Biodistribution and Targeting of anti-5T4 Antibody-Drug...

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Biodistribution and Targeting of anti-5T4 Antibody-Drug Conjugate using

Fluorescence Molecular Tomography

Anand Giddabasappa1, Vijay R Gupta1, Rand Norberg1, Parul Gupta1, Mary E. Spilker2, Joann Wentland2, Brian Rago2, Jeetendra Eswaraka1, Mauricio Leal2, Puja Sapra3. 1Global Science & Technology – Comparative Medicine, 2Pharmacokinetics Dynamics and Metabolism, 3Oncology Research Unit, Pfizer Inc. 10724 Science Center Drive, San Diego, CA 92121, USA.

Corresponding author: Anand Giddabasappa, Global Science and Technology (WCM), Pfizer Global Research and Development. 10724 Science Center Dr., San Diego, CA 92121. Phone: 858- 622-7522; Fax: 858-526-4119; E-mail: [email protected]

Running Title: Biodistribution of anti-5T4-ADC by FMT Imaging.

Key Words: 5T4-ADC, Biodistribution, Cancer, FMT, Imaging.

Abbreviations: 2-D – two dimensional; 3-D – three dimensional; Ab – antibody; ADC –

antibody drug conjugate; anti-5T4-Antibody – 5T4-Ab; 5T4-mcMMAF – 5T4-ADC;

ELISA - enzyme-linked immunosorbant assay; FMT – fluorescence molecular

tomography; LC-MS – liquid chromatography/mass spectrometry; %ID – percentage

injected dose; NIR – near-infrared; PET – positron emission tomography; PK –

pharmacokinetics; ROI – region of interest; SEC-HPLC – size exclusion

chromatography-high performance liquid chromatography; SPECT - Single-photon

emission computed tomography; VivoTag®680XL – VT680.

on July 27, 2018. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 27, 2016; DOI: 10.1158/1535-7163.MCT-15-1012

http://mct.aacrjournals.org/

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Financial Information: The funding for this research was provided by Pfizer Inc.

Disclosure of Potential Conflicts of Interest All authors are either current or former employees (full time or contract) of Pfizer Inc. Text: 4953 words

Abstract: 246 words




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Abstract:

Understanding a drug’s whole-body biodistribution and tumor targeting can

provide important information regarding efficacy, safety and dosing parameters.

Current methods to evaluate biodistribution include in vivo imaging technologies

like positron electron tomography and single-photon emission computed

tomography or ex vivo quantitation of drug concentrations in tissues using

autoradiography and standard biochemical assays. These methods use

radioactive compounds or are cumbersome and do not give whole-body

information. Here, for the first time we show the utility of fluorescence molecular

tomography (FMT) imaging to determine the biodistribution and targeting of an

antibody-drug conjugate (ADC). An anti-5T4-Antibody (5T4-Ab) and 5T4-ADC

were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680).

Both conjugated compounds were stable as determined by SEC-HPLC and

plasma stability studies. Flow cytometry and fluorescence microscopy studies

showed that VT680 conjugated 5T4-ADC specifically bound 5T4 expressing cells

in vitro, and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-

ADC. In vivo biodistribution and tumor targeting in an H1975 subcutaneous

xenograft model demonstrated no significant differences between accumulation

of VT680 conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor.

Additionally, quantitation of heart signal from FMT imaging showed good

correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT

imaging) may be a surrogate for plasma drug clearance. These results




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demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change

the behavior of native biologic and FMT imaging can be an useful tool to

understand biodistribution and tumor targeting kinetics of antibodies, ADCs and

other biologics.




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Introduction:

The biodistribution of a therapeutic agent provides significant insights regarding

the drug’s therapeutic efficacy, on- and off-target potential toxicities, clearance

mechanisms, half-life and metabolism as well as information for designing next-

generation therapeutic agents with superior safety and efficacy (1-5).

Traditionally biodistribution studies are performed using radio-labeled

compounds in rodents and collecting tissues at various time points post-injection

to quantitate the drug concentration. This method is labor intensive, requires the

synthesis of radio-labeled compounds, specialized facilities to perform studies

and the use of a large number of animals. A major disadvantage of this method is

that not every tissue or compartment of the body can be evaluated. In vivo

imaging like Positron Emission Tomography (PET), Single-Photon Emission

Computed Tomography (SPECT) and optical imaging can address some of these

shortcomings (1, 2, 4-6). PET and SPECT have been used to determine

biodistribution of small molecules and biologics (4, 5, 7-9), but they too require

radio-labeled compounds and specialized facilities to perform studies. The radio-

labels decay with time and thus have a relatively short shelf life. Additionally, the

use of multiple radiotracers in PET or SPECT imaging is challenging due to the

crosstalk between energies of multiple windows (5).

An alternative method to study biodistribution of biologics is fluorescence-based

imaging. Labeling of biologic molecules with fluorescent probe is relatively easy,

efficient and can be performed in most research facilities. Multiple two-




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dimensional (2-D) and three-dimensional (3-D) fluorescence imaging

technologies are currently available for in vivo evaluation and have been utilized

to qualitatively evaluate biomarkers, biodistribution and targeting of biologic

molecules (8, 10-12). Among these technologies, fluorescence molecular

tomography (FMT) imaging is the most advanced, permitting acquisition of 3-D

tomographic data for localization, detection and quantitation of the fluorescently

labeled drug in multiple organs and at site of interest. FMT technology uses the

near-infrared (NIR) spectral region (600 – 900 nm) which reduces the auto-

fluorescence by physiological molecules and skin (8, 13-15). The wide spectral

window in FMT allows for the use of multiple fluorophores simultaneously.

Additionally, FMT imaging is non-invasive and the same animal can be monitored

longitudinally, thereby reducing the number of animals while maintaining or even

improving the relevance and statistical significance of a study.

In this study, we exploited this capability (namely, longitudinal imaging) and

evaluated the biodistribution and tumor targeting of a 5T4-antibody (5T4-Ab) and

its companion antibody-drug conjugate (ADC: 5T4-ADC) using FMT imaging. An

ADC is composed of an antibody that targets a specific antigen at the site of

action (i.e. tumor) and is conjugated via a linker to a cytotoxic agent (payload)

that elicits cell death (16, 17). Anti-5T4 antibody conjugated to

monomethylauristatin F (MMAF), here after referred to as 5T4-ADC, has shown

promising results in various in vitro and in vivo cancer models (18, 19). Our goal

in this study was twofold, first to develop a systematic method to evaluate the




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biodistribution of biologic drugs using FMT and second to evaluate whether

conjugation of mcMMAF (the linker-payload) had any effect on the biodistribution

of the 5T4 antibody. To accomplish these goals we labeled the naked-antibody or

the antibody arm of the ADC with NIR fluorophore VivoTag®680XL (here after

referred to as VT680) and showed that VT680 conjugation did not change the

stability, binding ability to the antigen nor the biological activity of 5T4-Ab or 5T4-

ADC. Additionally, no significant difference was observed between 5T4-Ab and

5T4-ADC pharmacokinetics (PK), biodistribution or tumor targeting. This is the

first study evaluating the biodistribution and tumor targeting of ADCs using FMT

imaging. Since the fluorophore can be conjugated to any protein/peptide, this

novel approach can become a platform technology in conducting biodistribution

studies of biologic drugs.




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Materials and Methods:

Fluorophore labeling: VT680 was labeled using a VivoTag®680XL protein

labeling kit as per the manufacturer’s instructions (Perkin Elmer, Waltham, MA).

The excitation and emission maximums (Exmax and Emmax) of VT680 were 668

nm and 688 nm, respectively. VT680 has the succinimidyl ester group that reacts

with the free amine groups present on lysine residues of the proteins. The

reactions were carried out in amine free buffer, i.e PBS. The antibodies and

ADCs used in this study were generated in-house (Pfizer Inc.) and the structural

information of 5T4-Ab and 5T4-ADC has been described before (18). Briefly, the

3 mg of Isotype control-ADC (control-ADC), 5T4-Ab and 5T4-ADC in PBS were

incubated with 150 µg of VT680 in bicarbonate buffer for 2 h (in dark) on a

rotating shaker. Following the conjugation reaction, the free fluorophore

molecules were removed using a protein purification column (Bio-Gel P-100).

Samples were quantified for the conjugation efficiency by measuring the

absorbance at 280 nm and 668 nm using a NanoDrop 8000® (Thermo Scientific,

Wilmington, DE), thereby providing the relative efficiency (degree of labeling,

DOL) of the VT680 conjugation to each antibody or ADC. Reagents with DOL

between 2-3 VT680 molecules/protein were used for this study. In order to

evaluate if the VT680 conjugation increased aggregation propensity of the

reagents, size-exclusion chromatography-HPLC (SEC-HPLC) was performed.

Briefly, Ab and ADCs in PBS (1 mg/ml) were separated on YMC-Pack Diol-200

column (Waters Corporation, Milford, MA) using HPLC (Agilent Technologies,




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Wilmington, DE). The mobile phase consisted of 0.02 M phosphate buffer and

0.4 M NaCl and system was run under isocratic conditions. The elution profiles of

separated proteins were detected by UV absorbance at 280 nm.

In vitro plasma stability of Ab and ADCs: Plasma was isolated from blood

collected from naïve nu/nu mice by terminal cardiac puncture procedure. In vitro

plasma stability studies were carried out by incubating a concentration equivalent

to a 10 mg/kg bolus dose of VT680 labeled 5T4-Ab (5T4-Ab-VT680) or 5T4-ADC

(5T4-ADC-VT680) with plasma. This mixture was incubated for different time

intervals (1, 24 or 48 h) at 37⁰C and 4⁰C. At the end of the incubation period an

aliquot equivalent to 1 µg of VT680 labelled Ab and ADC was separated on the

4-12% Bis-Tris gradient gels under reducing conditions to evaluate their

degradation profiles. VT680 fluorophore at a concentration corresponding to the

loaded protein (equivalent to DOL) was also run in a separate lane as a control.

The gels were imaged using an Odyssey Infrared Imaging System (LI-COR,

Lincoln, NE) using 700 nm laser.

Flow cytometry studies: H1975 and MDA-MB-468 cells were procured from

ATCC (Manassas, VA) and cell culture was performed as per their instructions.

No cell authentication was separately performed in our laboratory, but we

evaluated for rodent pathogens (IMPACT profiling by IDEXX BioResearch,

Columbia, MO) before in vivo studies. Cell surface binding of 5T4-ADC-VT680

and control-ADC-VT680 was evaluated in MDA-MB-468 and H1975 cell lines

using flow cytometry. Briefly, suspended cells (7.5 x 105 cells/mL) were prechilled




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to 4°C on ice and incubated for 1 h at 4°C with different concentrations of VT680

labeled ADCs in PBS (pH 7.4). After incubation, cells were washed, re-

suspended in ice cold PBS and immediately analyzed using a BD LSRII flow

cytometer. For all experiments, the geometric mean fluorescence intensities were

recorded for individual treatments. The experiments were performed in triplicates

and repeated twice.

Cell binding and internalization studies by fluorescence microscopy: The

cell surface binding and internalization of the ADC was evaluated by epi-

fluorescence microscopy. Briefly, MDA-MB-468 cells (5 X 104 cells/mL) were

cultured in Nunc™ Lab-Tek™ II Chamber Slide™ System in triplicates. Chamber

slides pre-chilled on ice were incubated for 4 h at 4°C with VT680 labeled ADCs

(10 µg/mL). For binding studies, incubated cells were washed twice with ice-cold

PBS and fixed using 4% paraformaldehyde at room temperature for 1 h. For

internalization studies, incubated cells were washed twice with ice-cold PBS

followed by incubation with pre-warmed medium at 37°C for 4 h to monitor

internalization of VT680 labeled ADCs. Later cells were fixed with 4%

paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X-100 in PBS

for 5 min. Cells were then washed and stained with anti-human FITC conjugated

secondary antibody (1:100 dilution) and mounted using Vectashield® mounting

medium containing DAPI (Vector Laboratories, Burlingame, CA). Images were

taken with a Nikon TE2000 microscope at 40X magnification.




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Cytotoxicity studies: The in vitro activity of VT680 labeled as well as unlabeled

5T4-ADC and control-ADC was tested in MDA-MB-468 cell line using CellTiter-

Glo Luminescent Cell Viability Assay (Promega Corporation, Madison, WI). Cells

(5x103 cells/well) were plated overnight in a 96 well plate followed by treatment

with different concentrations (0-100 µg/mL) of 5T4-ADC and control-ADC. After

72 h, Celltitre-Glo reagent was added and cell viability was determined as per

manufacturer’s instructions. The assay was repeated at least twice in triplicates.

In vivo studies: All procedures performed on animals in this study were in

accordance with established guidelines and regulations, and were reviewed and

approved by the Pfizer Institutional Animal Care and Use Committee. Animals

were housed under standard 12:12 light:dark cycle in ventilated racks at a room

temp of 72ºF and RH between 30-70%.

Five million H1975 cells in matrigel were injected into the SQ flanks of the female

nu/nu mice (8 weeks old). When the tumors were ~400 mm3 the animals were

injected with 5T4-Ab-VT680, 5T4-ADC-VT680, control-Ab-VT680 or VT680 alone

via the intravenous route. The dose was normalized to 2 nmol of VT680, as this

dose was determined to give good in vivo signal (data not shown) and also at this

dose 5T4-ADC showed moderate anti-tumor activity (18). Imaging was

performed using an FMT 4000 fluorescence molecular tomography imaging

system (Perkin Elmer Inc, Boston, MA). During imaging the animals were

anesthetized using a mixture of isoflurane and oxygen and placed in an imaging

cassette. Throughout the imaging session the animals were maintained under




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anesthesia in a thermally regulated (37°C) chamber. FMT imaging (whole-body)

was performed at 5 min and at 6, 24, 48, 96 and 240 h post-injection of probes.

Blood samples (20 μl) were also collected at each time point prior to imaging for

PK evaluation. The signal observed during in vivo imaging is the sum of signal

from the tumor or tissues due to drug accumulation and signal from the

vasculature within the tissue due to drug in circulation.

In order to minimize the fluorescence signal from the circulating drug and better

determine the amount of drug that has accumulated in the tissues, ex vivo

evaluation was done on a subset of animals. At 48, 96 and 240 h after dosing,

selected animals were subjected to whole-body perfusion (via intra-cardiac route)

with heparinized PBS/Saline and euthanized prior to collecting tissues. The

tissues (tumor, liver, brain, spleen, kidney and lungs) were imaged ex vivo by

FMT. The FMT data was reconstructed and analyzed using TrueQuant® software

(Perkin Elmer Inc, Boston, MA). To validate the 3-D quantitation of the probe by

FMT, we determined a standard curve using known concentrations (0 - 2000

pmol) in a phantom. Linear regression analysis of 3-D quantitation revealed an

R2 value of 0.97 showing a good linearity of detection (Supplemental Figure 1).

The 3-D region of interest (ROI) was drawn for the whole-body, tumor and the

organs of interest on the in vivo and ex vivo images. Total fluorescence (in pmol)

was obtained under a no thresholding setting to minimize the loss of low-

fluorescence signals. Percentage injected dose (%ID) for each time point and

organ of interest was calculated using the total signal from the whole-body at 5




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min (100% injected dose) from each mice. For determining the %ID/g for ex vivo

organs, the fluorescence values from each organ were normalized to its weight

and the whole-body signal at 5 min. After ex vivo imaging, the tumor/liver tissues

at the 48 h time point was flash frozen for quantitation of the Ab, ADC and

released payload using bioanalytical methods. The data represented is the

average (± SEM) of 3-6 mice/time point, for each treatment group.

Pharmacokinetic Analysis: Quantitation of the 5T4-Ab and 5T4-ADC

concentrations in mouse plasma was performed by enzyme-linked

immunosorbant assay (ELISA) as described earlier (18). Briefly, 5T4 protein was

coated on to 96 well-plates to capture the 5T4-Ab and 5T4-ADC. The captured

reagents were detected using biotinylated goat anti-human IgG for the antibody

and biotinylated anti-MMAF antibody for the ADC. The absorbance values were

determined using a spectrophotometer. The released payload (cys-mc-MMAF)

on 5T4-ADC was quantified by LC-MS/MS assay using a 550 Qtrap and C18

column with cys-mcMMAD as the internal standard (18). The PK (including the

terminal half-lives) of 5T4-Ab and 5T4-ADC from the FMT imaging and plasma

were determined by non-compartmental analysis using Phoenix WinNonlin 6.3

(Certara L.P.). The 3-D FMT imaging quantitation of heart (pmol/ml of VT680)

was obtained from TrueQuant® software and converted into equivalent protein

concentrations (µg/ml) for comparison with plasma concentrations.




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Results:

Biochemical characterization of fluorophore conjugated 5T4-Ab and 5T4-

ADC: Prior to evaluating the biodistribution and targeting of 5T4-Ab and 5T4-

ADC in an in vivo tumor model, we followed a stepwise in vitro quality control

assessment process (Figure 1). The compounds of interest were labeled with the

NIR fluorophore – VT680. In order to minimize the effects of fluorophore

conjugation to the 5T4-Ab or 5T4-ADC, we developed a conjugation protocol that

would yield a DOL of ~3 VT680 molecules per antibody. Supplemental Figure 2A

shows the absorbance spectrum of VT680, 5T4-ADC and 5T4-ADC-VT680. The

VT680 alone curve showed a major peak at ~670 nm whereas 5T4-ADC showed

a major peak at ~280 nm. The 5T4-ADC-VT680 conjugate showed two peaks at

~280 nm and ~670 nm representing the absorbance maximum of antibody

(protein) and fluorophore (VT680), respectively. Similar to absorbance

characteristics, we saw fluorescence of VT680 and the 5T4-ADC-VT680

conjugate at ~690 nm, whereas the 5T4-ADC alone had no fluorescence at 690

nm (data not shown).

The labeled and the unlabeled biologic molecules had similar elution profiles on

SEC-HPLC. SEC-HPLC of 5T4-ADC and 5T4-ADC-VT680 had a major peak

representing the monomers and a minor peak probably representing protein

aggregates (Supplemental Figure 2B). Area under the curve (AUC) of the major

peak was 98.2% for 5T4-ADC and 97.7% for 5T4-ADC-VT680, whereas the AUC

of the minor peak was 1.1% for 5T4-ADC and 1.8% for 5T4-ADC-VT680. This




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data suggested that labeling of 5T4-ADC with VT680 did not increase the

aggregation propensity. Similar elution profiles were also obtained with 5T4-Ab

and control-Ab/ADC labeled with VT680 (data not shown).

In order to evaluate the stability of VT680 conjugated biologic molecules in PBS

or plasma, in vitro stability studies were performed. Either 5T4-Ab-VT680 or 5T4-

ADC-VT680 were incubated with PBS or mouse plasma at 4⁰C or 37⁰C for 1, 24

and 48 h. After incubation the samples were separated on a gradient gel in

reducing conditions to evaluate any degradation or released VT680 from biologic

molecules. Since the 5T4-Ab and 5T4-ADC were labeled with a 680 nm

fluorophore, we directly imaged the gels using an infrared imaging system. Two

distinct bands representing the heavy chain and light chain of the antibody were

observed in 5T4-Ab-VT680 and 5T4-ADC-VT680 samples (Supplemental Figure

2C). Interestingly the heavy chain fragments in the plasma samples migrated

faster than the PBS samples. This may be due to the excess protein present in

the plasma samples which in turn is reducing the number of SDS molecules per

protein. No degradation bands reflecting the cleaved VT680 were observed in

5T4-Ab-VT680 or 5T4-ADC-VT680 samples incubated in PBS or plasma. VT680

alone showed a distinct band at the lower end of the gel (Supplemental Figure

2C). These results indicate that VT680 formed a stable conjugation to the Ab and

ADC.

Evaluation of binding and functional properties of VT680 conjugated

reagents: To determine if the conjugation of VT680 to biologic molecules




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perturbed their binding property, we used flow cytometry and epi-fluorescence

microscopy. Previous studies showed that H1975 is a relatively low 5T4

expressing cell line and MDA-MB-468 is a high 5T4 expressing cell line (18).

Concentration dependent binding of 5T4-ADC-VT680 and control-ADC-VT680

was compared using flow cytometry (Figure 2A). 5T4-ADC-VT680 bound to both

H1975 and MDA-MB-468 cells in a concentration dependent fashion, whereas

control-ADC-VT680 had minimal binding. Mean fluorescence intensity showed

that 5T4-ADC-VT680 binding was higher in MDA-MB-468 cells compared to

H1975 cells (Figure 2A). The antibody binding capacity, a measure of relative

receptor number on each cell surface revealed that the expression was

approximately 31,000 and 63,400 for H1975 and MDAMB-468, respectively

(Supplemental Figure 3). In order to further confirm the cell surface binding of

5T4-ADC-VT680, we employed epi-fluorescence microscopy. Figure 2B shows

the localization of 5T4-ADC-VT680 at the cell surface. The line intensity profile

for VT680 (Figure 2B, red line) showed two sharp peaks depicting high and

specific binding at the cell surface. No significant cell surface binding was

observed with control-ADC-VT680, which was confirmed by the scattered line

intensity profile. The line intensity profile for DAPI (nuclear marker, blue line)

showed a single broad peak inside the cell membrane for both 5T4-ADC and

control-ADC-VT680 (Figure 2B).

To further determine whether VT680 conjugation to the 5T4-ADC had any effect

on the cellular uptake or activity, internalization of the ADC was assessed using




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epi-fluorescent microscopy, while a cytotoxicity assay was used to verify activity.

Internalization was visualized both by directly imaging VT680 fluorophore and

indirectly by detection with an anti-human secondary antibody conjugated to

FITC. Punctate labeling inside the cytoplasm was observed in both the channels,

indicating localization probably in the lysosomes (Figure 2C). Line intensity

profiles confirmed the overlap of VT680 (red) and FITC (green) channels,

suggesting the specificity of the signal to 5T4-ADC (Figure 2C). These data also

suggest that the VT680 signal observed was from the VT680 conjugated to the

5T4-ADC and not free VT680 fluorophore.

To further evaluate the cell killing ability of VT680 labeled 5T4-ADC, we

performed cytotoxicity assay in MDA-MB-468 cells. Unlabeled 5T4-ADC showed

a concentration dependent effect on the viability of the cells (Figure 2D). The

cytotoxicity profile of 5T4-ADC-VT680 overlapped with that of the unlabeled 5T4-

ADC. Although the control-ADC had very minimal effect on the viability of cells,

the cytotoxicity profile of unlabeled control-ADC and control-ADC-VT680

overlapped each other (Figure 2D). These results show that the VT680 labeling

of biologic molecules at 2-3 DOL did not change their functional activity. This is

an important observation as it then discards the notion that a relatively large tag

interferes with ADC functional activity and opens up their usability.

In vivo biodistribution, clearance and plasma PK of 5T4-Ab and 5T4-ADC: In

vivo biodistribution and tumor targeting was evaluated in the H1975 mouse

xenograft model. In vivo whole-body (torso) FMT imaging was performed at




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various time points (5 min and 6, 24, 48, 96 and 240 h) post-injection. Blood was

also collected and processed to obtain plasma before each imaging session. A 3-

D ROI was created for selected organs of interest as per the protocol described

by Vasquez et al., (8). Since, there was a minimal signal (<5%) observed in the

head region at all time-points, the torso ROI was used to depict signal from the

whole-body. The whole-body data represents the signal from various

tissues/organs in the torso region and also the probe in the circulation. Initially,

FMT imaging showed minimal changes in the whole-body signal, which likely

reflects the compound’s distribution into the tissues, while after 48 h, the

compound’s systemic clearance becomes evident (Figure 3A). The FMT profile

of both 5T4-Ab-VT680 and the 5T4-ADC-VT680 overlapped at all the time points

suggesting no difference in clearance due to linker-payload (mcMMAF)

conjugation over the 10 day period. Plasma concentrations determined by

detecting the antibody from 5T4-Ab-VT680 and 5T4-ADC-VT680 samples by

ELISA also showed an overlapping clearance profile (Figure 3B). In all cases an

initial distribution phase was observed followed by a sustained rate of clearance

at later time-points. Interestingly, the payload’s profile as measured by LC/MS

assay showed a faster initial distribution compared to the antibody profile (Figure

3B). A 3-D ROI around the heart, which was assumed to primarily reflect the

blood profile was also evaluated (Figure 3C). Similar to the plasma PK data, 5T4-

Ab-VT680 and 5T4-ADC-VT680 profiles in heart tissue overlapped suggesting

that the payload conjugation did not change the elimination kinetics.




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Furthermore, the kinetic profiles obtained from plasma samples and the heart

ROI from FMT imaging were in good agreement with one another, supporting the

assumption that this ROI reflects blood clearance (Figure 3C).

Kinetics of tumor targeting and accumulation in the H1975 xenograft

model: Figure 4A shows the whole-body FMT images of the same animal at

various time points from either the 5T4-Ab-VT680 or 5T4-ADC-VT680 groups.

The image panel also illustrates that the distribution and clearance profiles of

5T4-Ab-VT680 and 5T4-ADC-VT680 from the whole-body were similar (Figure

4A). As anticipated, liver (being a major site for clearance) and tumor ( containing

the target antigen) were the two major sites of accumulation, while other tissues

showed minimal accumulation. The images also reveal that the accumulation and

elimination of 5T4-Ab-VT680 and 5T4-ADC-VT680 from the liver followed similar

profile. Since imaging was performed over a 10 day period, the tumor size was

measured according to a standard schedule (twice a week). No tumor growth

inhibition was observed in the 5T4-Ab-VT680 group, whereas in the 5T4-ADC-

VT680 group there was trend showing tumor growth inhibition after day 3 post-

injection (Figure 4B). This further confirms our in vitro data that the fluorescent

labeling do not change the functional activity of the 5T4-ADC or the 5T4-Ab

within the 10 day observation period.

To determine the kinetics of targeting and accumulation of 5T4-Ab-VT680 and

5T4-ADC-VT680, the fluorescence signal was quantified and normalized to the

tumor size. The peak accumulation was observed at 24-48 h post-injection,




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although significant accumulation was observed even at 96 h post-injection

(Figure 4C). There was no statistically significant difference between 5T4-Ab-

VT680 and the 5T4-ADC-VT680 group, however a trend towards increased

accumulation in the 5T4-Ab-VT680 group was observed. These differences may

be due to the tumor growth inhibition or cell killing observed in the 5T4-ADC-

VT680 group but not in the 5T4-Ab-VT680 group. After 96 h, there was a

consistent decline in the signal from both the groups although there was still a

significant fluorescent signal observed at 240 h post-injection of drugs suggesting

a prolonged retention of the biologic drug in the tumor. Ex vivo imaging of tumors

and other organs was performed at 48, 96 and 240 h after whole-body perfusion.

The highest accumulation in tumor for 5T4-Ab-VT680 and 5T4-ADC-VT680 was

observed at 48 h with %ID/g of 9.89 (SEM ± 1.51) and 7.38 (SEM ±2.02),

respectively followed by a decline at 96 and 240 h (Figure 4D and E). 5T4-Ab-

VT680 was significantly higher than 5T4-ADC-VT680 only at 240 h (4.54 ±0.16

vs 2.99 ±0.77 %ID/g). In order to confirm that these findings were specific to 5T4-

Ab and 5T4-ADC (rather than a VT680 fluorophore-based artifact), we evaluated

the tumor accumulation of control-Ab conjugated with VT680 and the free VT680

fluorophore. There was no significant accumulation of control-Ab or VT680 in the

tumors compared to the 5T4-Ab or 5T4-ADC (ex vivo imaging at 48 h;

Supplemental Figure 4A).

Kinetics of accumulation and clearance in the liver and other organs: FMT

imaging showed that the liver was one of the major sites for accumulation of the




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VT680-labeled biologics. The FMT imaging profile for clearance of 5T4-Ab-

VT680 and 5T4-ADC-VT680 from liver overlapped each other (Figure 5A).

Although the in vivo concentration profiles in liver tissue followed a similar trend

to whole-body clearance up to 48 h (Figure 3A), 5T4-Ab-VT680 and 5T4-ADC-

VT680 cleared slightly faster from liver after 48 h time point (Figure 5A). Figures

5B and C show the ex vivo images and quantitation of the signal from liver. Both

groups (5T4-ADC-VT680 and 5T4-Ab-VT680) had good liver signal suggesting

accumulation in liver at 48, 96 and 240 h. Since liver has no 5T4 expression and

the antibody does not cross-react with mouse, this is likely non-specific

accumulation or the signal from metabolized VT680. In order to confirm this non-

specific accumulation of 5T4-Ab-VT680 and 5T4-ADC-VT680 in liver, a separate

ex vivo study (48 h) evaluated the accumulation of both the control-Ab-VT680

and VT680 fluorophore alone. The accumulation of 5T4-Ab-VT680 and 5T4-

ADC-VT680 in liver was similar to control-Ab-VT680, whereas the free VT680

fluorophore had very minimal accumulation in the liver (Supplemental Figure 4B).

Additionally, we determined the amount of released payload (cys-mcMMAF) from

5T4-ADC in the tumor and liver by an LC-MS/MS method and found ~15 fold

higher payload concentrations in tumor than liver (Figure 5D). Ex vivo imaging

was also performed and quantified for a few selected organs: brain, spleen, lungs

and kidneys (Table 1). There was minimal accumulation (<1.6 %ID/g) in these

tissues, compared to tumor and liver. Quantitation revealed no meaningful

differences between 5T4-ADC-VT680 and 5T4-Ab-VT680 in these organs.




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Discussion:

The goal of this study was to develop a method to evaluate the biodistribution

and tumor targeting of an antibody or ADC using FMT imaging. Since FMT

acquires 3-D isotropic data we hypothesized that FMT can provide quantitative

information of the fluorophore or its conjugates (8). Structural modification of

antibodies can result in dramatic effects on the three dimensional arrangement,

PK properties, immunogenicity, binding affinity and thus their function (20, 21).

Hence, attachment of any additional entity like a payload, fluorophore or

radiolabel to the antibody could affect its binding and functional activity. Multiple

studies have shown the utility of conjugating either radiolabel, fluorophore or both

to antibodies or their fragments for biodistribution studies. CD105 (endoglin)

monoclonal antibody was conjugated with PET probe 89Zr and NIR probe CW-

800 and found no difference in binding or specificity between conjugated or non-

conjugated antibody (22). Hudson et al., conjugated NIR probe CF-750 to EGF

ligand for multispectral optoacoustic imaging and demonstrated that it did not

affect the biological activity significantly (23). Connor et al., showed that

conjugation of IRdye-800 to mouse monoclonal IgG1 (8C2) significantly changed

the PK property of this antibody (1). They adapted whole-body sectioning

accompanied by two dimensional fluorescence imaging for quantitation. Such

dissimilarities in PK can be related to the differences in modality used for

quantitation. These conflicting results also suggest that each antibody, peptide or




23

the fluorophore can have unique outcomes. In order to address these concerns,

we adapted a stepwise approach to our biodistribution studies using FMT and

performed qualitative in vitro assessment before the in vivo evaluation (Figure 1).

We labeled 5T4-Ab, 5T4-ADC or Isotype control-Ab/ADC with NIR fluorophore

VT680 and demonstrated by in vitro studies that conjugating VT680 at a DOL < 3

does not affect the stability or functional property (i.e. binding, internalization and

cytotoxicity) of 5T4-Ab or 5T4-ADC. In vivo biodistribution and tumor targeting of

5T4-Ab and 5T4-ADC was compared in an H1975 subcutaneous xenograft

model after administering a single intravenous bolus dose of compound. This

model was selected because H1975 cells express 5T4 antigen, the tumor growth

in vivo is at a moderate rate and previous studies have shown that 5T4-ADC

localizes to the tumor and inhibits the tumor growth (18, 24). The whole-body

biodistribution profile from FMT and the plasma concentration profiles (from LBA

assays) were overlapping for both 5T4-Ab-VT680 and 5T4-ADC-VT680

suggesting that payload or VT680 conjugation had no major effects on

accumulation and clearance of 5T4-Ab. Interestingly, the whole-body FMT signal

and the plasma PK profile appear to provide complementary information on the

distribution and elimination of the compounds. The whole-body FMT data

includes the signal from the probe in circulation and also accumulation in the

peripheral tissues. The compounds distribution into the extravascular tissue

space is evident in the early flat whole-body signal that occurs within the same

timeframe as the distribution phase observed in the plasma concentrations (0 –




24

48 h). Beyond 48 h, both samples show similar clearance profiles with estimated

terminal half-lives ranging from 3.4 to 4 days for the 5T4-Ab-VT680 and 3.3 to

3.6 days for the 5T4-ADC-VT680 measured in plasma samples or by whole-body

FMT. These half-lives were consistent with the previously reported plasma PK

values in naïve mice and MDA-MB-435/5T4 tumor model using the 5T4-ADC

which was not conjugated to VT680 (24).

Previously it has been shown that plasma input functions and blood time-activity

correlation can be determined by non-invasive PET imaging (25, 26). Since FMT

data provides 3D quantitative data we presumed that the signal from a ROI

encompassing the heart could be compared to the plasma concentration profile.

Given that 5T4-Ab-VT680 or 5T4-ADC-VT680 are not expected to have

significant uptake in the heart, the majority of the heart ROI signal can be

attributed to the blood fraction. Visual comparison of the plasma concentration

profiles and the FMT heart imaging profiles shows good agreement, suggesting

that the heart ROI is a reasonable surrogate for the plasma kinetics (i.e., the

separate plasma PK evaluation may not be needed in future studies).

Tumor growth was also monitored during the imaging period. We observed tumor

growth inhibition in the 5T4-ADC-VT680 group after day 4, although statistically

not significant due to the relatively small group size (p > 0.1). This suggests that

VT680 conjugated ADC retained in vivo tumor regression ability corroborating the

similar observations made with in vitro cytotoxicity data. Tumor accumulation

profiles by FMT imaging showed peak accumulation for both 5T4-Ab-VT680 and




25

5T4-ADC-VT680 groups (as the ratio of %ID/tumor size) was at 24-48 h post-

injection. There was an increased accumulation in the 5T4-Ab-VT680 group (p >

0.08), which might be attributed to the bigger tumor size in this group.

Additionally, the regression of the tumor size in the 5T4-ADC-VT680 group may

have added to the complexity in targeting and quantitation. Previous studies have

demonstrated that the growth rate and doubling time may affect the drug

exposure in the tumor (27, 28). To eliminate the contribution from the

fluorescence probe in the circulating blood, we performed ex vivo imaging after

whole-body perfusion. Results from the ex vivo imaging confirmed that significant

amounts of 5T4-Ab-VT680 and 5T4-ADC-VT680 were accumulated within the

tumor, whereas minimal accumulation was observed for the control-Ab. As

anticipated liver was a major site for non-specific accumulation and clearance of

VT680 conjugated 5T4-Ab, 5T4-ADC and control-Ab. Evaluation of released

payload by LC-MS/MS analysis from the ex vivo tumor and liver samples showed

that the tumor had ~15 times higher accumulation, an indication that the 5T4-

ADC went through the target mediated internalization and subsequent release of

the payload. This data was consistent with the previous observation that 5T4-

ADC and its released payload have a propensity for accumulating in the tumor

compared to other tissues (24). While this observation (the ability to differentiate

released payload from ADC in tumor and liver samples) was possible by using

LC-MS/MS methodology, our lab is working on methods that would allow for dual

fluorophore labeling of ADC and be able to differentiate the profiles of antibody




26

and released payload by FMT imaging. Additionally, the liver imaging data also

suggests that VT680 signal observed in the liver may have a significant

contribution from free VT680 after cleavage or from VT680 conjugated to

proteolysed Ab/ADC (metabolism) and not entirely by VT680 conjugated 5T4-Ab

and 5T4-ADC. It will be interesting to further understand if VT680 has any

residualizing property, similar to some of the NIR and radiolabeled probes (6,

29).

Conclusion:

Overall in this study we demonstrated that FMT is an useful imaging modality to

evaluate the biodistribution and tumor targeting of biologics. Using in vitro tools,

we showed that VT680 conjugation to 5T4-Ab or 5T4-ADC did not change the

stability, binding nor cytotoxic activity. In addition, in vivo FMT imaging and

plasma concentration analysis showed that both 5T4-Ab-VT680 and 5T4-ADC-

VT680 had similar biodistribution and clearance profiles and that both 5T4-Ab-

VT680 and 5T4-ADC-VT680 accumulated significantly in the H1975 tumors.

Since labeling of fluorophore to antibodies is simpler than radiolabeling and the

technology does not need a special facility, we believe FMT is a good tool to

evaluate biodistribution and targeting of biologic drugs. Recently, multiple NIR

fluorophores have become available that exhibit various chemistries, as well as

better properties for conjugation and in vivo imaging. It will be interesting to

expand upon the initial proof of principle demonstrated herein and assess the




27

utility of these fluorophores for biodistribution studies allowing for longitudinal

FMT imaging to evaluate not only ADC but also the released payload.

Acknowledgements: The authors acknowledge Seattle Genetics Inc. and

Oxford BioMedica for access to technology. We also thank the Pfizer –

Comparative Medicine (La Jolla) technical staff, Kush Lalwani (GST – CM, Pfizer

Inc) and Rachel C Roach (CTI-Pfizer Inc) for their assistance in the experiments.

Disclosure of Conflict of Interest: All authors are or were full time or part-time

employees of Pfizer Inc.




28

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32

Table1. Ex vivo quantitation of tissues and tumor by FMT imaging (%ID/g).

% ID/g5T4-ADC-

VT6805T4-Ab-VT680

5T4-ADC-VT680

5T4-Ab-VT680

5T4-ADC-VT680

5T4-Ab-VT680

5T4-ADC-VT680

5T4-Ab-VT680

5T4-ADC-VT680

5T4-Ab-VT680

5T4-ADC-VT680

5T4-Ab-VT680

48 h Mean 25.37 19.28 0.24 0.4 0.38 0.013 0.6 1.68 0.26 0.06 7.16 8.01SEM 6.28 1.15 0.23 0.09 0.19 0.013 0.23 0.14 0.26 0.05 4.79 0.79

96 h Mean 28.84 19.21 1.57 0.39 0.18 0.215 1.6 0.37 0.42 0.01 4.76 4.52SEM 6.12 2.27 0.79 0.17 0.06 0.058 0.55 0.05 0.4 0.01 1.86 1.43

240 h Mean 9.29 14.12 0.12 0.62 0.09 0.248 0.26 0.63 0 0.01 2.99 4.54SEM 0.88 2.18 0.03 0.39 0.03 0.108 0.08 0.11 0 0.01 0.77 0.16

Liver Spleen Kidney Lungs Brain Tumor




33

Figure Legends:

Figure 1: Schematic representing the stepwise protocol followed in

evaluating biodistribution and targeting of biologic drugs using FMT

imaging. Ab/ADC (Biologic drug) was labeled with VT680 by NHS ester reaction.

After labeling, the VT680 conjugated Ab/ADCs were qualitatively and/or

quantitatively evaluated for VT680 labeling, stability, binding to the antigen and

cytotoxic activity by in vitro methods. After these quality control evaluations, in

vivo biodistribution and tumor targeting was determined longitudinally by FMT

imaging and PK analysis of blood and tissues.

Figure 2: In vitro evaluation of binding and activity of 5T4-ADC-VT680. (A)

Flow cytometric evaluation of cellular binding of 5T4-ADC-VT680 and control-

ADC-VT680. H1975 and MDA-MB-468 cells were incubated with different

concentrations of VT680 labeled 5T4-ADC or Isotype control-ADC at 4⁰C for 1 h

and analyzed by flow cytometry. Note a dose-dependent increase in binding of

5T4-ADC in both cell lines. MDA-MB-468 cells had higher 5T4-ADC binding than

H1975 cells. No significant specific binding was observed from control-ADC in

both cell lines. The inset graphs show the histogram of control-ADC-VT680 and

5T4-ADC-VT680 at 100 µg/ml concentration. (B) Evaluation of cellular binding

5T4-ADC-VT680 and control-ADC-VT680 in MDA-MB-468 cell line using

fluorescence microscopy. Punctate signal was observed on the surface of cells

labeled with 5T4-ADC-VT680, but a weak non-specific signal was observed in




34

control-ADC-VT680 cells. Pixel intensity along the line on the microscopic image

is represented by the line intensity profile. The red line in the line intensity profile

represents the VT680 signal whereas the blue line represents DAPI (nucleus)

signal. Two sharp peaks were observed for VT680 signal in 5T4-ADC-VT680

labeled cells, but scattered lines were observed in control-ADC-VT680 labelled

cells. The line intensity profile for nucleus (DAPI) was similar (single broad peak)

for both treatments. (C) Evaluation of cellular internalization VT680 labeled 5T4-

ADC in MDA-MB-468 cell line using fluorescence microscopy. Red – VT680

signal; Green – FITC (anti-human secondary antibody); Blue – DAPI (nucleus).

Punctate labeling in the cytoplasm was observed by both VT680 and FITC

signals suggesting cellular internalization of 5T4-ADC. Yellow overlay in the

microscopy image shows the co-localization of VT680 (direct detection) and FITC

(indirect detection) labeling. The line intensity profile shows a good overlap of red

line (VT680) with green line (FITC) signal suggesting co-localized signal by direct

and indirect labeling. (D) Comparison of cytotoxicity of VT680 labeled 5T4-ADC

and control-ADC with unlabeled ADC. All experiments were repeated at least

twice; ± SEM is represented.

Figure 3: Whole-body profile of VT680 labeled 5T4-Ab and 5T4-ADC by FMT

imaging and Plasma PK. Female nu/nu mice bearing H1975 xenografts (~400

mm3) were dosed with 5T4-Ab or 5T4-ADC (normalized to 2 nmol VT680) and

imaged at 5 min, 6, 24, 48, 96 and 240 h post injection. Blood/plasma was also




35

collected at simultaneous time points for PK analysis. (A) Whole-body FMT

profile of 5T4-Ab-VT680 and 5T4-ADC-VT680, (B) Traditional plasma PK profile

of antibody for 5T4-Ab-VT680 and 5T4-ADC-VT680 groups and payload for 5T4-

ADC-VT680 group, (C) Comparison of heart signal from FMT imaging as a

plasma PK surrogate with traditional plasma PK profile for 5T4-Ab-VT680 and

5T4-ADC-VT680. n=5-6/group, ± SEM is represented in the graphs.

Figure 4: Tumor profile of 5T4-Ab-VT680 and 5T4-ADC-VT680. (A) Whole-

body FMT images at 5 min, 6, 24, 48, 96 and 240 h post-injection showing the

tumor targeting and clearance of 5T4-Ab-VT680 and 5T4-ADC-VT680. Arrows

show the location of tumor (T) and liver (L). (B) Tumor growth curve (measured

by calipers) of 5T4-Ab-VT680 and 5T4-ADC-VT680 groups during the imaging

phase. (C) FMT 3-D quantitation of tumor signal showing the in vivo profile of

accumulation of 5T4-Ab-VT680 and 5T4-ADC-VT680. FMT quantitation was

normalized to tumor size as determined by caliper measurements. (D) Ex vivo

FMT images showing the signal of 5T4-Ab-VT680 and 5T4-ADC-VT680 in tumor

(T) and brain (B) at 48, 96 and 240 h. (E) 3-D quantitation of ex vivo FMT images

of tumor from 5T4-Ab-VT680 and 5T4-ADC-VT680 at 48, 96 and 240 h. -n=5-

6/group, ± SEM is represented in the graphs; * = p-value < 0.05.

Figure 5: FMT imaging and quantitation of Liver and other tissues. (A) In

vivo FMT profile for liver at 5 min, 6, 24, 48, 96 and 240 h post-injection showing




36

the clearance of 5T4-Ab-VT680 and 5T4-ADC-VT680. (B) Ex vivo FMT images

showing the signal of 5T4-Ab-VT680 and 5T4-ADC-VT680 in spleen (Sp), liver

(Liv), kidney (K), lungs (Lu). (C) 3-D quantitation of ex vivo liver fluorescence at

48, 96 and 240 h. (D) Quantitation of released payload (cys-mcMMAF) in tumor

and liver by LC-MS method at 48 h post-injection. n=3-6/group, ± SEM is

represented in the graphs; * represents p-value < 0.05.



















Published OnlineFirst July 27, 2016.Mol Cancer Ther Anand Giddabasappa, Vijay R Gupta, Rand Norberg, et al. Conjugate using Fluorescence Molecular TomographyBiodistribution and Targeting of anti-5T4 Antibody-Drug

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