Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells

Abstract

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4 + T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4 + T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.

The self-reactive FVIII T cell repertoire in healthy individuals relies on a short set of epitopes and public clonotypes

“…While this approach does not permit phenotype analyses, it offers the opportunity for cell frequency estimation ( 19 ), epitope identification, and TCR analyses through CDR3β sequencing of AIM-expressing T cells. CD137 (4-1BB) and CD134 (OX-40) used in this study are cell surface markers, which are specifically induced by TCR engagement and expressed by antigen-specific T cells upon antigen recognition ( 29 – 33 ).…”

The immunogenicity of human-origin therapeutic antibodies are associated with V gene usage

“…The immunogenicity risk assessment usually happens after the affinity optimization. Multiple methods are used to assess the immunogenicity risk, including in silico methods (MHC-II binding prediction and "humanness" assessment), in vitro methods (DC loading assay, MAPPS assay and T cell activation assays) and in vivo animal models (rat and non-human primates) (33)(34)(35)(36)(37)(38)(39)(40). Molecules with high immunogenicity risk may require additional modifications to reduce the potential immunogenicity.…”

Reducing Immunogenicity by Design: Approaches to Minimize Immunogenicity of Monoclonal Antibodies

“…If the mAb is immunogenic, the DCs activate the CD4 T cells leading to the production and release of IFN-γ, which is detected by ELISPOT [ 101 ]. Flow cytometric measurement of CD4 T cell activation surface markers, CD134 and CD137, accurately assessed the immunogenicity of multiple mAbs, although this assay was limited in predicting the immunogenicity risk of mAbs that inhibit TNFα, which itself is an immune-activating cytokine [ 102 ].…”

“…Flow cytometric methods using incorporation of the synthetic nucleoside analog of thymidine bromodeoxyuridine (BrdU) during the DNA synthesis phase of cell division, staining for the proliferation marker Ki67, or carboxyfluorescein diacetate succinimidyl ester (CFSE) staining provide less hazardous and more selective approaches to evaluate T cell proliferation. By combining proliferation markers with detection of the presence of CD4 + -selective markers, such flow cytometry-based methods provide single-cell resolution, making them more suitable for studying heterogeneous cell populations [ 102 , 104 , 105 ]. CFSE is a cell-permeable dye that distributes evenly amongst all cells, and its concentration reduces by half with each cell division, enabling detection of up to ten rounds of cell division [ 105 ].…”