(W1030-06-33) Development of Sensitive Free and Total Assays for the Quantification of Soluble CD200R1 in Human Plasma Samples to Assess Drug-Target Engagement

Purpose: CD200R1 and its ligand, CD200, are Type 1 transmembrane proteins that contribute to immunosuppressive signaling pathways. 23ME-00610, a humanized monoclonal antibody targeting human CD200R1 inhibits this pathway and could potentially enhance immune-cell activity within the tumor microenvironment as an immune-oncology (IO) therapy. To check whether the soluble form of the receptor exists and its baseline in human circulation, and to assess target engagement and determine dose selection in clinical trials, two highly sensitive ELISA methods were developed respectively to accurately quantify free and total soluble CD200R1 in human plasma samples.

Methods: The free assay measures only free soluble CD200R1 using a non-competing anti-CD200R1 antibody paired with a biotinylated 23ME-00610. The total assay captures free and drug-bound forms of soluble CD200R1 with the same non-competing antibody, followed by saturation with unlabeled 23ME-00610 and detection with a biotinylated type II anti-idiotype antibody against the 23ME-00610. Recombinant CD200R1 spiked buffer samples and human plasma samples were spiked with and without unlabeled 23ME-00610 to check specificity of both assays. Normal and disease human plasma samples were screened with the method for free soluble CD200R1 to determine the baseline of soluble CD200R1, and 6 plasma samples were cross-checked with both methods to compare quantification comparability in both assays.

Results: The free assay calibration range is from 15.625 to 1000 pg/mL, with a minimum required dilution (MRD) of 20x for samples. The specificity results showed that stoichiometrically-excessive amount of 23ME-00610 was able to completely deplete the assay signal. Total assay calibration range is from 20 to 2500 pg/mL, with the same MRD of 20x. The method can tolerate up to 1000 ug/mL of unlabeled 23ME-00610 without interference in human plasma. Prevalence screening results showed the soluble CD200R1 baseline level of 2740±708.3 pg/mL in normal human plasma and 2480±903.9 pg/mL in disease human plasma. Both assays demonstrated comparable baseline levels of CD200R1 in the absence of the drug. Clinical data showed expected results with target saturation seen at higher dose and dose dependent accumulation of total CD200R1.

Conclusion: With the free soluble CD200R1 method, we observed for the first time that CD200R1 was present and was measurable in human periphery, with limited variability in individuals. The development of the free and total assays provides essential tools for monitoring the target engagement of 23ME-00610 and effectively evaluating dosing regimens in clinical settings (clinical trial information: NCT05199272).