Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1

Our plain-English summary of Mechanism of Collaborative Enhancement of Binding of Paired Antibodies to Distinct Epitopes of Platelet Endothelial Cell Adhesion Molecule-1, by R. Kiseleva, C.F. Greineder, C.H. Villa, et al (PloS One, 2017).

The study

• Authors: R. Kiseleva, C.F. Greineder, C.H. Villa, E.D. Hood, V.V. Shuvaev, J. Sun, A.M. Chacko, V. Abraham, H.M. DeLisser, V.R. Muzykantov
• Journal: PloS One
• Published: 2017;12(1):e0169537
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Monoclonal antibodies (mAbs) directed to extracellular epitopes of human and mouse Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) stimulate binding of other mAbs to distinct adjacent PECAM-1 epitopes. This effect, dubbed Collaborative Enhancement of Paired Affinity Ligands, or CEPAL, has been shown to enhance delivery of mAb-targeted drugs and nanoparticles to the vascular endothelium. Here we report new insights into the mechanism underlying this effect, which demonstrates equivalent amplitude in the following models: i) cells expressing a full length PECAM-1 and mutant form of PECAM-1 unable to form homodimers; ii) isolated fractions of cellular membranes; and, iii) immobilized recombinant PECAM-1. These results indicate that CEPAL is mediated not by interference in cellular functions or homophilic PECAM-1 interactions, but rather by conformational changes within the cell adhesion molecule induced by ligand binding. This mechanism, mediated by exposure of partially occult epitopes, is likely to occur in molecules other than PECAM-1 and may represent a generalizable phenomenon with valuable practical applications.

In plain English

This laboratory study examined the mechanism of "Collaborative Enhancement of Paired Affinity Ligands" (CEPAL), in which one monoclonal antibody bound to PECAM-1 (CD31) increases binding of a second antibody to an adjacent, distinct epitope. The authors measured binding of radioiodinated antibodies Mec13.3 and 390, given alone ("solo") or with 25 nM of the unlabeled partner antibody ("paired"), in three systems: live REN human malignant mesothelioma cells stably expressing full-length mouse PECAM-1 or the homodimer-deficient K89A mutant, cell membrane homogenates, and immobilized recombinant mouse PECAM-1. In live cells, mAb 390 lowered the Kd of labeled Mec13.3 0.48 ± 0.03-fold in RmP cells (8.8 to 4.2 nM) and 0.60 ± 0.05-fold in RmPK89A cells (9.1 to 5.5 nM), while Mec13.3 did not significantly change the binding affinity (Kd) of 390. Maximum binding sites (Bmax) rose in every setting, including for 390 paired with Mec13.3 (1.12 ± 0.05-fold in RmP and 1.47 ± 0.03-fold in RmPK89A cells). The authors concluded that the effect persisted without cell signaling or homophilic binding, consistent with ligand-induced conformational change exposing partly hidden epitopes.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory binding study (radioimmunoassay) in cultured cells, cell membrane homogenates and immobilized recombinant protein
• Who was studied: REN human malignant mesothelioma cells stably expressing recombinant mouse PECAM-1 (RmP) or the K89A mutant (RmPK89A), with wild-type REN cells as control; membrane homogenates of these cells; and purified recombinant mouse PECAM-1 extracellular domain
• Exposure: Radioiodinated anti-mouse PECAM-1 monoclonal antibodies Mec13.3 and 390 applied alone or together with a fixed 25 nM concentration of the unlabeled partner antibody
• Main results:
  • In live cells, unlabeled mAb 390 reduced the Kd of [125I]-mAb Mec13.3 0.48 ± 0.03-fold in RmP cells (8.8 to 4.2 nM) and 0.60 ± 0.05-fold in RmPK89A cells (9.1 to 5.5 nM), both P<0.001
  • The effect on binding affinity was not reciprocal in live cells: unlabeled Mec13.3 had negligible effect on the Kd of [125I]-mAb 390 (RmP: 3.6 nM paired vs 4.2 nM solo)
  • Bmax for [125I]-mAb Mec13.3 with paired mAb 390 rose 1.79 ± 0.09-fold in RmP cells and 2.00 ± 0.08-fold in RmPK89A cells (P<0.001)
  • In live cells, Bmax for [125I]-mAb 390 paired with Mec13.3 rose 1.12 ± 0.05-fold in RmP cells and 1.47 ± 0.03-fold in RmPK89A cells over solo binding (P<0.001)
  • In cell membrane homogenates, paired unlabeled antibodies increased Bmax 2.69 ± 0.36-fold for [125I]-mAb Mec13.3 and 1.30 ± 0.02-fold for [125I]-mAb 390 (P<0.001)
  • With immobilized purified recombinant PECAM-1, Bmax increased about 1.5-fold for both antibody pairings (p<0.001), and mAb 390 changed the Kd of [125I]-mAb Mec13.3 0.53 ± 0.03-fold
• The authors' conclusion: The authors concluded that collaborative enhancement does not depend on cell signaling or on PECAM-1 homophilic binding, and suggested it may occur with other immunoglobulin-superfamily adhesion molecules
• Limitations the authors state:
  • The authors noted that the membrane homogenate preparation might still contain residual machinery for cellular activation, prompting the further test with purified protein
  • Transgene expression levels differed between the cell lines carrying the different constructs
  • The authors noted that previously reported in vivo mouse experiments showed a mutual (opposite) pairing effect, unlike these in vitro findings
  • The authors stated that crystal structures and conformational changes still need to be determined to build a detailed model

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Monoclonal Antibody.

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