Quick and Economical Method of Determining the Specificity of Antibody Drug Lead and Target-binding Moiety for the Target

Antibody drugs and target-binding moiety of a chimeric antigen receptor (CAR) transgene need to be highly specific for the target they recognized, otherwise off-target toxicity is expected. Off-target refers to adverse effects as a result of modulation of other targets; these may be related biologically or totally unrelated to the target of interest [1]. Monoclonal antibody (mAb) and single chain Fv (scFv) that is most commonly used in CAR transgene as the target-binding moiety (TBM), being derived from mAb, are usually highly specific for the target. Albeit a rare event, off-target toxicity of mAbs is observed ^[2-6].

Proteins that belong to the same superfamily as the target, being the most similar to the target in sequence and structure, are most likely to react with the mAb and scFv, hence likely to be the cause of off-target toxicity. Therefore, validation of target specificity is essential for novel drug lead or a scFv used as TBM of a CAR transgene with potential clinical application.

To confirm the specificity of a lead molecule, one need to get all the superfamily member proteins and test the binding between mAb / scFv and these proteins using binding assays, like ELISA (enzyme-linked immunosorbent assay), SPR (surface plasmon resonance), and FACS (fluorescence activated cell sorting) if the target is a membrane bound protein. Thing is with some targets, the number of superfamily members is huge. For instance, BCMA (B-cell maturation antigen) is a member of the TNF receptor superfamily (TNFRSF). According to a recent search on uniprot.org, the number of superfamily members is as many as about 100 proteins. To get all these protein would cost a lot. Also some of the proteins may not be readily available. Furthermore, to test the bind of these proteins to the lead binder molecule in an assay as sensitive as SPR assay which may not be accessible in some labs, would cost much for consumables like the sensor chip.

To overcome this difficulty, some scientists in Memorial Sloan Kettering Cancer Center and Albert Einstein College of Medicine employed an ingenious idea of determining the specificity of a lead molecule. They developed an automated flow cytometry cell-cell conjugation assay in which a cell population expressing the lead molecule or a query molecule as they call it, is mixed with cells expressing a library of cell surface molecules to identify potential query-target interactions ^[7].

In this assay, the query molecule is a panel of anti-BCMA scFvs, and the library component targets are a library of 479 combined Ig super family and TNFR superfamily constructs. The assay was carried out as follows:

• To construct the target library, Ig superfamily full-length extracellular domains were cloned into a vector containing an erythropoietin (EPO) signal peptide and murine PD-L1 transmembrane domain with GFP fused on the C-terminal cytoplasmic side. In addition, full-length native TNFR superfamily constructs was fused to a C-terminal cytoplasmic GFP. These plasmids were used to transfect HEK293 cells individually. Only targets displaying high GFP expression and correct membrane localization were used for the target library, leaving 395 targets.
• Similarly, the anti-BCMA scFv query sequences were cloned into a Clontech N1 mCherry vector upstream of an inserted transmembrane domain (murine PD-L1) with mCherry fused to the C-terminal cytosolic side. The resulting plasmids were used to transiently transfect HEK293 cells as well as the target library constructs.
• Two days post transfection, binding reactions were setup in 96-well V-bottom plates by mixing equal volumes of query cells expressing anti-BCMA scFvs and library expressing cells.
• After binding, cell-cell conjugates were analyzed by flow cytometry. The percent bound was calculated as the number of double positive events (GFP & mCherry) divided by the total number of cells. High percent bound suggest the possibility of non-specific off-target binding.

This approach has the advantage of allowing both the query and library component targets to be presented in the context of the mammalian plasma membrane, with close to native post-translational modifications and interactions. It also saves the time and cost for the producing a large number of proteins and testing the reactivity of these proteins to the lead molecule, and should be widely used to assess the specificity of a novel molecule with potential clinical application. That being said, the potential for non-specific binding of the lead molecule can be further evaluated using a variety of essential normal tissue types by Immunohistochemistry (IHC).