(M1530-09-50) Design of Total VEGF Assay Provides Simple Data Interpretation of Anti-VEGF Therapeutic Study

Purpose: Vascular Endothelial Growth Factor (VEGF) is an angiogenic factor associated with an array of diseases, including several cancers and retinopathy, making this protein both a valuable biomarker and therapeutic target. The development of a VEGF assay in the presence of an anti-VEGF therapeutic could be informative, indicating therapeutic efficacy by measuring “free” VEGF, or tracking the expression of VEGF throughout disease progression with “total” VEGF. This work details the development of a serum total VEGF assay in the presence of anti-VEGF therapeutics using the same antibody pair as the paired free VEGF assay with added acid dissociation and quenching steps. Here we demonstrate sensitive and specific measurement of total VEGF using electrochemiluminescence (ECL) in the presence of anti-VEGF therapeutics in serum with minimal sample manipulation and dilution.

Methods: The total VEGF assay was developed using commercially available antibodies. Briefly, diluted serum samples were acid-treated for 10-15 minutes with a small volume of HCl, then neutralized with a Tris-based solution containing a vast molar excess of biotinylated anti-VEGF antibody. The samples were transferred after incubation to a streptavidin-coated plate and allowed to bind for one hour. After a wash, a SULFO-tagged anti-VEGF antibody was added to label captured VEGF and ECL detection on the MSD platform was performed.

Results: The total VEGF assay demonstrated sufficient tolerance to the anti-VEGF therapeutic in serum. Testing of patient samples, revealed high ( >1000 pg/mL) VEGF concentration in the Total VEGF assay ( acid-treated samples) independent of the patient anti-VEGF treatment status. VEGF levels in the “Free” assay ranged between 1.2 and 167-fold lower than the total assay, suggesting a high degree of VEGF sequestration. Treatment of samples with an excess of VEGF scavenger before acid treatment showed a decrease in VEGF detection in the total VEGF assay, in some samples to below the limit of detection (LOD) of the assay, demonstrating the selectivity of the assay. Testing of the bound fraction of sample eluate processed through IgG-depletion columns showed VEGF was present in the bound fraction as well.

Conclusion: The total VEGF assay revealed interfering substances not attributable to the drug of interest, making the Free/Total paired assays less indicative of therapeutic efficacy. The high concentration of total VEGF in some clinical samples adds another dimension to the data analysis of the clinical samples for using VEGF as a biomarker for therapeutic efficacy and disease progression. The detection of VEGF in IgG bound fractions suggests the possibility of higher order molecular immune complexes present in serum, potentially confounding VEGF measurements or tracking with an immune response. This approach allowed for the same critical reagents in the total and free assays with only a few additional steps to dissociate and re-bind the target for detection, a strategy that can be employed with other Free/Total paired assays. Although this approach has its limitations in drug tolerance, it minimizes the impact of sample processing and dilution on the assay bias and sensitivity, allows for detection and quantitation of samples through disease progression and treatment, and provides more direct data analysis of clinical samples.

Acknowledgments: Funding for this work was provided under a fee for service contract with a Sponsor. The Sponsor had verified that the patient informed consent documents permitted use of the samples in the manner described.

Total VEGF Assay Format



VEGF Recovery vs. Anti-VEGF Drug Concentration



VEGF Sequestration in Clinical Serum Samples, pg/mL