(T1230-02-10) Development of a Highly Sensitive Immunoassay to Measure Circulating Levels of a Nanobody-Based Imaging Agent

Purpose: Cytotoxic CD8+ T cells are key players in the antitumor responses of cancer immunotherapies. Non-invasive, whole-body imaging can be used to visualize the distribution and accumulation of cytotoxic T cells at tumor sites using tracers specific for CD8, which can give an indication of tumor infiltration. An anti-CD8 immuno Positron Emission Tomography (iPET) agent was developed as a clinical imaging tool to quantify cytotoxic T cells and guide clinical development of cancer immunotherapy programs. The imaging agent consists of a humanized single variable domain on a heavy chain (VHH)/ nanobody targeting human CD8a, conjugated to a radioisotope-chelated linker.

Purpose: This work describes the development and qualification of a clinical pharmacokinetic (PK) assay to measure the concentration of an anti-CD8 VHH imaging agent in serum. High sensitivity is required due to the predicted fast clearance of the VHH from circulation. This assay is currently implemented in a Phase I trial to inform the recommended dose for the anti-CD8 iPET agent.

Methods: Four monoclonal antibodies (mAbs) directed to the VHH were generated in rabbits. Multiple pairwise combinations of these mAbs and/or the soluble target CD8a were evaluated as capture and detection reagents in different assay formats, with a target sensitivity of 1ng/mL. The assay platforms that were evaluated for this PK assay were ELISA and Gyrolab^® (Gyros Protein Technologies AB, Uppsala, Sweden).

Results: ELISA experiments aimed to achieve acceptable sensitivity, dynamic range and selectivity. While capture with CD8aFc and detection with an anti-VHH mAb did not lead to a suitable standard curve, other formats using anti-VHH mAbs as capture had acceptable curves with good dynamic range (Figure 1). However, selectivity experiments failed in those formats due to high background signals observed in a number of unspiked commercial samples from patients of different disease indications and healthy individuals (Figure 2). Because of the unacceptable selectivity, an alternative immunoassay platform had to be evaluated. Next, we assessed the Gyrolab^® platform, which offers the advantages of a broad standard curve range and, due to its proprietary compact disc (CD) technology which uses affinity flow-through, can reduce background signals and improve sensitivity. Different formats using the Gyrolab^® platform were explored. The final Gyrolab^® format that achieved the desired assay performance consists of CD8aFc as capture and an anti-VHH mAb as detection, with an MRD= 1/20 and LLOQ= 1.15 ng/mL. This assay format met all the qualification acceptance criteria, including accuracy and precision over 9 runs, selectivity and dilutional linearity (Figure 3).

Conclusion: We successfully developed and qualified a clinical PK assay using the Gyrolab^® platform to detect low levels of an anti-CD8 iPET imaging agent in serum. This PK assay is currently implemented in a Phase I dose escalation clinical trial to help determine the optimal dose of the tracer.

Comparison of 2 ELISA formats using different capture reagents

An ELISA format using an anti-VHH as capture leads to high assay signal for a number of unspiked samples

Acceptable standard curve reproducibility and assay control recovery over 9 runs