GREEN et al ASTATINE‐211 RIT ERADICATES DISSEMINATED LYMPHOMA SupplementalMethods:
Granta519‐fireflyluciferasecellline(Granta‐519Luc):
The luciferase expressing cell line was generated by retroviral transduction to stably
express firefly luciferase for in vivo bioluminescent imaging (BLI). In brief, Phoenix cells
wereutilizedtoproduceretrovirusencodingfireflyluciferase(FFLuc),neomycinresistance
(Neo), and the cell surface marker Thy 1.1 (rat CD90). Retroviral particles were
concentratedfromculturesupernatantsusingPEG‐it(SystemBiosciences#LV810A‐1)and
co‐incubated with Granta‐519 cells in the presence of the cationic polymer polybrene to
enhance transfection efficiency. The resultant Granta‐519 FFLuc cells were subjected to
antibiotic selection (G418 Sulfate, CalBiochem #345812) and sorted by fluorescence‐
activatedcellsortingforThy1.1expression(BD#554898,mouseanti‐ratThy1.1PE).This
populationofcellswasdesignatedGranta‐519Lucandcellswerefreshlythawedbeforeeach
experiment.
Bioluminescenceimaging:
BLIwasmeasuredbeginningonthedayoftumorchallengewithanIVISSpectrumimager
(PerkinElmer)atFHCRC.Priortoimaging,micewereadministeredani.p.injectionof150
mg/kg D‐luciferin (potassium salt diluted in PBS to 15 mg/mL, sterile filtered, aliquoted,
stored at ‐20°C, protected from light, BioSynth Chemistry and Biology, Cat#L8220), and
subsequentlyanesthetizedwithinhalationofisofluorane,2.5%.Acquisitionandanalysisof
images was performed using Living Image V4.3.1 software (Perkin Elmer). Images were
gated for region of interest that included the whole body of each mouse from both dorsal
andventralimages.DatawerecalibratedandreportedasTotalBLI(photons/sec).
Antibody‐B10labelingwith211At:
A 50‐100 µL solution of 500 mM sodium phosphate, pH 6.8 was combined with 95 µL of
1F5‐B10at5.2mg/mLinphosphatebufferedsaline(PBS).Tothis,50‐150µLof 211At(1.4‐
2.8mCiatneutralpHinwater)wasaddedfollowedby5µLof0.1mg/mLchloramine‐Tin
water. After 1 min at room temperature, the reaction was quenched by adding 5 µL of 1
mg/mLsodiummetabisulfiteinwater.TheentiremixturewasthenrunoveraPD‐10(G25)
columnandcollectedinPBS.
1 GREEN et al ASTATINE‐211 RIT ERADICATES DISSEMINATED LYMPHOMA Alphacameraimaging:
FemaleathymicnudemicewithsubcutaneousRamostumors(generatedusingtheprocess
described under “biodistribution studies” above) received either [211At]1F5‐B10 (210 µg;
100 µCi) or [211At]HB8181‐B10 (210 µg; 100 µCi)co‐injected with 400 µg of an irrelevant
IgG2a Ab (HB8181) to block non‐specific Fc receptor binding of 1F5. Serial 10‐µm
cryosections of tumors were processed for imaging, histology (H&E staining), and
immunohistochemistry (IHC; mCD31 & mCD34 for blood vessels; hCD20 for lymphoma
cells). Cryosectioned tissues were placed on the scintillator and images acquired. Pixel
intensityintheacquiredimagesislineartoradioactivity(numberofdecaysperunittime),
enablingquantificationofthe 211Atactivityintheimagedspecimens.Framesweredigitally
processed(e.g.noisereductionandresolutionrecovery)andimportedintoImageJsoftware
(version 1.39; NIH) where “regions of interest” were created to analyze the intra‐tumoral
variationsof 211Atactivity.Αlpha‐imagesweresuperimposedwithconsecutiveH&EorIHC
sections (to confirm location of tumor [hCD20] and vessels). Tumor radioactivity was
quantified using the direct proportionality between radioactive content and ‐camera
imagepixelintensity;inadditionabsorbeddosestodifferentregionswithinthetumorwere
estimated.
Supplementalfigure:
MAb
FigureS1.
Isothiocyanato‐phenethyl‐ureido‐closo‐decaborate(2‐), (B10‐NCS) is an amine‐reactive
bifunctionallabelingreagentcontainingaboroncagesuitableforstable 211At‐labeling.The
211Atatomisattachedtoaboronatom(likelyavertexatom)ontheboroncagemoietyas
depicted.Theinvivostabilityof 211Atlabeledcompoundsiscriticaltothesuccessofalpha
2 GREEN et al ASTATINE‐211 RIT ERADICATES DISSEMINATED LYMPHOMA particleRIT.Theinstabilityof 211At‐carbonbondsfoundinmanylabeledcompoundsleads
torelease offree[211At]astatideresultingintoxicityinanimal models.The boron‐astatine
bond formed on the anionic aromatic boron cage moiety shown above has demonstrated
increasedstabilityandreducedinvivodeastatination.1
References: 1. Wilbur DS. [211At]Astatine‐Labeled Compound Stability: Issues with Released [211At]Astatide and Development of Labeling Reagents to Increase Stability. Current Radiopharmaceuticals. 2008;1(3):144‐176. 3