Preclinical efficacy of hK2 targeted [<sup>177</sup>Lu]hu11B6 for prostate cancer theranostics
Authors: Oskar Vilhelmsson Timmermand, Jörgen Elgqvist, Kai A. Beattie, Anders Örbom, Erik Larsson, Sophie E. Eriksson, Daniel L.J. Thorek, Bradley J. Beattie, Thuy A. Tran, David Ulmert, Sven-Erik Strand
Journal: Theranostics (2019)

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops.In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression.Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease.The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway.In a previous study, hu11B6 conjugated with Actinium-225 ( 225 Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy.We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 ( 177 Lu) and investigated whether similar tumor killing and AR-enhancement is produced.Moreover, single-photon emission computed tomography (SPECT) imaging of 177 Lu is quantitatively accurate and can be used to perform treatment planning.[ 177 Lu]hu11B6 therefore has significant potential as a theranostic agent.Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice.Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied.Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [ 177 Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging.Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom.Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-β-gal activity (senescence) and analyzed by digital autoradiography (DAR).Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg).Tumor accumulation of [ 177 Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen.However, SPECT imaging with therapeutic amounts of [ 177 Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177 Lu and decreasing tumor volumes.For [ 177 Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor IvyspringInternational Publisher volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection.Tumor volume reduction correlated with injected activity and the absorbed dose.IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA-β-gal activity (14 d).Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake.AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017).Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177 Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects.The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level.Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225 Ac labeled hu11B6, however emerging at a later timepoint.