CloneID: NI-0101
Antigen Long Description: The original antibody was generated through a humanization process involving CDR grafting of the original mouse anti-TLR4 clone 15C1 onto selected human germline sequences and performing framework optimization.
Origin Pub PMID: 26013163
Buffer Composition: PBS only.
Chimeric Use Statement: This is a chimeric antibody made using the variable domain sequences of the original human IgG1 format. It was created to improve compatibility with existing reagents, assays, and techniques.
Uniprot Accession No.: O00206
Specificity Statement: This antibody is specific for human TLR4 and binds the LRR 9 domain (residues 328-YN-329, 349-KLK-351); it engages both FcγRI and FcγRIIA, but not FcγRIII.
Application Notes (Clone): 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 Fcγ receptors and TLR4, showing strong binding to FcγRI (47.3 ± 1.3 nM) and FcRn (1.82 ± 0.09 nM) and moderate binding to FcγRIIA 131R (0.151 ± 0.015 µM), FcγRIIA 131H (0.311 ± 0.037 µM), and FcγRIIB (0.870 ± 0.038 µM). 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 Fcγ 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 FcγRI and FcγRIIA engagement, disrupting TLR4 dimers and blocking TLR4 activation, with higher concentrations completely blocking TLR4-FcγR clustering, thus extending the duration of TLR4 inhibition (Shang et al., 2014; PMID: 24737331). The relative contribution of this antibody's arms on its avidity was explored by testing various formats, revealing that its increased potency on monocytic cell lines is primarily mediated by Fcγ receptor engagement, notably through FcγRI binding (Loyau et al., 2014; PMID: 25484053). This antibody's 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).
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