Use of Retrogenix cell microarray technology to confirm specificity of diagnostic molecules created using the NanoMab platform

High specificity to a primary target receptor is not only important when developing novel therapeutics, it may also be critical in the development of reliable diagnostic tools. Uniquely, for diagnostics that are intended to be physically administered to patients in order to detect and quantify disease in situ, polyspecificity also raises the risk of adverse reactions in addition to potentially hampering diagnostic effectiveness.

NanoMab is an innovative developer of diagnostics and precision cancer therapies. The company is currently running human trials of novel in vivo diagnostics for specific tumours. Radioactive isotopes that are conjugated to tumour targeting moieties are injected into the patient. Whole-body imaging scans then provide information on both primary and metastatic tumours and can also be used to select patient and monitor responses to cancer treatment. Here we discuss a NanoMab lung cancer diagnostic which is now commencing Phase 2 studies1. Demonstrating specificity to the primary target – PD-L1 – was one of the key steps in the preparation of the regulatory package that was required prior to advancement into the clinic.

NanoMab sdAbs

NanoMab’s technology is built around camelid single domain antibodies (sdAb). These are the smallest naturally derived, single-domain, antigen-binding fragments and have many advantages over other commonly used vectors, including stability and good tumour penetration and retention. To confirm the specificity of their candidate PD-L1-targeted sdAb, NanoMab provided a dual His- and HA-tagged version of the molecule (the “test protein”) for screening using the Retrogenix cell microarray platform. A variant of the protein which lacked a HA tag was also supplied to be used as a screening control.

Initial investigation showed low levels of background binding of the test protein to fixed, untransfected HEK293 cells. The test protein was then screened for binding against cells individually expressing 5,484* full-length human plasma membrane and secreted proteins using both an anti-His and an anti-HA detection approach. This revealed nine hits, but only one – CD274 (PD-L1), the known primary target – was specific to the test protein.

Each hit, along with a series of control targets, was re-expressed and re-tested with: the test protein; the control (related) protein; and other positive and negative control treatments. After removing hits that were non-specific, two specific interactions remained for NanoMab’s test protein, which were seen with both detection approaches (figure 1). These were CD274 isoforms 1 and 2 (PD-L1). The same interactions were observed with the related control protein. No off-target interactions were identified for the test protein, indicating monospecificity for its primary target.

Figure 1. Confirmation screen results. Top left: spotting pattern of expressed library hits and control targets detected via ZsGreen1. Top right: anti-HA detection of binding for the dual-tag test protein. Bottom panel: anti-His detection of binding for the dual-tag test protein and various controls. The test protein bound to its known primary target (two isoforms); no additional specific (off-target) interactions were detected.   

Impact and future developments

The Retrogenix screen showed both versions of the NanoMab test protein to be highly specific to their primary (intended) target, PD-L1. This provided data that supported the case for the regulatory approval that has allowed NanoMab to progress into clinical investigation.

NanoMab’s strategy of targeted radiolabelling to visualise and monitor tumours in situ not only provides much-needed, non-invasive diagnostic tools, it also paves the way for the development of novel therapeutics. In these “theranostics”, the diagnostic radioisotopes are replaced with therapeutic radioisotopes that are suitable for molecular targeted radiation therapy. This has the potential to provide novel treatment options for patients who have currently untreatable forms of cancer.

 

We thank NanoMab for permission to use their screening data and discuss their exciting platform technology. For more information please visit the Nanomab website

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