(T1330-01-05) CTLA4 SPLEX Assay Validation and Analysis on Disease and Healthy Donor Sample

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. An In house developed standard sensitivity MSD assay for CTLA4 measurement lacked adequate sensitivity, the Lower limit of Quantitation (LLOQ) was 1pg/mL and, in multiple cases, sample were below this limit, so a higher sensitivity assay was requested and developed on the MSD SPLEX format. Here we describe the screening of assay reagents along with validation of the best pair in the High Sensitivity MSD SPLEX format intended for exploratory clinical use. Proof of utility was evaluated using normal and diseased serum samples from NSCLC and Melanoma.

Methods: The CTLA4 antibodies were evaluated with calibrator proteins generated In-house. The best performing pair was optimization for best sensitivity. The final pair was evaluated for dilution linearity, parallelism, and matrix interference using serum samples. The assay was developed on the MSD SPLEX format and appears to have very good sensitivity and specificity for the intended needs, with a calibration curve range of 2 to 4000 fg/mL. The assay was optimized and taken into validation for accuracy precision, recovery and stability in fit for purpose validation in preparation for exploratory clinical use.

Results: The best pair of antibodies had very good sensitivity, performance, and specificity. The minimum required dilution was 2-fold allowing for dilution of plasma interference and keeping good sensitivity. The CTLA4 SPLEX 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 are different mean measured level of CTLA4 in different diseases. In healthy donors CTLA4 was 38.5 fg/mL (range 27.5 to 53.6 fg/mL, n=8) and in disease types tested the average levels (10 donors each) was 92.1 fg/mL in NSCLC, 154 fg/mL in Melanoma.

Conclusion: The CTLA4 SPLEX assay was successfully developed and taken into validation. The assay had good performance and passed the validation criteria for exploratory clinical use. The CTLA4 SPLEX assay had a LLOQ of 20 fg/mL, by contrast the standard CTLA4 assay had an LLOQ of 1pg/mL. The CTLA4 SPLEX assay was used for analysis of normal and various diseased donor samples. The results show that CTLA4 levels were different in serum from the different disease types tested with no samples falling below the assay limit, suggesting that the assay is superior in sensitivity to the standard in-house CTLA4 assay and suitable for exploratory clinical use. The assay will be used in assessing plasma levels of CTLA4 in support of the development of novel oncology immunotherapies.


CTLA4 SPLEX Assay Typical Calibration Curve


CTLA4 Levels in Normal and Diseased Samples