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Threetypes of metal-chelating polymers (MCPs) with hydrazide endgroups were synthesized. (1) The first set of polymers (the F-series)was synthesized with a furan end group, and all of the pendant groupsalong the chain carried only a diethylenetriaminepentaacetic acid(DTPA) metal-chelating functionality. The hydrazide was introducedvia 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-localizationpeptide sequence (NLS peptides, CGYGPKKKRKVGG, harboring the NLS fromthe simian virus 40 large T-antigen) in addition to the DTPA metal-chelatinggroups. (3) The third type of polymer (the P-Py series) was a variationof the P-series polymers but with the introduction of a small numberof pyrene chromophores along the backbone to allow for UV measurementof the incorporation of the MCPs into trastuzumab (tmab). These hydrazide-terminatedpolymers were site-specifically conjugated to aldehyde groups generatedby NaIO4oxidation of the pendant glycan in the Fc domainof tmab. The immunoconjugates were radiolabeled with 111In and analyzed by SE–HPLC to confirm the attachment of thepolymer to the antibody. HER2 binding assays demonstrated that neitherthe MCPs nor the presence of the NLS peptides interfered with specificantigen recognition on SK-Br-3 cells, although nonspecific bindingwas increased by polymer conjugation. Our results suggest that MCPscan be site-specifically attached to antibodies via oxidized glycansin the Fc domain and labeled with 111In to construct radioimmunoconjugateswith preserved immunoreactivity. [ABSTRACT FROM AUTHOR]

We describe the synthesis of metal chelating polymersbased onpolyaspartamide and polyglutamide backbones as carriers for 111In in radioimmunoconjugates. These polymers [PAsp(DTPA), PGlu(DTPA)]have a biotin end group and diethylenetriaminepentaacetic acid (DTPA)chelators attached to the primary amines of the diethylenetriamine(DET) pendant groups of biotin-poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] andof biotin-poly{N′-[N-(2-aminoethyl)-2-aminoethyl]glutamide}[PGlu(DET)]. Like Asn-containing proteins and polypeptides, polyaspartamidesundergo uncatalyzed degradation under model physiological conditions(10 mM phosphate buffer, pH 7.4, 150 mM NaCl). We studied the uncatalyzeddegradation of the polyaspartamide polymers by size exclusion chromatographyand found that the degradation rate was sensitive to the nature ofthe pendant groups. The metal-free polymer underwent somewhat slowerdegradation than the corresponding polymers in which the DTPA groupswere saturated with Eu3+or In3+, but even after14 days, substantial fractions of the polymers survived. We concludethat these polymers undergo negligible degradation on the time scale(24–48 h) of radioimmunotherapy treatment of tumors with 111In. From a mechanistic perspective, we note that these degradationrates are on the order of the deamidation rates reported [J. Peptide Res. 2004, 63,426] for Asn-containing pentapeptides, with half-times on the orderof 10 days, but much slower than the rapid decay (hours) reportedrecently [Biomaterials2010, 31, 3707] for poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} itself. This variationin degradation rate can be explained in terms of the influence ofpositive charges on the pendant group enhancing the acidity of theside-chain amide nitrogen of the aspartamide repeat unit. The DETpendant group is positively charged at pH 7, but in indium-loadedPAsp(DTPA) this charge is offset by the net negative charge of theDTPA-In complex. [ABSTRACT FROM AUTHOR]