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

Abstract

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-aryl hydrazone. 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 (RT, pH 6, 1 h) in the presence of a catalyst (aniline) to allow for rapid generation of the covalent bis-aryl hydrazone, 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 × 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 × anti-MLC1 bispecific antibody may be used in large animal studies of acute myocardial infarction and may provide a starting point for clinical studies.

Understanding and targeting cancer stem cells: therapeutic implications and challenges

“…An anti-CD90 antibody has also been reported to protect bone marrow-derived multipotent stromal cells from differentiation into chondro-, osteogenic, or adipo-lineages 66 . In addition, CD133 is highly expressed in many types of CSCs, including lung cancer, breast cancer, and glioma.…”

MSCs: Delivery Routes and Engraftment, Cell-Targeting Strategies, and Immune Modulation

“…This cancer treatment was tested in vivo, yielding reduced tumor size and a significantly increased rate of survival [118]. In vitro, a recent report describes the use of an anti-CD90/anti-myosin light chain kinase to bind MSCs and increase their resistance to shear in a parallel plate assay [119]. …”

Construction of homogeneous antibody–drug conjugates using site-selective protein chemistry

“…NHS esters have been used to introduce bioorthogonal functionalities, such as azides and hydrazones, for subsequent modification through Staudinger ligation and hydrazone exchange, respectively. 22 – 24 NHS esters can also be used to modify maleimide-crosslinkers. These crosslinkers can then be used to conjugate antibodies to different molecules bearing a nucleophilic sulfhydryl functional group, such as drugs, quantum dots, and even DNA barcodes.…”

“… 32 An activated β-lactam bearing an analogue of an anti-viral drug was used to modify mAb 38C2 at a Lys residue as well. 33 Furthermore, fluorophenyl ester drug analogues were used to modify an anti-CD90 antibody (5 × 10 10 ), 22 while isothiocyanates were used to introduce a radioactive label in anti-CD45 mAb through conjugation to the amine side chains of Lys residues. 34…”