Purpose: Validation of an immunoassay for measuring cytotoxic T-lymphocyte-associated protein 4 (CTLA4) was requested as an exploratory biomarker. CTLA4 is also a therapeutic target in immune-response diseases. CTLA4 is expressed in regulatory T cells and upregulated in conventional T cells after activation. CTLA4 acts like an off switch when bound to surface receptors on antigen presenting cells. This response is particularly of note in cancer. Commercial kits are available for CTLA4 measurement, but they lack sensitivity and in some cases adequate specificity. Here we describe the screening of commercial reagents along with validation of the best pair in an electrochemiluminescent format assay intended for exploratory clinical use. Proof of utility was evaluated using normal and diseased plasma samples from various immune diseases.
Methods: Commercial CTLA4 antibodies were evaluated with calibrator proteins from different sources. The best binding antibodies were selected for pairing and optimization. The final pair was evaluated for dilution linearity and matrix interference using plasma samples. The assay was developed on the MSD platform and appears to have adequate sensitivity and specificity for the intended needs, with a calibration curve range of 1 to 2500 pg/mL. The assay was optimized and taken into validation for accuracy precision, recovery and stability in fit for purpose format in preparation for exploratory clinical use.
Results: The best pair of antibodies were selected based on sensitivity, performance and specificity. The minimum required dilution was 2-fold allowing for dilution of plasma interference and keeping good sensitivity. The CTLA4 assay met all validation acceptance criteria parameters, with accuracy and precision CV’s of < 20%. Storage and Freeze-thaw stability were also within acceptance limits with stability up to 3 F/T cycles. Analysis of samples from normal and various immune diseases demonstrated that there is different mean measured level of CTLA4 in different diseases. In healthy donors CTLA4 was 22.1 pg/mL (range 1.2 to 31.6 pg/mL, n=10) and in various disease types the average levels (10-14 donors each) were 90.5 pg/mL in Ulcerative Colitis, 7.1 pg/mL in Chron’s, 7039.1 pg/mL in Arthritis, 83.1 pg/mL in Psoriasis, 107.5 pg/mL in Melanoma, 3.8 pg/mL in Renal Cancer, 58.3 pg/mL in NSCLC and 10.6 pg/mL in Sepsis.
Conclusion: The assay development was successful, antibodies were identified and taken into validation. The assay had good performance and successfully passed the validation criteria for exploratory clinical use. The assay was used for analysis of normal and various diseased donor samples. The results show that CTLA4 levels were different in plasma from the different disease types tested, suggesting that the assay is sensitive enough for exploratory clinical use. The assay is expected to be used in assessing plasma levels of CTLA4 in support of the development of novel oncology immunotherapies.