Evaluating CD133 as a Radiotheranostic Target in Small-Cell Lung Cancer

Despite decades of work, small-cell lung cancer (SCLC) remains a frustratingly recalcitrant disease. Both diagnosis and treatment are challenges: low-dose computed tomography (the approved method used for lung cancer screening) is unable to reliably detect early SCLC, and the malignancy’s 5 year survival rate stands at a paltry 7%. Clearly, the development of novel diagnostic and therapeutic tools for SCLC is an urgent, unmet need. CD133 is a transmembrane protein that is expressed at low levels in normal tissue but is overexpressed by a variety of tumors, including SCLC. We previously explored CD133 as a biomarker for a novel autoantibody-to-immunopositron emission tomography (PET) strategy for the diagnosis of SCLC, work that first suggested the promise of the antigen as a radiotheranostic target in the disease. Herein, we report the in vivo validation of a pair of CD133-targeted radioimmunoconjugates for the PET imaging and radioimmunotherapy of SCLC. To this end, [89Zr]Zr-DFO-αCD133 was first interrogated in a trio of advanced murine models of SCLC—i.e., orthotopic, metastatic, and patient-derived xenografts—with the PET probe consistently producing high activity concentrations (>%ID/g) in tumor lesions combined with low uptake in healthy tissues. Subsequently, a variant of αCD133 labeled with the β-emitting radiometal 177Lu—[177Lu]Lu-DTPA-A″-CHX-αCD133—was synthesized and evaluated in a longitudinal therapy study in a subcutaneous xenograft model of SCLC, ultimately revealing that treatment with a dose of 9.6 MBq of the radioimmunoconjugate produced a significant increase in median survival compared to a control cohort. Taken together, these data establish CD133 as a viable target for the nuclear imaging and radiopharmaceutical therapy of SCLC.


Serum Stability Studies
The stabilities of the radioimmunoconjugates were interrogated by incubating [ 89 Zr]Zr-DFO-αCD133 or [ 177 Lu]Lu-DTPA-A"-CHX-αCD133 in human serum on a ThermoMixer at 500 rpm and 37 °C for 6 days.
Every 24 h, the radiochemical purity of the radioimmunoconjugates were determined in triplicate via radio-iTLC measurements with an eluent of 50 mM EDTA, pH 5.0.

Cell Saturation Assay with DFO-αCD133
The immunoreactivity of [ 89 Zr]Zr-DFO-αCD133 was determined using a cell saturation assay.Briefly, 15 × 10 6 H82 (CD133+) cells were washed 3´ with ice-cold media, centrifuged (600 g, 2 min), and the supernatant was discarded.Ice-cold media (200 μL) with 1 μL of [ 89 Zr]Zr-DFO-αCD133 (1 μg/mL in Chelex-PBS + 1% BSA, pH 7.4) was added to the cell pellet, mixed thoroughly, and allowed to incubate for 1 h.After the incubation period, the cells were centrifuged, and the supernatant was reserved.The cells were washed 2´ with ice-cold media and the supernatants were each reserved in a separate microcentrifuge tube.The samples were then measured on an 89 Zr-calibrated gamma counter, with the activities (counts/minute) background-and decay-corrected to the start of the run.The immunoreactivity was expressed as a percentage by comparing the activity remaining in the cells to the total activity (cells + supernatant + washes).For the blocking experiments, the assay was run identically, but 5 μg of unlabeled αCD133 were co-incubated with the cells along with [ 89 Zr]Zr-DFO-αCD133.

Bead-Based Assay with DTPA-A"-CHX-αCD133
For the DTPA-conjugated mAb, a bead-based immunoreactivity assay was used as described by Sharma, et al.. 1 Briefly, 20 µL of HisPur TM Ni-NTA magnetic beads were washed twice with PBS + 0.05% Tween-20 (PBS-T).After each wash, the tubes were placed on a magnetic rack and the supernatant was discarded.Next, 200 µL PBS-T + 10 µL CD133 antigen (0.1 mg/mL in Chelex-PBS + 1% BSA, pH 7.4) were added to the beads and the solutions were mixed thoroughly.The tubes were incubated for 15 min at room temperature on a rotating platform.Following incubation, the beads were washed twice with PBS-T.One cohort of beads did not receive any antigen to serve as a negative control.Next, 1 ng of [ 177 Lu]Lu-DTPA-A"-CHX-αCD133 (1 µg/mL in Chelex-PBS + 1% BSA, pH 7.4) was added.The samples were allowed to react for 30 min on a rotating platform at room temperature.Following incubation, the supernatants were collected.The beads were then washed twice with PBS-T, and the supernatants of each wash were collected.All samples were then measured on a 177 Lu-calibrated gamma counter, with the activities (counts/minute) background-and decaycorrected to the start of the run.The immunoreactivity was expressed as a percentage by comparing the activity remaining in the beads to the total activity (beads + supernatant + washes) (n = 3).Blocking studies were performed as described in the cell saturation assay above.

Surface Plasmon Resonance
The affinity of αCD133 for the CD133 antigen was measured using surface plasmon resonance experiments.Briefly, protein A was immobilized onto an activated carboxyl sensor using Nicoya OpenSPR kit as per the manufacturer's instructions.The mAb ¾ αCD133 or DFO-αCD133 diluted in running buffer (HBS + 0.05% P-20 + 0.1% BSA) ¾ was captured onto the protein A sensor (25 µg/mL over 300 s).A multicycle kinetics experiment was performed by flowing 1.23, 3.4, 11, 33, and 100 nM HER2 antigen solutions (prepared in running buffer) over the sensor for 300 s.Glycine HCl (10 mM, pH 1.5) was used as a regeneration solution between each antigen injection to strip the mAb from the protein A prior to the subsequent antigen concentration injection.Blank buffer runs were subtracted from the results and the kinetics were determined using TraceDrawer.

Preparation of Tissue Slides
From the Orthotopic Xenograft Model Following the final PET imaging timepoint, the orthotopic mice were sacrificed via CO2(g) asphyxiation.
The lungs were first perfused through the right ventricle of the heart with 3 mL PBS using a 28-gauge needle.
Then, the lungs were inflated with ~2 mL of a 50:50 formalin:OCT mixture via a 23-gauge needle inserted into the trachea.The inflated lungs were immediately submerged in formalin and allowed to incubate for 24 h.Afterwards, the lungs were washed with 20% sucrose overnight, and the left and right lungs were separated.
The left lungs and the inferior lobe of the right lungs were then embedded in a cryomold with OCT and immediately cryogenically frozen.The next day, 10 μm slices of the tissue were cut using a cryostat microtome and collected onto slides.

From the Metastatic Xenograft Model
Following the final PET imaging timepoint, the metastatic mice were sacrificed via CO2(g) asphyxiation.
A portion of the left liver lobe was removed from the mouse, rinsed with water, and dried thoroughly with a paper towel.The livers were then embedded in a cryomold with OCT and flash-frozen with dry ice.The next day, 10 μm slices of the tissue were cut using a cryostat microtome and collected onto slides.

Bioluminescence Imaging
To monitor the growth of the orthotopically and metastatically implanted H82-luc cells, bioluminescence images of the mice were collected using an IVIS Spectrum-CT instrument.To this end, 100 μL of 30 mg/mL firefly D-luciferin (IVISBrite Xenolight) in PBS was administered to the mice via an intraperitoneal injection.
Subsequently, the mice were anesthetized with 2% isoflurane/O2(g).At 15 min post-injection, the mice were imaged in the prone and lateral positions.All images were analyzed with Living Image ® .

Autoradiography of Tissue Slides
Following the protocols described in Preparation of Tissue Slides, the slides were placed into a cassette with a clean phosphor imager plate (FujiFilm Imaging Plate, BAS-MS) and the cassette was stored in the dark for 48 h.Following this, the radioactivity on the phosphor imager plate was scanned using Typhoon FLA 7000 instrumentation.

H&E Staining of Tissue Slides
Histology was performed on the tissue slides using a hematoxylin and eosin staining kit from Abcam (ab245880).The tissue slides were fixed with formalin prior to the staining; the lungs were fixed immediately after ex vivo extraction, and the flash-frozen liver slides were fixed in a Coplin jar prior to staining.To begin, the slides were rinsed with PBS for 3 min to remove the OCT and air dried.The slides were dipped in the hematoxylin stain (2 min for the lung tissue slides; 4 min for the liver tissue slides), followed by 3 washes in DI water.Then, the slides were dipped in bluing reagent for 10-15 s, followed by two DI water washes.After the second wash, the slides were quickly dipped in 100% ethanol and air dried.Finally, the slides were stained with the eosin (2 min for the lung tissue slides; 1 min for the liver tissue slides) and washed 3´ with 100% ethanol.After the slides had completely air dried, they were mounted and sealed with a coverslip.Images were acquired by the Memorial Sloan Kettering Cancer Center Imaging and Image Analysis Core.

Dosimetry
Human/murine organ time-integrated activity coefficients (TIACs units of h or MBq×h/MBq) for [ 177 Lu]Lu-DTPA-A"-CHX-αCD133 were estimated from the PET images of [ 89 Zr]Zr-DFO-αCD133 in mice bearing subcutaneous H82 xenografts (see PET Imaging methods in the main text).
The %ID/g murine organ uptake values were converted to standardized uptake values (SUVs; normalized by total body mass) for estimation of organ-level absorbed doses a murine computational phantom.The percentage of injected dose in phantom organ I, %IDI, was obtained from the equation below, which assumes SUV is independent of body mass: where SUVi is the measured standardized uptake value for mouse organ i, m I is the mass of corresponding phantom organ I, and mTB is the total phantom mass.The %IDI at each timepoint was subsequently multiplied by a corresponding radioactive decay factor.To obtain TIACs, the resultant activity-time curves were integrated by the trapezoidal method over interval spanning the time-of-injection to the last measured timepoint (144 h); beyond the last measured timepoint, clearance was assumed to occur via radioactive decay only, and the analytical expression for the integral was used.The TIAC for the rest-of-body was obtained by subtracting the individual organ TIACs from that of the total body.The TIAC for the total body was computed as t1/2/ln(2) (i.e.under the assumption that no biological excretion occurred), where t1/2 is the physical halflife of the radionuclide.
Using the derived TIACs, normal organ absorbed dose coefficients were computed for the 25 g reference mouse (MOBY phantom) using PARaDIM 1.0/PHITS version 3.20.To enable tumor absorbed dose estimates, the phantom was modified to include a 100 mm 3 tumor on the left flank.

Gel Electrophoresis
αCD133, DFO-αCD133, and degly DFO-αCD133 samples were prepared for SDS-PAGE according to the manufacturer's instructions (NuPAGE TM , ThermoFisher).Briefly, 2 µg of each antibody were reduced using 10´ reducing agent, denatured using 4´ LDS buffer, and diluted with DI water.The reduced antibody samples were then placed on a ThermoMixer at 85 ºC and 300 rpm for 15 min.Next, 20 µL of each sample was added to the wells of a Novex 4-12% SDS Page Gel, and the gel box was filled with NuPAGE TM MOPS running buffer.A Novex TM Sharp pre-stained protein ladder was also added on each side of the samples.The gel was allowed to run at 70 V for 2.5 hours and washed 3´ with DI water.Enough SimplyBlue TM SafeStain was added to cover the gel, and the gel was allowed to stain for 90 minutes on a shaker.Following staining, the gel was washed 3´ with DI water.Finally, the gel was imaged using a LI-COR Odyssey CLx instrument and analyzed using Image Studio TM Acquisition Software.

Flow Cytometry
2 × 10 6 H82 cells were aliquoted per sample and washed 3´ (650 rcf, 2.5 min) with ice-cold PBS.50 µL of either αCD133, DFO-αCD133, or a non-specific hIgG1 isotype control (6 µg/mL) were added to their respective tubes (n = 3).The cells were incubated on ice for 30 min and washed 3´ with ice-cold PBS.After the final wash, 50 µL of goat anti-human IgG Alexa Fluor TM 488 antibody (6 µg/mL) were added to the tubes and the samples were incubated for 30 min on ice.The samples were then washed 3´ with ice-cold PBS and the cell pellets were resuspended in FACS buffer (PBS + 0.05% FBS + 2 mM EDTA).Finally, all samples were measured using FACS Caliber instrumentation and the data was analyzed using FlowJo TM software.All samples were performed in triplicate.Cells that did not receive any primary or secondary antibody were used as a non-stained control.S2. unexpectedly died during the collection of the 120 h PET image and is not shown thereafter.These data correspond with that described in Table S2.Coronal slices are shown on the left, while MIPs are shown on the right.These data correspond with that shown in Table S3.S3.S3.

Figure S4 .
Figure S4.Representative PET MIPs of two mice bearing metastatic tumors in which the color and gain of the images have been adjusted to better illustrate the focal uptake of the radiotracer in the liver.The black arrows on the images (right) denote areas of significant radiotracer uptake, signifying the presence of metastatic SCLC lesions.

Table S5 .
Survival results for each of the mice in the longitudinal therapy study.