Synthesis of Polyglutamide-Based Metal-Chelating Polymers and Their Site-Specific Conjugation to Trastuzumab for Auger Electron Radioimmunotherapy

Abstract

Three types of metal-chelating polymers (MCPs) with hydrazide end groups were synthesized. (1) The first set of polymers (the F-series) was synthesized with a furan end group, and all of the pendant groups along the chain carried only a diethylenetriaminepentaacetic acid (DTPA) metal-chelating functionality. The hydrazide was introduced via a Diels-Alder reaction between the furan and 3,3'-N-[ε-maleimidocaproic acid] hydrazide (EMCH). (2) The P-series polymers was designed to carry several copies of a nuclear-localization peptide sequence (NLS peptides, CGYGPKKKRKVGG, harboring the NLS from the simian virus 40 large T-antigen) in addition to the DTPA metal-chelating groups. (3) The third type of polymer (the P-Py series) was a variation of the P-series polymers but with the introduction of a small number of pyrene chromophores along the backbone to allow for UV measurement of the incorporation of the MCPs into trastuzumab (tmab). These hydrazide-terminated polymers were site-specifically conjugated to aldehyde groups generated by NaIO4 oxidation of the pendant glycan in the Fc domain of tmab. The immunoconjugates were radiolabeled with (111)In and analyzed by SE-HPLC to confirm the attachment of the polymer to the antibody. HER2 binding assays demonstrated that neither the MCPs nor the presence of the NLS peptides interfered with specific antigen recognition on SK-Br-3 cells, although nonspecific binding was increased by polymer conjugation. Our results suggest that MCPs can be site-specifically attached to antibodies via oxidized glycans in the Fc domain and labeled with (111)In to construct radioimmunoconjugates with preserved immunoreactivity.

Functionalization of Cellulose Nanocrystals with PEG-Metal-Chelating Block Copolymers via Controlled Conjugation in Aqueous Media

“…The mPEG-PBLG- t Boc polymers were then treated with an excess of ethylenediamine (EDA) at room temperature (RT) to convert the benzyl groups to aminoethylamide pendant groups. 44 This reaction was carried out for different times ranging from 3 to 5 h to optimize both the amine conversion and preservation of the t Boc end group. By comparing the integration of the t Boc protons at 1.44 ppm to that of the signal at 4.34 ppm (backbone methine), the surviving t Boc functionality for mPEG-PGlu(EDA) 18 -Boc in this step stayed the same for the reaction times of 3 h ( 1 H NMR spectrum not shown) and 5 h ( Figure S8 ).…”

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 1: Cysteine Residues and Glycans

“…The authors found that the resulting site-specifically Tc-99m-labeled radioimmunoconjugate proved more stable than an analogous, traditionally conjugated variant. More recently, in an effort to develop In-111-labeled immunoconjugates for Auger electron radiotherapy, Lu et al site-specifically conjugated polymers bearing ~30 DTPA each to trastuzumab via oxidation with NaIO 4 , reaction with the hydrazide-bearing polymers, and reduction with NaBH 3 CN [ 86 ]. The resulting constructs were shown to have approximately 1.2 polymers/mAb and could be successfully labeled with 111 In in high yield and at higher specific activities than traditional, lysine-conjugated trastuzumab-DTPA.…”

Immune activity of sweet potato (Ipomoea batatas L.) glycoprotein after enzymatic and chemical modifications

“…Considering that NaIO 4 could oxidize the carbohydrate portion of SPG-1, 35 these findings may indicate that the carbohydrate portion of SPG-1 is the major immune active site. Therefore, NaIO 4 oxidation remarkably decreased the T cell immune response of SPG-1.…”