RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward alphavbeta3-expressing endothelial cells.
To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both alphavbeta3 and CD3. Monocyclic RGD peptides (cRGDfK) were covalently coupled to an anti-CD3 monoclonal antibody at different peptide:protein ratios. The resulting RGDpep-anti-CD3 conjugates bound specifically to alphavbeta3-expressing endothelial cells. Displacement studies with radiolabeled alphavbeta3 ligand demonstrated that the alphavbeta3 binding affinity of RGDpep-anti-CD3 conjugates was elevated as compared to the non-conjugated RGDpep. IC50 values ranged from 150-1.1 nM, depending on the amount of coupled RGDpep molecules per antibody molecule. RGD modification did not affect the ability of anti-CD3 antibodies to bind to CTL. Furthermore, RGDpep-anti-CD3 was fully capable of activating T cells upon CD3 binding as was shown in a Jurkat/NFAT reporter-gene activation assay. All RGDpep-anti-CD3 conjugates were able to induce RGDpep, CD3-dependent lysis of human primary endothelial cells by anti-CD3/IL-2 activated human peripheral blood mononuclear cells (PBMC), with a significant induction of cytotoxicity observed at an E/T ratio as low as 10. Redirecting cytolytic activity reached up to 50% cytotoxicity using the conjugate with the highest RGD peptide load. Combining the good accessibility of tumor blood vessel endothelium for CTL with the efficiency of target cell killing warrants further investigations on anti-tumor effects of this type of conjugates in vivo.
Schraa
Astrid J
AJ
Department of Pathology and Laboratory Medicine, Medical Biology Section, Groningen, the Netherlands.
Kok
Robbert J
RJ
Botter
Sander M
SM
Withoff
Sebo
S
Meijer
Dirk K F
DK
de Leij
Lou F M H
LF
Molema
Grietje
G
D000906:Antibodies / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology*; D042783:Endothelial Cells / Q000276:immunology*; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D039302:Integrin alphaVbeta3 / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D009842:Oligopeptides; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000906:Antibodies; D017252:CD3 Complex; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid