N-acetylgalactosamino dendrons as clearing agents to enhance liver targeting of model antibody-fusion protein

Abstract

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.

Figure 1

Glycodendron Clearing Agents (1–4), α-thio-GalNAc Endgroup (5) and Biotin Core.

Figure 2

HPLC Analysis of Dendrons 10 (G⁰), 11 (G¹), 12 (G²), 13 (G³), 14 (G⁴). Analysis conditions used: gradient of 45–95% acetonitrile (in water, 0.05% TFA) in 25 minutes; reversed phase C4 column (300 Å, 4.6 × 50 mm).

Figure 3

HPLC Analysis of Clearing Agents 1–4. Analysis conditions used: gradient of 20–40% acetonitrile (in water, 0.05% TFA) in 20 min; reversed phase C4 column (300 Å, 4.6 × 50 mm).

Figure 4

Blood Clearance of ¹³¹I-scFv-CC49-SAv. Time activity curve for ¹³¹I-activity in blood determined by serial blood collection up to 120 min following injection of ¹³¹I-scFv-CC49-SAv (t = −22 h to −24 h) and compounds 1–4 (t = 0) in biotin-starved mice. Cohorts given vehicle or 100 μg of biotinylated-dextran were included as controls. Data is shown as mean ± SEM, *P<0.05, **P <0.005. Note: scale on y-axis is 50–100%.

Figure 5

Glycodendrons Enhance Liver Targeting/Uptake of ¹³¹I-scFv-CC49-SAv. Tissue-to-blood ratios determined from biodistribution obtained 24 hr p.i. of vehicle (Veh), glycodendrons (1, 2, 3, 4), or biotindextran (Dex) (Figure S29). Data is shown as mean ± SEM.

Scheme 1

Synthesis of Amino-diethyl-hexanoate (a) Δ, KI, DIEA, overnight (70–80%); (b) PdC, 110°C, 5 h (60%)

Scheme 2

Synthesis of Generation 0 (G⁰) Dendron (a) Conc. HCl, reflux, 48 h (99%); (b) CH₂Cl₂, di-tert-butylcarbonate, 60 min (95%); (c) DMF, 7, HATU, TEA, 20 min (Crude 95%)

Scheme 3

Synthesis of Dendron Scaffold 11–13 (a) Ethyl Ester Deprotection: 1 N NaOH, 40°C, 1–2 h; (b) Coupling: 7, HATU, DIEA in DMF, 20–30 min (Crude yields are shown).

Scheme 4

Synthesis of Dendron Scaffold 14 (a) BOC Deprotection: TFA, 10 min; (b) Coupling: 9, HATU, TEA in DMF, 20 min; (c) Ethyl Ester Deprotection: 1 N NaOH, overnight; (d) Coupling: 7, HATU, TEA in DMF, 20 min.

Scheme 5

Synthesis of tert-Butyl (5-Bromoxypentyl)carbamate 16 (a) TEA, TsCl, DMAP (cat.), 0°C; overnight at RT (80%); (b) ACN, NaBr, Bu₄NBr, reflux overnight (83%).

Scheme 6

Synthesis of N-tert-Butyloxycarbonyl-S-(2-Acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-Dgalactopyranosyl)-5-Thiopentylamine Trifluroacetic Acid Salt 19 (a) Toluene, Lawesson’s reagent, reflux 2.5 h; (b) DMF, DIEA, 16 (two steps 89%); (c) TFA/DCM (2:5, v:v), 2 h.

Scheme 7

Synthesis of Clearing Agents 1–4