Fluorescent Image-Guided Surgery wi th an Anti-Prostate ... · Prostate Cancer Xenografts in Real-Time Geoffrey A. Sonn1, Andrew S. Behesnilian1, ... Los Angeles, CA, USA 2Molecular

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Fluorescent Image-Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real-Time Geoffrey A. Sonn*1, Andrew S. Behesnilian*1, Ziyue Karen Jiang*1, Kirstin A. Zettlitz2,3, Eric J. Lepin2,3, Laurent A. Bentolila4,5, Scott M. Knowles2,3, Daniel Lawrence2, Anna M. Wu2,3, Robert E. Reiter1 * These authors contributed equally to this work Affiliations: 1Department of Urology, University of California, Los Angeles, CA, USA 2Molecular & Medical Pharmacology, University of California, Los Angeles, CA, USA 3Crump Institute for Molecular Imaging, University of California, Los Angeles, CA, USA 4Deparment of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA 5California NanoSystems Institute, University of California, Los Angeles, CA, USA Running title: Fluorescent Image-Guided Surgery with an Anti-PSCA Diabody Keywords: Prostate cancer; Real time imaging; Antibody fragment; Fluorescent image guided surgery; Prostate stem cell antigen Financial Support: This work was supported by the National Cancer Institute P50CA092131 UCLA SPORE in Prostate Cancer, and R01CA174294 Multifunctional ImmunoPET Tracers for Pancreatic and Prostate Cancer

Robert E. Reiter, MD, MBA Department of Urology David Geffen School of Medicine at UCLA 300 Stein Plaza Los Angeles, CA 90095 Phone: 310-794-7224 Fax: 310-206-5343 E-mail: [email protected] Conflicts of interest: All authors declare that they have no conflicts of interest. Word Count: 4228 Total Number of Figures: 4

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Translational Relevance The ability to visualize cancer in real-time using molecularly targeted fluorescent probes has the

potential to transform the modern practice of cancer surgery. In the case of prostate cancer,

the inability to differentiate cancer from normal surrounding structures contributes both to

incomplete cancer removal and surgical side effects. Technology for fluorescence imaging in

humans during robotic surgery is commercially available, but its application is limited by the

lack of prostate-specific optical probes. We report the development and validation of a novel

targeted optical imaging probe using an antibody fragment against a cell surface marker of

prostate cancer. Such probes will be useful for maximizing resection of primary and metastatic

cancers while minimizing damage to critical adjacent tissues.




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Abstract Purpose: The inability to visualize cancer during prostatectomy contributes to positive margins,

cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the

potential for real-time intra-operative imaging. The goal of this study was to develop a probe

for image-guided prostate cancer surgery.

Experimental Design: An antibody fragment (cys-diabody, cDb) against prostate stem cell

antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the

probe to PSCA was confirmed by gel electrophoresis, size exclusion and flow cytometry,

respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the

biodistribution and in vivo kinetics, while an invasive intramuscular model was utilized to

explore the performance of Cy5-cDb-mediated fluorescence guidance in representative surgical

scenarios. Finally, a prospective, randomized study comparing surgical resection with and

without fluorescent guidance was performed to determine if this probe could reduce the

incidence of positive margins.

Results: Cy5-cDb demonstrated excellent purity, stability and specific binding to PSCA. In vivo

imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA+ and -

dual xenografts, the mean fluorescence ratio of PSCA+/- tumors was 4.4:1. In surgical resection

experiments, residual tumors <1mm that were missed on white light surgery were identified

and resected using fluorescence guidance, which reduced the incidence of positive surgical

margins (0/8) compared to white light surgery alone (7/7).

Conclusions: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer

xenografts in mice, and facilitates more complete tumor removal than conventional white light

surgery alone.




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Introduction Radical prostatectomy remains the most common definitive treatment option for the >250,000

men newly diagnosed with localized prostate cancer each year (1), with up to 85% of all

prostatectomies in the United States performed robotically (2-4). Early extracapsular extension

of prostate cancer is rarely visible during prostatectomy, and proximity of the prostate to the

rectum, urinary sphincter, and erectile nerves precludes wide local excision. As such, positive

surgical margin rates range from 6.5-32% in contemporary series (5), and are directly correlated

with poor cancer control (6, 7). The posterolateral prostate and prostatic apex remain the most

common sites for positive surgical margins (8), which are intimately associated with the

neurovascular bundle controlling erectile function and urinary sphincter, respectively. As

urinary incontinence and sexual dysfunction remain major issues postoperatively (9), surgeon

reluctance to remove healthy tissue in an attempt to reduce these side effects likely account for

these higher rates. The ability to visualize small foci of extracapsular extension of prostate

cancer at the time of surgery may reduce the incidence of positive surgical margins while

reducing damage to critical adjacent structures.

Several research teams have endeavored to develop cancer specific optical imaging agents that

use fluorescence to highlight cancer in real-time during surgery. Intraprostatic free Indocyanin

Green (ICG) has been utilized clinically in prostatectomy as a lymphangiographic agent in the

detection of sentinel lymph nodes and for delineation of prostate by limited diffusion (9, 10).

However, use of free ICG is limited by the lack of biochemical specificity to prostate or prostate

cancer, and suffers from dye spillage from handling or manipulating fluorescent tissue. Cancer

specific optical agents that overcome these limitations are currently being developed by various

groups in the pre-clinical stage. These include activatable cell-penetrating peptides (ACPPs) that

are specifically activated by metalloproteinases expressed in a number of solid tumors (11, 12),

as well as prostate specific membrane antigen (PSMA) targeted small molecules (13), ligands

(14), and intact antibodies labeled with fluorescent dyes (15-17).




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While the exquisite specificity of antibodies offers great promise in the targeted delivery of

fluorescent dyes to cancer cells in vivo, the large size of intact antibodies (~155 kDa) results in

slow tumor penetration and long circulating half-life. The use of antibody fragments enables

preservation of cancer specificity with faster tumor targeting kinetics and potentially greater

intratumoral diffusion (18, 19). Indeed, the rapid penetration of the fragments may enable

imaging within 2 hours of injection (20). Recombinant humanized antibody fragments

(minibodies and diabodies) have been developed that retain their specificity to prostate cancer

and have been used successfully to image soft tissue and bone metastases using positron

emission tomography (PET) in both transgenic and xenograft models (21, 22).

In the present study, we employed diabodies that target the prostate stem cell antigen (PSCA),

a glycosyl phosphatidylinositol-anchored glycoprotein that is expressed on the cell surface of

virtually all prostate cancers with minimal background in normal prostate or surrounding

tissues (23). Higher levels of PSCA have been correlated with poor prognosis and metastatic

disease (24, 25). PSCA is also upregulated in a substantial percentage of bladder and pancreatic

cancers (26, 27). It is both a promising therapeutic and imaging target (21, 28). Therapeutic

monoclonal antibodies against PSCA have been evaluated in Phase I and II clinical trials. A

clinical trial using an engineered humanized anti-PSCA minibody has recently begun patient

enrollment (29).

With a goal of offering molecular imaging-guided surgery to men undergoing prostatectomy,

we employed a humanized anti-PSCA cys-diabody fragment, A2, and site-specifically labeled it

with a far red fluorescent dye, Cy5, via maleimide chemistry. The purity and specificity of the

probe was evaluated in vitro. Optimal in vivo imaging parameters were determined by imaging

human prostate cancer xenograft-bearing mice. We performed real-time fluorescently guided

surgery to remove invasive mouse xenografts and elucidate the potential clinical utility of this

probe in detecting small foci of residual prostate cancer. We also performed a prospective

randomized study to assess the ability of fluorescently guided surgery to reduce positive

surgical margins using an intramuscular model that yields difficult to resect tumors.




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Materials and Methods Reagents

The 2B3 A2 cys-diabody, (cDb, 50 kDa) was developed and validated for preclinical in vivo

targeting of PSCA at UCLA (30). It was derived by yeast affinity maturation of a humanized

monoclonal anti-PSCA antibody, 2B3, and engineered to contain a C-terminal free cysteine that

forms an inter-chain disulfide bond stabilizing dimerization. Upon mild reduction this disulfide

bond can be broken and free thiols are available for site-specific labeling away from the antigen

binding site using e.g. maleimide chemistry. A2 cDb was purified from mammalian cell culture

supernatant using immobilized metal affinity chromatography. Protein concentrations were

determined photometrically and purity was analyzed by SDS-PAGE. Detailed biodistribution

data for the A2 cDb was previously determined (21). Non-specific binding was not seen.

Fluorescent signals were present in liver, kidney and bladder due to the metabolism and urinary

excretion of the probe. Cy5 Maleimide (649 nm absorbance, 670 nm emission) was purchased

from GE Healthcare (Piscataway, NJ).

Synthesis of Cy5-cDb probe

To achieve optimal conjugation efficiencies, the diabody was first concentrated using an

Amicon® Ultra-0.5mL (10K) Centrifugal Filter Device (Millipore, Carrigtwohill, County Cork,

Ireland) to a concentration greater than 2.8 mg/mL. Then, 50 μM diabody was reduced in 40-

fold molar excess of TCEP for 2 hours at room temperature. A 20-fold molar excess of Cy5

Maleimide dissolved in dimethylformamide was then added to the reduced diabody and the

mixture was incubated for 2 hours at room temperature. After incubation, excess dye was

removed using a 2 mL Zeba Desalt Spin Column (Thermo Scientific). Cy5 and diabody

concentrations were then measured using a spectrophotometer at 650 nm and 280 nm,

respectively. The ratio of Cy5 to diabody was calculated to confirm the number of fluorophore

molecules conjugated to each diabody molecule.

Size Exclusion




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Size exclusion chromatography (SEC) was performed using a Superdex 75 HR 10/30 column (GE

Healthcare Life Sciences) on an AKTA Purifier and PBS as mobile phase at a flow rate of 0.5

mL/minute. Both A280 for protein detection and A650 for fluorophore detection were monitored

during elution. Retention time was compared to following standard proteins: bovine serum

albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) (Sigma-Aldrich,

Saint Louis, MO, USA).

Cell culture

CWR22Rv1 cells that express minimal levels of endogenous PSCA were obtained from American

Type Culture Collection and cultured in RPMI 1640 medium containing 10% fetal bovine serum

(FBS), 1X sodium pyruvate and 1% Penicillin-Streptomycin-Glutamine (PSG). A PSCA expressing

lentivirus was used to transduce these cells to generate a 22Rv1-PSCA+ line, as previously

described (31). Quantitative flow cytometry with the murine 1G8 anti-PSCA antibody previously

showed little or no expression of PSCA on 22Rv1 cells, with 2.2 × 106 PSCA antigens on 22Rv1-

PSCA+ cells (31). LAPC-9 cells that endogenously express PSCA were passaged in vivo and

explanted tumors were processed into single cell suspension before injection into nude mice as

described previously (28).

Flow cytometry analysis

Both parental and PSCA+ 22Rv1 cells were dissociated with glucose-Ethylenediaminetetraacetic

acid (EDTA) and stained with the Cy5-cDb probe (1 µg per 1×106 cells) on ice for 1 hour followed

by 3x washes with PBS+2% FBS. A murine anti-human PSCA antibody, 1G8 (28), was used as

positive control followed by Alexa Fluor 647-goat-anti-mouse IgG as secondary antibody

(Invitrogen). Fluorescence signal was acquired and analyzed using a FACSCalibur flow cytometer

(BD Biosciences, San Jose, CA).

Measurement of affinity using an Attana Cell 200TM C-Fast system

The antigen, human PSCA-mouseFc fusion protein, was immobilized on an LNB-carboxyl sensor

chip by amine coupling at a concentration of 50 µg/ml according to the manufacturer’s protocol,




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resulting in a frequency shift of 200 Hz. Binding experiments were performed with HBS-T (10

mM HEPES, 150 mM NaCl, 0.005% Tween20, pH 7.4) as running buffer and the temperature

was controlled at 22°C. Varying concentrations of the antibody fragments (300 – 3 nM,

quadruplicates) were analyzed for binding and the chip surface was regenerated using 10 mM

glycine pH 2.5 between each sample. Buffer injections prior to each sample were used as

internal reference and an activated/deactivated (no antigen) surface chip was used for external

reference. Data were collected and analyzed using the Attana Attaché software and the binding

curves were fit using a mass transport limited binding model.

Xenograft models

All procedures were approved by the UCLA Animal Research Committee. 5- to 8- week old male

athymic nude mice were used for all experiments. Mice were fed irradiated, alfalfa-free food

(Harlan Laboratories, Indianapolis, IN) to reduce non-specific fluorescent signal. For

subcutaneous models, 1.5×106 22Rv1 or LAPC-9 cells were mixed with Cultrex (1:1, v/v,

Trevigen®, Inc) and injected into the subcutaneous space overlying the shoulder on the dorsal

surface of the mice. Particularly, in 22Rv1 experiments, the parental and the PSCA-

overexpressing lines were implanted in the same mice, with the parental tumor on the left

shoulder and the PSCA+ tumor on the right. When combined tumor size approached 15 mm, in

vivo imaging was performed (typically 10-14 days after injection of 22Rv1, 21 days after

injection of LAPC-9) and animals euthanized. For the intramuscular model, cells were injected

into either the bilateral flank muscles (in the preliminary surgical resection experiments) or into

the right thigh muscles (in the prospective randomized study).

In vivo imaging

Imaging was performed using the IVIS 200 In Vivo Imaging System (Xenogen). Cy5 was excited

using a 615-665 nm bandpass filter and emission was measured at 695-770 nm. These settings

are optimized for the dye Cy5.5, which has peak absorbance and emission at wavelengths

approximately 25 nm longer than Cy5. Exposure time was 1 second and binning was set at

medium. Prior to imaging, the probe was prepared by mixing the Cy5-cDb, 4 µL of 10% human




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serum albumin , and sterile normal saline to bring the injected volume to 100 µL. 25 µg or

indicated doses of diabody were used in imaging experiments. The probe was intravenously

injected via the lateral tail vein. Animals were anesthetized with 2% isoflurane during imaging

sessions. Imaging was performed at 6 hours or indicated time points after probe administration.

When multiple timepoints were used, animals were allowed to awaken after imaging, and then

re-anesthetized for each time point. Following the final time point for each mouse, mice were

sacrificed by isoflurane overdose and cervical dislocation. The skin was then removed and the

mice were again imaged with the whole-body mouse imaging system. Regions of Interest (ROI)

were delineated over the tumor sites with reference to white light. Maximal fluorescence

intensity in the ROIs was analyzed using Living Image® software. In mice with 2 tumors (22Rv1

experiments), the fluorescence intensity of the PSCA+ tumor was compared to the PSCA- tumor.

In mice with 1 tumor (LAPC-9 experiment) the fluorescence intensity of the tumor was

compared to the body (background).

Surgery

For all surgery experiments, 25 µg of Cy5-cDb was injected intravenously on the day of

operation. Mice were imaged in vivo on the IVIS at 5 hours to confirm fluorescence detection in

the tumor. Surgery was performed at 6 hours. Animals were sacrificed immediately before

surgery in accordance with our Animal Research Committee protocol to reduce unnecessary

duress. Tumors were resected using a fluorescence dissecting microscope (Leica M205 FA, Leica

Microsystems, Buffalo Grove, IL) at 10× magnification under white light or with excitation and

emission parameters for Cy5 (Excitation 620/60 nm and Emission 700/75 nm). Images of the

fluorescence signal were captured with a Leica DFC360 FX monochrome camera and displayed

on an adjacent computer monitor. After surgery, any residual fluorescent tissue and/or tumor

margins were surgically collected, fixed in formalin and sent to the UCLA pathology core facility

for hematoxylin and eosin (H&E) staining and histological analysis. In the feasibility experiment,

a margin of remaining non-fluorescent tissue was also removed and processed as control.




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Results Development and characterization of Cy5-cys-Db

The engineered anti-PSCA cDb contains a C-terminal cysteine residue that can be reduced to

enable site specific labeling (Figure 1A) (32). After reduction, the dimeric diabody is held

together solely by non-covalent inter-chain interactions. Therefore, completely reduced

diabody migrates on SDS-PAGE as a 25 kDa monomer band, whereas the non-reduced diabody

migrates as a 50 kDa band (Figure S1A). Following reduction, the cysteine moiety is available for

binding to the Cy5 dye via maleimide chemistry (Figure 1A). We investigated a variety of

different concentrations of dye to maximize site-specific conjugation. Optimal conjugation was

obtained using a 15-fold molar excess of Cy5 relative to the cys-diabody (Figure S1B). To assess

reproducibility of the diabody-Cy5 conjugation, the dye:protein ratio was checked (n=8). The

mean dye:protein ratio was 1.31 (SD = 0.04). To evaluate purity and integrity of the conjugate, a

size exclusion experiment was performed. This demonstrated good purity (major peak

representing a Cy5-cDb molecule) with no sign of protein aggregation and only a small amount

of unbound dye (late elution peaks) (Figure 1B). Both the unconjugated diabody and the Cy5-

cDb eluted with similar retention times (22.4 and 22.5 min, respectively) indicating that

reduction of the cys-diabody and conjugation of the Cy5 did not interfere with dimer

maintenance.

To determine if the Cy5-cDb conjugate maintains its binding to PSCA in vitro, a series of flow

cytometry experiments were performed using PSCA over-expressing 22Rv1 cells (Figure 1C).

Specific binding of Cy5-cDb to PSCA was demonstrated, although a low level of binding to

22Rv1-PSCA- cells was also observed due to low endogenous levels of PSCA expression. We

further confirmed these results in an endogenous PSCA-expressing pancreatic cell line, Capan-1

(data not shown). Furthermore, to investigate whether Cy5 labeling had affected the binding

affinity quantitatively, quartz crystal microbalance (QCM) measurements were conducted using

an Attana Cell 200TM C-Fast system to assess the interaction of Cy5-cDb to recombinant PSCA

protein, which was immobilized on an LNB carboxyl sensor chip, in comparison to the mock-

treated cDb control. As shown in Figure 1D, both Cy5-cDb (0.42 nM) and mock-labeled cDb




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(1.96 nM) had apparent affinity in the low nanomolar range confirming that reducing the C-

terminal disulfide bridge and site-specific conjugation of maleimide-Cy5 did not impair binding

of the cys-diabody to PSCA. Small differences in KD might be attributed to minor inaccuracy in

protein concentration due to the presence of BSA in the samples, which was required during

the purification procedure to stabilize the diabody.

In vivo tumor imaging

To determine if the Cy5-cDb probe binds to PSCA-expressing tumors in vivo, we first performed

imaging experiments with subcutaneous xenografts. Four mice, each bearing PSCA+ and PSCA-

22RV1 xenografts, were imaged on the IVIS 200 (Excitation 615-665nm, Emission 695-770nm)

at 6 hours after intravenous injection of 25 µg of the probe. This revealed fluorescence in the

PSCA positive tumor and lack of fluorescence in the non-PSCA expressing tumor (Figure 2A-B).

Strong auto-fluorescence was detected from the skin and intestines at this wavelength, and

fluorescence was also identified in the kidneys due to renal excretion of the probe. We

repeated the experiment using the endogenously PSCA expressing LAPC-9 xenograft in order to

rule out any model-specific effects. We consistently observed positive fluorescent signal only in

PSCA-positive tumors, with a mean tumor-to-muscle ratio of 4.48 (SD 0.52; Figure S2). Further,

to confirm that the positive signal observed in PSCA positive tumors was attributable to Cy5-

cDb and not preferential uptake of free Cy5 dye, we imaged mice bearing both PSCA+ and

PSCA- 22Rv1 xenografts with 5 µg of free Cy5. At 4 hours after injection, PSCA- tumors (n=4)

exhibited greater fluorescence than their PSCA+ counterparts. Mean PSCA+ to PSCA- ratio was

0.55 (SD 0.18; data not shown).

Dose Determination. To determine the optimal dose for in vivo imaging, mice bearing PSCA+

and PSCA- 22Rv1 xenografts were injected with 2.4 µg, 12 µg and 25 µg (n=5) of the probe and

imaged at 2, 4, and 6 hours using the IVIS 200. PSCA+ tumors fluoresced more than PSCA-

tumors at each dose. Post mortem PSCA+ to PSCA- ratios at 6 hours were 1.58 (SD 0.52), 2.43

(SD 0.58), and 4.47 (SD 1.63) for the 2.4 µg dose, 12 µg dose, and 25 µg dose, respectively




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(Figure 2C). Based upon this result, subsequent imaging experiments were performed using the

25 µg dose.

Optimal time for imaging. To evaluate the optimal interval from probe injection to in vivo

imaging, mice with PSCA+ and PSCA- 22Rv1 xenografts were administered 25 µg of Cy5-cDb and

serially imaged at 1, 2, 4, 6, 8, and 24 hours (n=5). Maximum fluorescence intensity and ratios

from PSCA+ and PSCA- tumors were determined at all time-points (Figure 2D-F). Maximal PSCA

positive to negative ratio and fluorescence intensity were reached at 6-hours post injection

(Figure 2E-F). To remove interference from skin auto-fluorescence, the experiment was

repeated at the 2, 6 and 24 hour time-points without skin overlying the tumor. Again, peak

PSCA+ to PSCA- fluorescence intensity ratio was achieved at 6 hours. The post mortem ratios

were 1.94 (SD 0.66) at 2 hours, 4.04 (SD 1.52) at 6 hours, and 3.53 (SD 1.58) at 24 hours

(p=0.06) (Figure S3).

Surgical resection of tumors under fluorescence guidance

In order to assess the feasibility and utility of optical imaging to aid complete tumor resection,

we established intramuscular xenografts of PSCA+ 22RV1 (n=7; Figure 3 and S4) and LAPC-9

(n=3; Figure S5), in which tumors are invasive and therefore difficult to resect under white light

alone. Upon tumor establishment, 25 µg of Cy5-cDb were administered intravenously and

surgery was performed six hours later. We first made a skin incision and exposed the surface of

thigh muscle (Figure 3A). We attempted to resect the tumor completely under white light alone

(Figure 3B, left). Next, the fluorescent mode was turned on to evaluate for residual signal

(Figure 3B, right). Areas with residual fluorescence were surgically resected, fixed and

examined histologically to confirm that the fluorescence was indeed coming from cancer cells

(Figures 3C). A final fluorescent image was taken after the secondary surgery (Figure 3D) and

tissues from the tumor bed were collected and examined histologically to confirm the absence

of residual tumor. As demonstrated in Figure 3A, these tumors were often indistinct compared

to muscle, and the extent and margins difficult to readily identify under white light. Of the 10




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tumors, 8 had post-resection sites (six 22Rv1 and two LAPC-9) that had detectable residual

fluorescence following white light surgery, some areas as small as <1-2 mm. In some cases,

there were multiple sites of residual fluorescence adjacent to a single tumor. Histology of

residual fluorescent foci was positive for cancer in all 8 cases (exemplified by Figure 3B and C).

Prospective comparison of surgical margins following resection with or without the aid of

fluorescence

In order to demonstrate the ability of fluorescent surgery to reduce positive surgical margin

rates in a controlled, objective manner, we next performed a prospective randomized study

comparing white light vs. white light and fluorescent surgery. As shown in Figure 4A, 17 nude

mice received intramuscular implantation of PSCA+ 22Rv1 xenografts on their thighs 21-22 days

before the i.v. injection of Cy5-cDb. Mice were then randomized into two cohorts to receive

either just a white light surgery or an additional fluorescence guided surgery. The surgeon was

blinded to the groupings while performing the first-round white light surgery in order to insure

that all 17 mice received the same unbiased operation, with the goal of removing as much

tumor as possible while preserving adjacent normal tissues (akin to radical prostatectomy). As

expected, the xenografts were invasive into the thigh musculature and demonstrated strong

fluorescence and contrast with adjacent nerves and normal tissue (Figure S6). Following first

stage white light resection, the fluorescent light was turned on to assess margin status; all mice

(n=17) had residual fluorescent signals (Figure 4B). At this point, mice randomized to the

fluorescence cohort underwent secondary surgery to remove residual fluorescing tissue, which

was then subjected to histological staining and analysis. Consistent with the previous pilot study,

all remaining fluorescent tissues collected from the 9 animals in this cohort contained residual

cancer (Figure 4C). Lastly, to determine final margin status, remaining thigh musculature was

harvested and examined for the presence of prostate cancer with the aid of an expert uro-

pathologist. Positive surgical margins were found in 8/8 mice in the white light only cohort but

0/9 mice assigned to fluorescent-image guided surgery (Figure 4D and Figure S7).




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Discussion The inability to visualize cancer during surgery leads to incomplete cancer removal and surgical

side effects. This is especially true in prostatectomy, where small foci of extracapsular extension

of prostate cancer can easily be overlooked, leading to positive surgical margins and

compromised onocologic control. This encourages surgeons to resect healthy tissue in an

attempt at good oncologic control, leading to substantial urinary and sexual side effects. The

ideal imaging agent is one that is specific to cancer (or the cancer-bearing organ where the

entire organ is to be extirpated), safe, sensitive enough to detect small amounts of residual

tumor, and has a half-life to allow visualization throughout the critical span of the operation. To

date, no such optical probe is clinically available.

Using an antibody fragment labeled with a fluorescent dye, we developed and validated an

optical imaging probe (Cy5-cDb) that specifically recognizes prostate cancer xenografts

expressing the cell surface glycoprotein PSCA. The far-red fluorescent dye Cy5 was chosen

because of the stability and ease of conjugation of cyanine dyes, the feasibility of performing

site-specific labeling of our cys-diabody, the lack of affinity change with Db-Cy5 conjugation,

and the availability of equipment for whole-body and microscopic imaging during surgery. The

probe enabled fluorescent imaging in real-time to guide surgical resection of tumors in mice.

The small size of the diabody (50 kDa, below the threshold for renal clearance) enabled

administration and imaging on the same day, and persisted throughout the course of surgery.

In vitro and in vivo characterization of the Cy5-cDb probe demonstrated high affinity to

recombinant PSCA protein as well as excellent specificity for PSCA-expressing cells. When

injected intravenously into mice, optimal imaging was achieved within six hours, and in many

cases, strong signal to background was present within two hours of administration. This

compares favorably to a study by Nakajima et al. in which prostate cancer xenografts were

imaged using an anti-prostate specific membrane antigen (PSMA) intact antibody conjugated to

ICG, where time from administration to imaging was 2 days (16).




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When using the probe to guide surgical resection of tumors in mice, the signal was sufficiently

strong to be seen easily and used to guide surgery at a video-quality frame rate. Further, using

fluorescence, residual tumor fragments <1 mm in size were often identified after standard

surgical resection with white light.

We demonstrated the ability of real-time fluorescent-image guided surgery to significantly

reduce positive surgical margin rates when compared to white light surgery alone in an

intramuscular model of prostate cancer. An intramuscular model was chosen to provide a

scenario in which complete removal of cancer would present a challenge for the surgeon under

white light, examining the hypothesis that our diabody probe can aid in identifying and

completely resecting small foci of residual cancer following white light surgery. While no small

animal model captures the complexities of radical prostatectomy, we believe our findings

highlight the potential utility in identifying small foci of extracapsular extension of cancer during

prostatectomy that would otherwise be missed. We envision this can play a role in the

posterolateral prostate during nerve sparing, and may aid in the apical dissection, which lacks

distinct capsule and surgical planes. Presence of fluorescence may prompt intraoperative frozen

sections when the exact extent of cancer is unclear in vital areas such as the neurovascular

bundle (33, 34).

Several other research groups have investigated similar strategies of targeted fluorescent

molecular imaging. Jiang et al. imaged tumor xenografts that overexpress matrix

metalloproteinases 2 and 9 using activatable cell penetrating peptides (ACPP) that are cleaved

by the MMPs, thereby activating fluorescence (11). The group injected their ACPPs

intravenously into mice bearing isografts two days prior to imaging, then resected the tumors

and demonstrated improved survival in mice whose tumors were removed using fluorescence

guidance (12). The ability of the ACPP to image prostate cancer has yet to be investigated.

Nakajima et al. developed an activatable antibody-fluorophore conjugate made of a humanized

anti-PSMA antibody (J591) linked to indocyanine green (ICG). They used this to perform in vivo

imaging of prostate cancer xenografts expressing PSMA (16). As with the study using ACPPs,




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this method is limited by the ≥2 days required from administration to imaging. The use of this

probe to guide surgical resection has not been published. The strategy of using an antibody

fragment to shorten the interval from administration to imaging was employed by Oliveira et al.

They used an anti-epidermal growth factor receptor (EGFR) antibody fragment (7D12

nanobody) labeled with the NIR dye IRDye800CW. This enabled tumor visualization as early as

30 minutes post-injection (20). Use of this probe in surgical resection has not been reported.

More recently, Neuman et al. reported the use of a low-molecular-weight NIR fluorescent agent

(YC-27) that targets PSMA in preclinical models of prostate cancer (13, 35). Survival surgery in a

mouse subcutaneous xenograft model comparing white light to real-time fluorescent was

performed 20 hours post injection, with no recurrence in the fluorescent group (0/8).

Investigators noted adequate tumor contrast with YC-27 within 6 hours of administration.

Although to date clinical studies of fluorescently-labeled prostate probes have not been

reported, Maurer et al recently described the use of a hand-held gamma camera to detect

signal from a gallium-labeled PSMA PET probe in metastatic lymph nodes intra-operatively (36).

One would predict that use of an optical label would improve the sensitivity of detection.

Consistent with this hypothesis, we are developing dual PET/optical tracers to enable PET

imaging followed by fluorescently guided-surgery.

While the results using our probe to resect tumors in mice are promising, several limitations are

acknowledged. First, imaging and surgery were performed in xenograft tumor models using

human prostate cancer cell lines. The humanized diabody does not recognize mouse PSCA.

Therefore, evaluating the signal to background that will be achieved for cancer surgery in

humans will require use of genetically engineered PSCA knock-in mouse models or an antibody

fragment capable of recognizing both human and murine PSCA. Second, while this proof of

concept study demonstrates the ability to find and resect small foci of residual cancer following

white light surgery and significantly reduce positive surgical margins, no small animal model

exists that recapitulates the complexities of prostatectomy, and as such the extent to which this

improves surgical outcomes will have to be determined clinically (12). Third, the use of a

fluorescent probe to improve detection and resection of lymph node metastasis is an exciting




17

potential application. We have yet to investigate the feasibility of this application by evaluating

lymph node imaging with our probe. Fourth, auto-fluorescence at the emission wavelength of

Cy5 is greater than that for near-infrared (NIR) dyes (37). This is particularly apparent in skin

and the gastrointestinal tract, although it can be reduced by dietary changes (38). We

acknowledge that the current trend for imaging applications is towards NIR dyes. Cy5 was

chosen given the ease of conjugation of cyanine dyes and the feasibility of performing site-

specific binding to our cys-diabody. In addition, Cy5 is compatible with commonly available light

sources and CCD cameras at our facility without significant alterations. We are currently

developing ICG and IR-800 labeled diabodies. Fifth, the differential fluorescence between

benign human prostate and prostate cancer has yet to be evaluated.

Notwithstanding these limitations, the development of novel targeted fluorescent probes has

tremendous translational potential. Optical probes are relatively easy to produce and imaging

technology is relatively inexpensive (20). In fact, a fluorescent camera is already available for

the Da Vinci robot (Intuitive Surgical, Sunnyvale, CA), the surgical system used to perform the

overwhelming majority of robotic prostatectomies in the world. To make use of this technology,

a method to selectively deliver the fluorescent dye to cancer cells is required. The Cy5-cDb

probe developed herein enables targeted detection and resection of cancers expressing PSCA

using an antibody fragment that enables same day administration and imaging. Efforts to

translate this probe into the clinic are ongoing.

Conclusion We successfully synthesized a monoclonal antibody fragment-fluorophore conjugate consisting

of a humanized anti-prostate stem cell antigen (PSCA) diabody linked to the fluorescent dye Cy5

that enables same day administration and in vivo imaging. This probe was used successfully to

resect residual cancer fragments in mice under real-time fluorescent guidance, and

demonstrated a significant reduction in positive surgical margins compared to white-light

surgery alone.




18

Acknowledgments We would like to thank Vadim Goldshteyn for his expert technical assistance with the study. In vivo fluorescence imaging was performed at the California NanoSystems Institute Advanced Light Microscopy/Spectroscopy and the Macro-Scale Imaging Shared Facilities at UCLA.

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Diabody. (A) Schematic of the conjugation reaction. Anti-PSCA cys-Diabody was first reduced by

TCEP to open up the inter-chain disulfide bond, revealing cysteine residues that can then be

conjugated to Cy5 via maleimide chemistry. (B) Size exclusion chromatography of purified

A2_cDb and Cy5-labeled A2_cDb. Retention time of standard proteins is indicated. (C) Flow

cytometry analysis comparing binding of Cy5-cDb to 22Rv1 cells that over express PSCA (left)

and the parental line (right) that expresses very low level of PSCA. Strong binding of Cy5-cDb to

PSCA (orange line) was evident compared to the positive control (red line). In the right panel, a

small amount of binding to the PSCA- 22Rv1 cells (orange line) was also present. (D) Quartz

crystal microbalance (QCM) measurements of the interaction between mock labeled cDb or




21

Cy5-cDb and recombinant PSCA. The table summarizes the on and off rates and Bmax for

indicated samples. Mock-treated cDb had an apparent affinity of 1.96 nM and Cy5-cDb 0.42 nM,

both in the low nanomolar range.

Figure 2. Determination of the optimal imaging parameters for in vivo detection of PSCA-

expressing tumors using Cy5-cDb. Fluorescent signal intensity increases from dark red to

yellow. (A-B) A mouse bearing 22rv1 PSCA+ (right shoulder) and PSCA- (left shoulder) tumors

was photographed (A) and fluorescent imaged (B) 6 hours post i.v. administration of the Cy5-

cDb probe. The skin was removed to avoid interference from autofluorescence. PSCA-specific

fluorescent signals were detected on the right shoulder. Background signal is present in the skin

and kidneys (probe excretion). (C) Dose determination. 2.4 μg, 12 μg, or 25 μg of Cy5-cDb was

i.v. administrated to PSCA (+) and (-) 22rv1 tumor bearing-mice (shown are 5 animals in each

group). Fluorescent imaging was performed at 6 hours after skin removal. Red arrow: PSCA (+)

tumor; green arrow: PSCA (-) tumor. Mean ratios of PSCA +/- tumors in each dose were also

indicated. (D-F) Timing determination. Five PSCA (+) and (-) 22rv1 tumor-bearing mice received

25 μg of Cy5-cDb intravenously and were then imaged at indicated time points. (D) Serial

images of a representative animal over time. Red arrow: PSCA (+) tumor; green arrow: PSCA (-)

tumor; yellow arrow: background signal from stomach. (E) Maximum fluorescence intensity

over time determined by measurement of respective PSCA (+) and (-) tumor regions of interest.

(F) PSCA (+) to PSCA (-) ratios over time calculated based on (E). As the skin was not removed

for (E) and (F), the levels and ratios shown will underestimate the true +/- ratio, though

provides the optimal time point for imaging.

Figure 3. Cy5-cDb enabled fluorescence-guided surgical resection of PSCA-expressing tumors.

(A) White light and fluorescent image of 22Rv1 PSCA (+) intramuscular tumor (arrow) prior to

resection. (B) Fluorescent image after white light surgical resection shows residual cancer that

is not clearly seen on white light. (C) H&E of residual fluorescent tissue from (B) demonstrating

tumor (*) and normal adjacent muscle. (D) Fluorescent image showing absence of signal

following fluorescence-guided surgical resection. H&E staining and systematic sectioning of

tissue from this area confirmed absence of tumor.




22

Figure 4. Cy5-cDb directed fluorescence guided surgery enabled complete removal of

infiltrative intramuscular prostate cancer. (A) Schematics of the study design. (B, C)

Representative white light (B) and fluorescent (C) images of a tumor bed after white light only

surgery revealing tumor that would otherwise have been missed. (D) The residual fluorescent

tissue from (B, C) proved to be cancer (*) by H&E staining (10X magnification). (E)

Representative H&E staining of tumor margins from the white light surgery alone (top panel;

with residual tumors [*]) and the fluorescence-guided surgery cohorts (bottom panel; no

residual tumors).




Cys-Diabody

TCEP

S

H

S

H

Cy5-

maleimide

S

|

Cy5

S

|

Cy5

A

22Rv1xPSCA 22Rv1 (PSCA neg)

Cells Only (negative control)

Cells + 2° Ab (negative control)

Cells + 1° and 2° Ab (positive control)

Cells + Cy5-cDb probe

C

B

Figure 1.

D Mock labeled cDb Cy5-cDb

kon (M-1s-1) koff (s-1) Bmax KD (nM)

Mock cDb 1.81 x 105 3.55 x 10-4 20.0 1.96

Cy5-cDb 2.16 x 105 9.05 x 10-5 19.1 0.42

Cy5-cDb cDb




2.4 μg Db Dose – Mean Ratio 1.58

12 μg Db Dose – Mean Ratio 2.43

25 μg Db Dose – Mean Ratio 4.47

0.00

0.50

1.00

1.50

2.00

1hr 2hr 4hr 6hr 8hr 23hr

PSCA+/- Tumor Ratio Over Time

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

1.20E+09

1hr 2hr 4hr 6hr 8hr 23hr

Maximum Fluorescence Intensity

PSCA+

PSCA-

1 hour 2 hour 4 hour 6 hour 8 hour 24 hour

C

D

E

F

Figure 2.




White Light Image Fluorescent Image

Pre-resection

Post white-light resection

A

B

*

C D

Post fluorescent resection

Figure 3.




T

T Fully grown tumor

White light

surgery

T

T positive margin

T

N=9

Secondary

fluorescent

surgery

T negative margin

N=17 Margin

evaluation by

histology

T

N=9

N=8

A

C

*

*

*

*

D

E

B

*

Figure 4. Research.




Published OnlineFirst October 21, 2015.Clin Cancer Res Geoffrey A Sonn, Andrew S Behesnilian, Ziyue Karen Jiang, et al. Mouse Prostate Cancer Xenografts in Real-TimeCell Antigen (PSCA) Diabody Enables Targeted Resection of Fluorescent Image-Guided Surgery with an Anti-Prostate Stem

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Fluorescent Image-Guided Surgery wi th an Anti-Prostate ... · Prostate Cancer Xenografts in Real-Time Geoffrey A. Sonn*1, Andrew S. Behesnilian*1, ... Los Angeles, CA, USA 2Molecular

Fluorescent Image-Guided Surgery wi th an Anti-Prostate ... · Prostate Cancer Xenografts in Real-Time Geoffrey A. Sonn1, Andrew S. Behesnilian1, ... Los Angeles, CA, USA 2Molecular