Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

Abstract

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Here we show that an intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (db BiTES, 300 kDa). The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of the 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES maintain high in vivo tumor targeting as demonstrated by PET imaging, and redirect dbBiTE coated T-cells (1 microgram/10 million cells) to kill CEA + target cells both in vitro, and in vivo in CEA transgenic mice.

T‐cell surface generation of dual bivalent, bispecific T‐cell engaging, RNA duplex cross‐linked antibodies (dbBiTERs) for re‐directed tumor cell lysis

“…Bromoacetamido-DBCO (Br-DBCO) was synthesized as previously described. [7] NHS-Alexa-488 was purchased from Thermo Fisher, DBCO-Alexa 488 from Click Chemistry Tools, murine anti-human CD3 antibody (OKT3) from BioXcell (Lebanon, NH). Humanized anti-human CD3 antibody (hOKT3) and humanized anti-CEA antibody hT84.66-M5A (M5A) was produced in house as previously described.…”

“…[21] Human breast carcinoma MDA-MB-231 cell line was transfected with CEA as previously described. [7]…”

“…Alkylation was confirmed by mass spectrometry on an Agilent 6520 QTOF mass spectrometer as previously described. [7] Similar conditions were used to alkylate hinge reduced M5A with either Br-DBCO (dbBiTER experiments) or bromoacetamido-PEG 5 -azide (on cell dbBiTE experiments). An aliquot (1 mg) of azido-M5A was further derivatized with NHS-Alexa-488 (molar ratio) for the on-cell dbBiTE experiments.…”

“…TEM was performed as previously described. [7] For immunogold staining, cells were fixed in 0.3% glutaraldehyde in PBS and stained with Nanogold anti-mouse IgG (Nanoprobes) and enhanced with HQ Silver enhancement kit (Nanoprobes).…”

“…Since the hinge regions are distant from the antigen combining regions, this approach preserves antibody activity. When the two antibodies are clicked together they form distinct 300 kDa particles we have dubbed dbBiTEs [7] for their retention of dual valency, a key feature of each original bivalent antibody designated as avidity. [8] Since the two antibodies are intimately connected at their hinge regions, they were visualized as six-lobed particles by EM analysis, [7] affirming the idea that two antibodies can fit together at their hinges without steric hindrance.…”