackson ImmunoResearch, #309-095-008). Data are represented as the mean of triplicates ±SE.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Figure 15. In vivo blood clearance studies of the [89Zr]Zr-DFO-scFv-Fc- CD44 in mice. Female CD-1 IGS mice (Charles River, strain 022) were intravenously injected with about 0.37 MBq (10 μCi) of [89Zr]Zr-DFO-scFv-Fc-CD44 via the tail vein. After each time point (1 h, 4 h, 1 d, 5 d, and 7 d), blood was collected by small cheek bleeds. Each sample was weighed, and radioactivity counted with a Beckman Gamma- 8000 counter. Blood uptake was calculated as percentage of injected dose per gram (%ID/g). Blood clearance parameter were analyzed by PKSolver 2.0 using the two- compartment analysis after IV bolus injection. Data represent the mean ±SE based on 89 n=5 animals. According this analysis, t1/2Alpha (serum distribution half-life) of [ Zr]Zr-DFO- scFv-Fc-CD44 in mice was 2.3 h and t1/2Beta (serum elimination half-life) was 77.2 h.

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 1. Biodistribution of [64Cu]Cu-NOTA-scFv-Fc-CD44 in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor-xenografts at different time points (1 h, 4 h, and 24 h) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on five mice (n=5) per group.

Organ 1 hour 4 hours 24 hours Tumor 3.3 ± 0.3 7.9 ± 1.5 22.7 ± 5.1 Blood 20.7 ± 1.7 14.1 ± 1.4 9.8 ± 0.3 Lung 9.5 ± 0.7 9.7 ± 0.9 6.5 ± 0.1 Liver 11.5 ± 0.5 12.8 ± 0.9 6.9 ± 0.4 Spleen 6.0 ± 0.6 7.4 ± 2.2 3.6 ± 0.1 Kidney 9.7 ± 0.6 11.3 ± 0.8 12.9 ± 1.3 Bladder 2.0 ± 0.1 4.9 ± 1.9 3.6 ± 0.3 Muscle 1.0 ± 0.1 1.5 ± 0.1 1.9 ± 0.1 Heart 5.6 ± 0.4 6.5 ± 0.4 4.2 ± 0.2 Bone 2.3 ± 0.3 3.1 ± 1.0 1.7 ± 0.1 Pancreas 2.1 ± 0.2 2.2 ± 0.2 2.0 ± 0.1 Stomach 0.9 ± 0.1 1.7 ± 0.3 1.4 ± 0.4

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 2. Biodistribution of [89Zr]Zr-DFO-scFv-Fc-CD44 in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor-xenografts at different time points (1 d, 3 d, 5 d, and 7 d) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on four mice (n=4) per group.

Organ 1 day 3 days 5 days 7 days Tumor 33.3 ± 6.4 43.9 ± 7.6 42.7 ± 6.9 55.5 ± 8.6 Blood 9.0 ± 2.3 6.3 ± 1.7 1.6 ± 0.7 2.3 ± 0.8 Lung 4.7 ± 0.8 4.4 ± 0.8 2.3 ± 0.2 2.6 ± 0.4 Liver 8.4 ± 2.4 7.2 ± 1.0 6.4 ± 0.3 7.1 ± 0.1 Spleen 7.9 ± 4.4 5.0 ± 0.9 4.4 ± 0.3 4.7 ± 0.7 Kidney 13.3 ± 1.2 15.0 ± 2.4 11.7 ± 1.6 15.1 ± 2.7 Bladder 3.7 ± 0.5 3.9 ± 0.3 2.3 ± 0.2 2.4 ± 0.2 Muscle 1.6 ± 0.3 1.4 ± 0.2 0.8 ± 0.1 0.7 ± 0.1 Heart 3.6 ± 0.5 2.9 ± 0.4 1.0 ± 0.1 1.3 ± 0.2 Bone 5.7 ± 2.4 9.0 ± 1.0 10.2 ± 1.6 11.9 ± 1.1 Pancreas 1.4 ± 0.1 1.3 ± 1.0 0.9 ± 0.0 0.9 ± 0.0 Stomach 0.9 ± 0.1 0.8 ± 0.1 0.4 ± 0.1 0.5 ± 0.1

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 3. Biodistribution of [64Cu]Cu-NOTA-scFv-Fc-GPA (control) in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor- xenografts at different time points (4 h and 24 h) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on five mice (n=5) per group.

Organ 4 hours 24 hours Tumor 4.6 ± 0.5 6.2 ± 0.8 Blood 13.5 ± 2.1 9.2 ± 0.6 Lung 5.6 ± 0.6 5.3 ± 0.3 Liver 18.9 ± 1.9 7.6 ± 0.7 Spleen 8.3 ± 2.5 3.6 ± 0.2 Kidney 7.2 ± 0.5 5.3 ± 0.4 Bladder 2.5 ± 0.5 3.6 ± 0.1 Muscle 1.2 ± 0.2 1.7 ± 0.1 Heart 4.7 ± 0.5 3.2 ± 0.2 Bone 2.8 ± 0.9 1.2 ± 0.1 Pancreas 1.5 ± 0.2 1.6 ± 0.1 Stomach 1.2 ± 0.1 0.8 ± 0.1

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 4. Biodistribution of [89Zr]Zr-DFO-scFv-Fc-GPA (control) in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor- xenografts at different time points (1 d and 7 d) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on five mice (n=5) per group.

Organ 1 day 7 days Tumor 8.1 ± 1.1 3.9 ± 0.5 Blood 7.9 ± 1.4 2.7 ± 0.2 Lung 4.5 ± 0.6 2.5 ± 0.1 Liver 9.1 ± 0.7 8.9 ± 0.8 Spleen 5.9 ± 0.9 4.3 ± 0.4 Kidney 8.1 ± 0.6 10.1 ± 0.8 Bladder 3.5 ± 0.3 2.3 ± 0.1 Muscle 1.6 ± 0.3 0.7 ± 0.0 Heart 3.2 ± 0.3 1.6 ± 0.1 Bone 4.7 ± 0.9 9.6 ± 0.4 Pancreas 1.4 ± 0.2 0.8 ± 0.0 Stomach 1.0 ± 0.0 0.4 ± 0.0

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 5. Biodistribution of [89Zr]Zr-DFO-scFv-Fc-CD44 in male NCI SCID/NCr mice (Charles River, strain 561) with PC3 tumor-xenografts at different time points (1 d and 7 d) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on five mice (n=5) per group.

Organ 1 day 7 days Tumor 20.7 ± 2.5 31.9 ± 3.1 Blood 16.0 ± 0.6 5.3 ± 0.1 Lung 8.2 ± 0.5 5.0 ± 0.1 Liver 7.2 ± 0.3 7.9 ± 0.4 Spleen 16.3 ± 0.2 16.4 ± 0.7 Kidney 10.0 ± 0.4 8.4 ± 0.2 Bladder 5.5 ± 0.5 3.0 ± 0.3 Muscle 2.6 ± 0.1 1.0 ± 0.0 Heart 6.3 ± 0.4 2.7 ± 0.1 Bone 6.4 ± 0.3 12.8 ± 1.0 Pancreas 2.4 ± 0.1 1.3 ± 0.0 Stomach 1.6 ± 0.1 0.8 ± 0.1

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 6. Biodistribution of [89Zr]Zr-DFO-scFv-Fc-GPA in male NCI SCID/NCr mice (Charles River, strain 561) with PC3 tumor-xenografts at different time points (1 d and 7 d) post injection. Data are reported as the percent of the injected dose per gram of tissue (mean ±SE) based on five mice (n=5) per group.

Organ 1 day 7 days Tumor 6.0 ± 0.3 3.5 ± 0.1 Blood 14.9 ± 0.4 4.5 ± 0.1 Lung 8.4 ± 0.4 4.7 ± 0.1 Liver 10.3 ± 0.4 11.2 ± 0.4 Spleen 33.6 ± 2.2 31.5 ± 1.4 Kidney 6.4 ± 0.4 6.8 ± 0.2 Bladder 3.9 ± 0.4 2.8 ± 0.2 Muscle 2.1 ± 0.1 1.1 ± 0.1 Heart 6.0 ± 0.3 2.7 ± 0.1 Bone 9.7 ± 0.4 19.7 ± 1.7 Pancreas 2.3 ± 0.0 1.3 ± 0.0 Stomach 2.3 ± 0.3 1.2 ± 0.1

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 7. Tumor-to-organ ratios (TTOs) of the [64Cu]Cu-NOTA-scFv-Fc- CD44 in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB- 231 tumor-xenografts at different time points (1 h, 4 h, and 24 h) post injection. Tumor- organ ratios are averages of five mice (n=5) per group (mean ±SE).

Organ 1 hours 4 hours 24 hours Blood 0.2 ± 0.0 0.6 ± 0.1 2.3 ± 0.6 Lung 0.4 ± 0.0 0.9 ± 0.2 3.5 ± 0.8 Liver 0.3 ± 0.0 0.6 ± 0.1 3.4 ± 0.1 Spleen 0.6 ± 0.1 1.3 ± 0.3 6.4 ± 1.4 Kidney 0.3 ± 0.0 0.7 ± 0.1 1.9 ± 0.6 Bladder 1.7 ± 0.2 2.6 ± 0.8 6.4 ± 1.3 Muscle 3.3 ± 0.3 5.4 ± 1.1 12.1 ± 3.1 Heart 0.6 ± 0.1 1.2 ± 0.2 5.6 ± 1.5 Bone 1.5 ± 0.2 3.2 ± 0.9 14.3 ± 4.5 Pancreas 1.6 ± 0.1 3.7 ± 0.7 11.3 ± 2.6 Stomach 4.1 ± 1.0 5.6 ± 1.5 19.3 ± 4.8

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 8. Tumor-to-organ ratios (TTOs) of the [89Zr]Zr-DFO-scFv-Fc-CD44 in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor-xenografts at different time points (1 d, 3 d, 5 d, and 7 d) post injection. Tumor- organ ratios are averages of four mice (n=4) per group (mean ±SE).

Organ 1 day 3 days 5 days 7 days Blood 4.5 ± 1.1 9.9 ± 3.2 39.4 ± 9.4 35.6 ± 11.0 Lung 7.2 ± 0.7 10.2 ± 0.6 18.8 ± 1.8 22.0 ± 1.6 Liver 5.1 ± 1.3 6.7 ± 1.5 6.7 ± 0.9 7.8 ± 1.0 Spleen 8.9 ± 2.8 10.3 ± 2.9 9.9 ± 1.8 13.1 ± 2.8 Kidney 2.5 ± 0.5 3.0 ± 0.3 3.6 ± 0.3 3.8 ± 0.4 Bladder 8.9 ± 1.1 11.2 ± 1.3 19.6 ± 4.0 22.9 ± 1.3 Muscle 21.0 ± 2.0 29.9 ± 1.9 51.8 ± 7.2 74.2 ± 5.9 Heart 9.2 ± 1.0 14.8 ± 0.7 41.2 ± 3.2 43.3 ± 4.4 Bone 9.1 ± 2.5 5.3 ± 1.2 4.4 ± 0.7 4.8 ± 0.7 Pancreas 23.2 ± 3.0 33.2 ± 4.9 48.6 ± 6.9 59.7 ± 9.7 Stomach 39.9 ± 9.3 53.5 ± 8.2 109.6 ± 17.6 151.0 ± 39.1

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 9. Tumor-to-organ ratios (TTOs) of the [64Cu]Cu-NOTA-scFv-Fc- GPA control in NCI female athymic NCr-nu/nu mice (Charles River, strain 553) with MDA- MB-231 tumor-xenografts at different time points (4 h and 24 h) post injection. Tumor- organ ratios are averages of five mice (n=5) per group (mean ±SE).

Organ 4 hours 24 hours Blood 0.4 ± 0.1 0.7 ± 0.1 Lung 0.8 ± 0.0 1.2 ± 0.2 Liver 0.3 ± 0.0 0.8 ± 0.1 Spleen 0.7 ± 0.1 1.8 ± 0.2 Kidney 0.7 ± 0.1 1.2 ± 0.1 Bladder 2.0 ± 0.2 1.7 ± 0.2 Muscle 4.0 ± 0.3 3.7 ± 0.6 Heart 1.0 ± 0.1 1.9 ± 0.2 Bone 2.2 ± 0.4 5.1 ± 0.5 Pancreas 3.3 ± 0.3 4.1 ± 0.5 Stomach 4.1 ± 0.5 8.0 ± 1.6

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 10. Tumor-to-organ ratios (TTOs) of the [89Zr]Zr-DFO-scFv-Fc-GPA control in female NCI athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB- 231 tumor-xenografts at different time points (1 d and 7 d) post injection. Tumor-organ ratios are averages of five mice (n=5) per group (mean ±SE).

Organ 1 day 7 days Blood 1.3 ± 0.3 1.4 ± 0.2 Lung 1.9 ± 0.3 1.6 ± 0.2 Liver 0.9 ± 0.1 0.5 ± 0.1 Spleen 1.5 ± 0.3 1.0 ± 0.2 Kidney 1.0 ± 0.1 0.4 ± 0.0 Bladder 2.3 ± 0.3 1.7 ± 0.2 Muscle 5.6 ± 1.1 5.4 ± 0.7 Heart 2.7 ± 0.5 2.5 ± 0.4 Bone 1.9 ± 0.4 0.4 ± 0.1 Pancreas 5.9 ± 0.9 4.6 ± 0.4 Stomach 8.0 ± 1.3 11.9 ± 2.4

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 11. Uptake ratios for specific ([64Cu]Cu-NOTA-scFv-Fc-CD44) versus non-specific antibody ([64Cu]Cu-NOTA-scFv-Fc-GPA) in NCI female athymic NCr- nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor-xenografts at different time points (4 h and 24 h) post injection. Ratios are based on the averages of five mice (n=5) per group.

Organ 4 hours 24 hours Tumor 1.7 3.7 Blood 1.0 1.1 Lung 1.7 1.2 Liver 0.7 0.9 Spleen 0.9 1.0 Kidney 1.6 2.5 Bladder 1.9 1.0 Muscle 1.3 1.1 Heart 1.4 1.3 Bone 1.1 1.4 Pancreas 1.5 1.3 Stomach 1.5 1.7

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 12. Uptake ratios for specific ([89Zr]Zr-DFO-scFv-Fc-CD44) versus non-specific antibody ([89Zr]Zr-DFO-scFv-Fc-GPA) in NCI female athymic NCr-nu/nu mice (Charles River, strain 553) with MDA-MB-231 tumor-xenografts at different time points (1 d and 7 d) post injection. Ratios are based on the averages of four (n=4, [89Zr]Zr- DFO-scFv-Fc-CD44) or five (n=5, [89Zr]Zr-DFO-scFv-Fc-GPA) mice per group.

Organ 1 day 7 days Tumor 4.1 14.3 Blood 1.1 0.8 Lung 1.0 1.0 Liver 0.9 0.8 Spleen 1.3 1.1 Kidney 1.6 1.5 Bladder 1.1 1.0 Muscle 1.0 1.0 Heart 1.1 0.8 Bone 1.2 1.2 Pancreas 1.0 1.1 Stomach 0.9 1.3

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Supplemental data: Preclinical Evaluation of an Engineered scFv-Fc Targeting Human CD44

Supplemental Table 13. Mean and maximum standard uptake values (1 or 7 days post injection) of the [89Zr]Zr-DFO-scFv-Fcs targeting CD44 or GPA (control) obtained from static PET imaging. Values were calculated from PET imaging with Inveon scanner from two pairs (n=2) of female NCI athymic NCr-nu/nu mice (Charles River, strain 553) and expressed as mean ±SE.

Organ 1 day: CD44 7 day: CD44 1 day: GPA 7 day: GPA

Tumor: SUVMean 8.5 ± 0.3 14.1 ± 1.0 4.00 ± 0.5 1.9 ± 0.3

Tumor: SUVMax 12.2 ± 0.2 20.3 ± 2.1 5.6 ± 0.6 2.9 ± 0.5

Muscle: SUVMean 0.8 ± 0.1 0.3 ± 0.0 0.6 ± 0.3 0.2 ± 0.1

Muscle: SUVMax 1.3 ± 0.2 0.8 ± 0.2 1.1 ± 0.1 0.7 ± 0.2

31