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. 2011 Aug 17;22(8):1706-14.
doi: 10.1021/bc200309h. Epub 2011 Aug 2.

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium

C William Gundlach 4th  1 , 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
Affiliations

Synthesis and evaluation of an anti-MLC1 × anti-CD90 bispecific antibody for targeting and retaining bone-marrow-derived multipotent stromal cells in infarcted myocardium

C William Gundlach 4th et al. Bioconjug Chem. .

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-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 A(354). 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.

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Figures

Figure 1
Figure 1
S-HyNic modification of anti-MLC1 antibody and retention of epitope specificity. (A) Reaction of anti-MLC1 antibody (mAb MLM508) with S-HyNic. The reaction products not shown include excess S-HyNic, N-hydroxysuccinimide (NHS), and unreacted mAb. S-HyNic and NHS were removed by buffer exchange before further testing and generation of the bispecific reagent. Modified antibodies are represented with one modification per molecule for simplicity. DMF - dimethylformamide; MES - 2-(N-morpholino)ethanesulfonic acid. (B) Reaction scheme followed to measure the molar substitution ratio (MSR). A portion of HyNic-MLM508 was incubated with 4-nitrobenzaldehyde (4-NB) to generate 4NB-HyNic-MLM508, which absorbs at 390 nm. During incubation at pH 6, the alkyl hydrazone hydrolyzes in situ to produce the free hydrazine. The calculated MSR was 0.9. (C) ELISA analysis of HyNic-MLM508 binding to immobilized substrate recombinant human MLC1. Modified MLM508 binding was detected with GAM-HRP. All antibodies were used at a concentration of 10 μg/mL. Results are presented as average A450 ± SD from triplicate wells. One of three experiments performed is shown.
Figure 2
Figure 2
Differentiation capacity of BMMSCs treated with anti-CD90 antibody (mAb 5E10). BMMSCs were treated with anti-CD90 mAb 5E10 (A-C) or isotype control mAb (D-E). Differentiation of cells into adipogenic (A, D; arrows indicate neutral lipid accumulation), osteogenic (B, E), and chondrogenic (C, F) lineages was examined.
Figure 3
Figure 3
Modification of anti-CD90 antibody (mAb 5E10) with 4FB(PEG)4-PFP and retention of antibody-binding specificity. (A) Reaction of mAb 5E10 with 4FB(PEG)4-PFP. Reaction products not shown include excess 4FB(PEG)4-PFP, pentafluorophenol, and unreacted antibody. Modified antibodies are represented with one modification per molecule for simplicity. 4FB(PEG)4-PFP and pentafluorophenol were removed by buffer exchange before generation of the bispecific reagent and further use. DMF, dimethylformamide. (B) Reaction scheme to determine the molar substitution ratio (MSR). A portion of 4FB(PEG)4-5E10 was incubated with 2-hydrazinopyridine dihydrochloride (2HP) to form 2HP-4FB(PEG)4-5E10, which absorbs at 354 nm. The calculated MSR was 2.2. (C) Flow cytometric analysis of 4FB-5E10 and 4FB(PEG)4-5E10 binding to pig BMMSCs. Unmodified 5E10 (solid, black), 4FB(PEG)4-5E10 (solid, light grey), 4FB-5E10 (solid, dark grey), and IgG1 control antibody (dotted, black) binding to BMMSCs was detected with GAM-FITC (10 μg/mL). 4FB-5E10 was synthesized in the same fashion as 2 (MSR = 3.0).The histograms are representative of three experiments.
Figure 4
Figure 4
Reaction of two independently modified antibodies (HyNic-MLM508 and 4FB(PEG)4-5E10) to form a bispecific construct mixture. The reactant-modified antibody mixtures are represented with one modification per molecule and without unmodified antibody for simplicity. Reaction products not shown include unmodified antibodies, 1, 2, heterodimers bearing multiple cross-links, and multimers.
Figure 5
Figure 5
Generation of the bispecific antibody as monitored by UV-vis spectroscopy. (A) Bis-aryl hydrazone (rectangle) generated in the reaction of 4FB(PEG)4-5E10 with HyNic-MLM508 (see Figure 4). (B) The progress of the cross-linking reaction shown in Figure 4 was monitored by the absorbance of the bis-aryl hydrazone at 354 nm. (dashed, t = 0; solid, t = 1 h). A354 plateaued 1 h after initiation.
Figure 6
Figure 6
Analysis of reaction products by nonreducing SDS-PAGE. Lane 1, unmodified MLM508 (2 μg); lane 2, unmodified mAb 5E10 (2 μg); lane 3, unmodified MLM508 and 5E10 combined (2 μg each, 4 μg total); lane 4, bispecific antibody reactant products (4 μg). Asterisk indicates bispecific antibody.
Figure 7
Figure 7
Antigen specificity of the bispecific antibody product (3). (A) Bispecific antibody (BiAb) highlighting the isotypes of the mAb of each respective arm. (B) ELISA analysis of BiAb binding to plastic immobilized recombinant human MLC1. MLC1 was immobilized at 10 μg/mL. After blocking with BSA, primary antibody was added at a concentration of 10 μg/mL. In the case of “excess MLM508,” unmodified MLM508 was added to the ELISA wells (20 μg/mL) 0.5 h before adding primary antibody. HRP-conjugated secondary antibody specific for IgG1 (the isotype of 5E10) was used for detection. Data are presented as average A450 ± SD from triplicate wells. One of three representative experiments is shown. (C) BiAb binding to pig BMMSCs as detected with anti IgG2a-FITC. To demonstrate specific BiAb binding, BMMSCs were pretreated with saturating levels of unlabeled mAb 5E10 (10 μg/mL) for 1 h before adding BiAb. Histograms are representative of three experiments.
Figure 8
Figure 8
Bispecific antibody–induced retention of BMMSCs on MLC1 under conditions of shear stress. Parallel-plate flow chambers were coated with MLC1 or BSA as a control. Cells were treated either with the bispecific antibody (BiAb; 5 μg/mL) or untreated (controls) and then loaded onto the substrate and incubated for 0.5 h at room temperature. Cell detachment was measured as shear stress increased. Data represent the average percent of cell adhesion. Error bars indicate the data range (n=2).
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