A recombinant triplebody with specificity for CD19 and HLA-DR mediates preferential binding to antigen double-positive cells by dual-targeting.
To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.
Department of Biology, University of Erlangen-Nuremberg, Erlangen, Germany. ischuber@biologie.uni-erlangen.de
Ingo
I
Schubert
Christian
C
Kellner
Christoph
C
Stein
Markus
M
Kügler
Michael
M
Schwenkert
Domenica
D
Saul
Bernhard
B
Stockmeyer
Christian
C
Berens
Fuat S
FS
Oduncu
Andreas
A
Mackensen
Georg H
GH
Fey
D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology; D018941:Antigens, CD19 / Q000276:immunology* / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D058851:GPI-Linked Proteins / Q000276:immunology / Q000378:metabolism; D057809:HEK293 Cells; D006684:HLA-DR Antigens / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D011485:Protein Binding; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism
D018033:Antibodies, Bispecific; D018941:Antigens, CD19; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D006684:HLA-DR Antigens; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies