A reporter gene assay for measuring the bioactivity of anti‐LAG‐3 therapeutic antibodies

Wang, Lan; Yu, Chuanfei; Wang, Kaiqin; Wang, Junzhi

doi:10.1002/bio.3905

Cited by 3 publications

(2 citation statements)

References 32 publications

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“…mediated by CHO cells ectopically expressing anti-CD3 molecules (CHO/aCD3) ( 36 , 37 ). Such non-specific TCR triggering of therapeutic antibodies targeting several checkpoints, such as TIGIT ( 17 ), PD-L1 ( 38 ), LAG-3 ( 39 ) has been extensively validated. Here, we show that the response of the Jurkat E7-TCR reporter system was comparable in magnitude to the one obtained through the standard CHO/aCD3 system, while offering a more physiological mechanism of triggering T cell activation in an antigen-specific manner and negating the need to manipulate target cells with the anti-CD3 scFv.…”

Section: Discussionmentioning

confidence: 99%

“…mediated by CHO cells ectopically expressing anti-CD3 molecules (CHO/aCD3) (36,37). Such nonspecific TCR triggering of therapeutic antibodies targeting several checkpoints, such as TIGIT (17), PD-L1 (38), LAG-3 (39) has been extensively validated. Here, we show that the response of the Jurkat E7-TCR reporter system was comparable in magnitude to the one obtained through the standard CHO/aCD3 system, while offering a Antigen-specific bioluminescence production by Firefly and Gaussia luciferase reporter Jurkat cells upon endogenous HPV16-E7 11-20 presentation (A) Percentage of increase in relative luminescence units (RLU) of Jurkat Fluc.E7-TCR cells upon co-culture with a panel of cell lines with different HLA-A2 and HPV16 expression.…”

Section: Discussionmentioning

confidence: 99%

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A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics

Freile¹,

Qi²,

Jacob³

et al. 2023

Front. Immunol.

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Investigations into the strength of antigen-specific responses in vitro is becoming increasingly relevant for decision making in early-phase research of novel immunotherapeutic approaches, including adoptive cell but also immune checkpoint inhibitor (ICI)-based therapies. In the latter, antigen-specific rapid and high throughput tools to investigate MHC/antigen-specific T cell receptor (TCR) activation haven’t been implemented yet. Here, we present a simple and rapid luminescence-based approach using the human papillomavirus 16 (HPV16) E711-20 peptide as model antigen and E7-TCR transgenic Jurkat.NFAT-luciferase reporter cells. Upon E7 peptide pulsing of HLA-A2+ cell lines and macrophages, an effector to target ratio dependent increase in luminescence compared to non-pulsed cells was observed after co-incubation with E7-TCR expressing Jurkat, but not with parental cells. Analogous experiments with cells expressing full-length HPV16 identified that E7-specific activation of Jurkat cells enabled detection of endogenous antigen processing and MHC-I presentation. As proof of concept, overexpression of established checkpoints/inhibitory molecules (e.g., PD-L1 or HLA-G) significantly reduced the E7-specific TCR-induced luminescence, an effect that could be restored after treatment with corresponding targeting antagonistic antibodies. Altogether, the luminescence-based method described here represents an alternative approach for the rapid evaluation of MHC-dependent antigen-specific T cell responses in vitro. It can be used as a rapid tool to evaluate the impact of the immunosuppressive tumor microenvironment or novel ICI in triggering effective T cell responses, as well as speeding up the development of novel therapeutics within the immune-oncology field.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics

Freile¹,

Qi²,

Jacob³

et al. 2023

Front. Immunol.

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Development and application of potency assays based on genetically modified cells for biological products

Liu

Zhou

Wang

2023

Journal of Pharmaceutical and Biomedical Analysis

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Dissociation of LAG-3 inhibitory cluster from TCR microcluster by immune checkpoint blockade

Hashimoto-Tane,

Bowman,

Sakuma

et al. 2024

Front. Immunol.

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Lymphocyte activation gene (Lag)-3 is an inhibitory co-receptor and target of immune checkpoint inhibitor (ICI) therapy for cancer. The dynamic behavior of Lag-3 was analyzed at the immune synapse upon T-cell activation to elucidate the Lag-3 inhibitory mechanism. Lag-3 formed clusters and co-localized with T-cell receptor microcluster (TCR-MC) upon T-cell activation similar to PD-1. Lag-3 blocking antibodies (Abs) inhibited the co-localization between Lag-3 and TCR-MC without inhibiting Lag-3 cluster formation. Lag-3 also inhibited MHC-II-independent stimulation and Lag-3 Ab, which did not block MHC-II binding could still block Lag-3’s inhibitory function, suggesting that the Lag-3 Ab blocks the Lag-3 inhibitory signal by dissociating the co-assembly of TCR-MC and Lag-3 clusters. Consistent with the combination benefit of PD-1 and Lag-3 Abs to augment T-cell responses, bispecific Lag-3/PD-1 antagonists effectively inhibited both cluster formation and co-localization of PD-1 and Lag-3 with TCR-MC. Therefore, Lag-3 inhibits T-cell activation at TCR-MC, and the target of Lag-3 ICI is to dissociate the co-localization of Lag-3 with TCR-MC.

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A reporter gene assay for measuring the bioactivity of anti‐LAG‐3 therapeutic antibodies

Cited by 3 publications

References 32 publications

A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics

A luminescence-based method to assess antigen presentation and antigen-specific T cell responses for in vitro screening of immunomodulatory checkpoints and therapeutics

Development and application of potency assays based on genetically modified cells for biological products

Dissociation of LAG-3 inhibitory cluster from TCR microcluster by immune checkpoint blockade

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