Abstract
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Introduction: Arylhalide albumin binders (ABs) were appended to the VLA-4 targeting LLP2A moiety to extend the blood circulation of [177Lu]DOTA-derivatives. This was aimed to investigate the potential of [177Lu]-LLP2A-AB conjugates as theranostic agents for VLA-4 overexpressing malignancies. Conjugates were modeled after [177Lu]DOTA-PEG4-LLP2A (Beaino, et al. Mol. Pharm. 2015, 12(6), p. 1929), yet contained a C-terminal lysine for coupling 4-(4-chlorophenyl)butyric acid (LLP2A-Cl), the iodo counterpart (LLP2A-I) or for acetylation (LLP2A-Ac). In vitro mouse serum albumin (MSA) binding and VLA-4 binding were assessed using natLu-LLP2A conjugates, while SPECT/CT imaging and biodistribution studies examined the interplay between prolonged blood circulation and tumor retention of [177Lu]-LLP2A tracers.
Methods: LLP2A precursors were synthesized on Rink (MBHA) amide resin via standard Fmoc-based protocols. Chelation of natLu3+ and 177Lu3+ was performed with either 0.5 M LuCl3 (aq) or [177Lu]LuCl3 (aq), respectively, in 0.1 M NaHCO3 (aq) pH 4.25. The natGa- and 68Ga-derivatives of LLP2A-I were prepared in 1 M GaCl3 (0.1 M NaOAc (aq)) or [68Ga]3+ (2 M HEPES (aq) pH 5). MSA binding was assessed by recording protein fluorescence quenching (λEx = 280 nm; λEm = 350 nm) by natLu-LLP2A conjugates (30 μM - 2 mM). VLA-4 competitive binding assays were performed with B16-F10 murine melanoma cells and natLu-LLP2A conjugates (1 pM - 10 μM). SPECT/CT and biodistribution experiments used B16-F10 tumor-bearing male C57BL/6J mice that were either imaged (~37 MBq injected) or sacrificed (2-4 MBq injected) at 1h, 3h, 24h or 120h p.i. (n = 4-5). Time-activity curves (TAC) and tumor:organ uptake ratios were then calculated.
Results: Each natLu-LLP2A conjugate was obtained in acceptable 19-29% yields while natGa-LLP2A-I gave an adequate 13% yield. MSA binding assays revealed Kd values of 80±1.9 µM, 53.5±1.9 µM and 40.9±1.0 µM for natLu-LLP2A-Ac, -Cl and –I, respectively. Competitive binding assays identified low-nM (IC50 = 2.05-2.34 nM) VLA-4 binding for each natLu-LLP2A. [177Lu]-LLP2A-Ac, -Cl and –I were produced within 73 ± 27min. to afford 613.1-824.1 MBq (decary-corrected radiochemical yield = 45-69%) of each tracer with radiochemical purity of 95-99% and Am = 370-396 GBq/µmol. SPECT/CT and biodistribution analyses showed tumor accumulation of each [177Lu]-LLP2A tracer at 1-24h p.i. (Ac: 3h = 17.09±3.41 %ID/g; Cl: 3h = 16.58±1.97 %ID/g; I: 3h = 12.55±1.73 %ID/g). However, sustained signals at 120h p.i. were observable only for the [177Lu]-LLP2A-AB tracers (Ac: 0.47±0.36 %ID/g; Cl: 3.32±0.66 %ID/g; I: 4.26±1.71 %ID/g). Calculation of the area under the time-activity curves gave ratios of 1: 29: 63 in the blood and 1: 2.95: 2.4 in tumors for [177Lu]-LLP2A-Ac, -Cl and –I, respectively. Further analysis revealed higher tumor:bone and tumor:spleen uptake ratios for [177Lu]-LLP2A-AB tracers compared to the control at 3-120h p.i.. Yet, [177Lu]-LLP2A-Ac exhibited notably greater tumor:heart, tumor:liver and tumor:lung uptake ratios at 1-24h p.i. versus the [177Lu]-LLP2A-AB tracers.
Conclusions: MSA binding assays showed that the selected arylhalides offered enhanced protein binding to the natLu-LLP2A-AB conjugates. Surprisingly, the LLP2A moiety itself contributed to MSA binding interactions. Competitive binding assays found that each natLu-LLP2A retained the expected low-nM IC50 binding to VLA-4. SPECT/CT and biodistribution experiments with [177Lu]-LLP2A tracers revealed that both AB moieties extended blood circulation and contributed to long-term tracer retention in tumors. It was noted, though, that the slower clearance and higher healthy organ uptake limited the benefits of the albumin binder strategy with these [177Lu]-LLP2A-AB tracers. Therefore, our studies suggest that this approach must be carefully employed with the LLP2A scaffold to achieve optimal molecular targeting and theranostics of VLA-4 associated malignancies.
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