DEVELOPMENT AND VALIDATION OF A METHOD FOR DETERMINING NEUTRALIZING ANTIBODIES TO PEMBROLIZUMAB IN HUMAN SERUM BASED ON INHIBITION OF BINDING OF PEMBROLIZUMAB TO ITS PD-1 TARGET BY ELISA

Abstract

Monoclonal antibodies (mAbs) have the potential to trigger undesired humoral immune responses in the patients and the formation of ADA (anti-drug antibody) to a drug protein.

Neutralizing anti-drug antibodies are one of the main factors affecting the safety and effectiveness of the therapy. If it is impossible to use cell-based tests to determine neutralizing antibodies, competitive ligand binding assay it can be used as an alternative.

Pembrolizumab is a broad-spectrum antitumor drug and is a humanized IgG4 kappa antibody to the programmed cell death receptor-1 (PD-1) that blocks the interaction of the receptor with its ligands PD-L1 and PD-L2. Due to the complex analysis, the use of a cell culture test for pembrolizumab is impossible, as there is a high risk of obtaining unreliable results.

Aim. Development and validation of a method for determining neutralizing antibodies to pembrolizumab in human serum based on inhibition of binding of pembrolizumab to its PD-1 target.

Material and methods. The experimental drug pembrolizumab RPH-075 (R-Pharm) was used in the study. Anti-Pembrolizumab antibodies KRIBIOLISATM Anti-Pembrolizumab (KEYTRUDA®) ELISA, India) were used as a positive control sample for neutralizing antibodies. The determination of antibodies was carried out by the ELISA method with using acid dissociation of the immune complex and the ACE technique (Affinity capture elution).

Results. The ELISA method was validated according to the following characteristic: selectivity, sensitivity, specificity, "hook" effect, drug tolerance, precision. Due to the use of sample pretreatment approaches (acid dissociation, ACE technique) for the analysis of neutralizing antibodies, a sensitivity of 100 ng/ml was achieved in the presence of pembrolizumab 40 μg/ml.

In this paper, a method for calculating the cut-point, sensitivity, and selectivity based on ROC analysis and floating exclusion limit (PSCP) through the average values of OD NC and LPC in each individual analytical cycle was substantiated.

Conclusion. The developed method for determining neutralizing antibodies to pembrolizumab can be used to assess the undesirable immunogenicity of the pembrolizumab at the stage of clinical trials.

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