Anti-TLR4 [NI-0101], Human IgG1, Fc Silent(tm), kappa

Absolute Antibody

Anti-TLR4 [NI-0101], Human IgG1, Fc Silent(tm), kappa

9

This antibody is specific for human TLR4 and binds the LRR 9 domain (residues 328-YN-329, 349-KLK-351); it engages both FcgammaRI and FcgammaRIIA, but not FcgammaRIII.

The original antibody (human IgG1) was generated and used for SPR, FACS, and various functional assays. SPR determined the binding affinity of Hu 15C1 with Fcgamma receptors and TLR4, showing strong binding to FcgammaRI (47.3 +/- 1.3 nM) and FcRn (1.82 +/- 0.09 nM) and moderate binding to FcgammaRIIA 131R (0.151 +/- 0.015 microM), FcgammaRIIA 131H (0.311 +/- 0.037 microM), and FcgammaRIIB (0.870 +/- 0.038 microM). FACS assays evaluated Hu 15C1 binding to CHO cells expressing human TLR4 and MD-2. Functional assays included human macrophage and U937 cell assays to measure IL-6 production, confocal microscopy, and FRET analysis to visualize TLR4 and Fcgamma receptor interactions, and LPS lung instillation in mice to assess cytokine and antibody levels in bronchoalveolar lavage fluid. Results indicated that Hu 15C1 enhances its inhibitory effect on TLR4 signaling through FcgammaRI and FcgammaRIIA engagement, disrupting TLR4 dimers and blocking TLR4 activation, with higher concentrations completely blocking TLR4-FcgammaR clustering, thus extending the duration of TLR4 inhibition (Shang et al., 2014; PMID: 24737331). The relative contribution of this antibodys arms on its avidity was explored by testing various formats, revealing that its increased potency on monocytic cell lines is primarily mediated by Fcgamma receptor engagement, notably through FcgammaRI binding (Loyau et al., 2014; PMID: 25484053). This antibodys KD value for human TLR4 was measured as 3.52 +/- 0.08 nM via SPR. This antibody exhibited an increased relative binding affinity (compared to clone 1E11) as measured in a competitive ELISA. The fab fragment of this antibody was crystallized and examined (Loyau et al., 2015; PMID: 26013163). This antibody was used in phase I and II clinical trials (Monnet et al., 2017; PMID: 27706798) (Monnet et al., 2020; PMID: 31892533).

Functional Assay, Flow Cytometry, ELISA, Neutralisation/Blocking, Other Application

Research Use Only

Monoclonal

NI-0101

TLR4

toll like receptor 4

ARMD10; CD284; homolog of Drosophila toll; hToll; TLR-4; TOLL; toll like receptor 4 protein; toll-like receptor 4

Human

IgG1

Toll-like receptor 4

This is a reformatted human IgG1 Fc Silent™ antibody, based on the original human IgG1 format, created for improved compatibility with existing reagents, assays and techniques.

Human

-20°C,2°C to 8°C

12352203