Attachment of Boronated Dendrimers to mAb

Dendrimers are one of the most attractive polymers that have been used as boron carriers due to their well-defined structure and large number of reactive terminal groups. Depending on the antigen site density, approximately 1000 boron atoms need to be attached per molecule of dendrimer and subsequently linked to the mAb. In our first study, second- and fourth-generation polyamido amino (PAMAM or "starburst") dendrimers, which have 12 and 48 reactive terminal amino groups, respectively, were reacted with the water-soluble isocyanato polyhedral borane [Na(CH3)3NB10 H8NCO] (3).63,64 The boronated dendrimer then was linked to the mAb IB16-6, which is directed against the murine B16 melanoma, by means of two heterobifunctional linkers, m-maleimido-benzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP).63,65 However, following i.v. administration, large amounts of the bioconjugate accumulated in the liver, and spleen and it was concluded that random conjugation of boronated dendrimers to a mAb could alter its binding affinity and biodistribution. To minimize the loss of mAb reactivity, a fifth-generation PAMAM dendrimer was boronated with the same polyhedral borane anion, and more recently it was site-specifically linked to the anti-EGFR mAb cutuximab (or IMC-C225) or the EGFRvlll-specific mAb L8A4 (Figure 6.3). Cetuximab was linked via glycosidic moieties in the Fc region by means of two heterobifunctional reagents, SPDP and N-(k-maleimidoundecanoic acid) hydrazide (KMUH).66,67 The resulting bioconjugate, designated C225-G5-B1100, contained approximately 1100 boron atoms per molecule of cetuximab and retained its aqueous solubility in 10% DMSO and its in vitro and in vivo immunoreactivity. As determined by a competitive binding assay, there was a less than 1 log unit decrease in affinity for EGFR ( + ) glioma cell line F98egfr, compared to that of unmodified cetuximab.66 In vivo biodistribution studies, carried out 24 h after intratumoral (i.t) administration of the bioconjugate, demonstrated that 92.3 mg/g of boron was retained in rats bearing F98EGFR gliomas, compared to 36.5 mg/g in EGFR ( —) F98 parental tumors and 6.7 mg/g in normal brain.67

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