Synthesis and Evaluation of an Anti-MLC1 脳 Anti-CD90 Bispecific Antibody for Targeting and Retaining Bone-Marrow-Derived Multipotent Stromal Cells in Infarcted Myocardium

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• 作者：C. William Gundlach IV ; Amy Caivano ; Maria da Graca Cabreira-Hansen ; Amir Gahremanpour ; Wells S. Brown ; Yi Zheng ; Bradley W. McIntyre ; James T. Willerson ; Richard A. F. Dixon ; Emerson C. Perin ; Darren G. Woodside
• 刊名：Bioconjugate Chemistry
• 出版年：2011
• 出版时间：August 17, 2011
• 年：2011
• 卷：22
• 期：8
• 页码：1706-1714
• 全文大小：980K
• 年卷期：v.22,no.8(August 17, 2011)
• ISSN：1520-4812

文摘

A key issue regarding the use of stem cells in cardiovascular regenerative medicine is their retention in target tissues. Here, we have generated and assessed a bispecific antibody heterodimer designed to improve the retention of bone-marrow-derived multipotent stromal cells (BMMSC) in cardiac tissue damaged by myocardial infarction. The heterodimer comprises an anti-human CD90 monoclonal antibody (mAb) (clone 5E10) and an anti-myosin light chain 1 (MLC1) mAb (clone MLM508) covalently cross-linked by a bis-arylhydrazone. We modified the anti-CD90 antibody with a pegylated-4-formylbenzamide moiety to a molar substitution ratio (MSR) of 2.6 and the anti-MLC1 antibody with a 6-hydrazinonicotinamide moiety to a MSR of 0.9. The covalent modifications had no significant deleterious effect on mAb epitope binding. Furthermore, the binding of anti-CD90 antibody to BMMSCs did not prevent their differentiation into adipo-, chondro-, or osteogenic lineages. Modified antibodies were combined under mild conditions (room temperature, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-arylhydrazone, which was monitored at A₃₅₄. We evaluated epitope immunoreactivity for each mAb in the construct. Flow cytometry demonstrated binding of the bispecific construct to BMMSCs that was competed by free anti-CD90 mAb, verifying that modification and cross-linking were not detrimental to the anti-CD90 complementarity-determining region. Similarly, ELISA-based assays demonstrated bispecific antibody binding to plastic-immobilized recombinant MLC1. Excess anti-MLC1 mAb competed for bispecific antibody binding. Finally, the anti-CD90 脳 anti-MLC1 bispecific antibody construct induced BMMSC adhesion to plastic-immobilized MLC1 that was resistant to shear stress, as measured in parallel-plate flow chamber assays. We used mAbs that bind both human antigens and the respective pig homologues. Thus, the anti-CD90 脳 anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.