An inhibitory epitope of human Toll‐like receptor 4 resides on leucine‐rich repeat 13 and is recognized by a monoclonal antibody

Summary Immune‐checkpoint blockade antibodies have been approved for the treatment of cancer. However, poorly immunogenic tumours are less responsive to such therapies. Agonistic anti‐Toll‐like receptor 4 (TLR4) monoclonal antibodies (mAbs) activate only cell‐surface TLR4; in contrast, lipopolysaccharide (LPS) activates both TLR4 and intracellular inflammatory caspases. In this study, we investigated the adjuvant activity of an anti‐TLR4 mAb in T‐cell‐mediated antitumour immunity. The anti‐TLR4 mAb induced the activation of antigen‐specific T‐cells in adoptive transfer studies. The growth of ovalbumin (OVA)‐expressing tumours was significantly suppressed by administration of OVA and the anti‐TLR4 mAb in combination, but not individually. The antitumour effect of anti‐PD‐1 mAb was enhanced in mice administered with OVA plus the anti‐TLR4 mAb. The OVA‐specific IFN‐γ‐producing CD8 T‐cells were induced by administration of OVA and the anti‐TLR4 mAb. The suppression of tumour growth was diminished by depletion of CD8, but not CD4, T‐cells. The inflammatory response to the anti‐TLR4 mAb was of significantly lesser magnitude than that to LPS, as assessed by NF‐κB activation and production of TNF‐α, IL‐6 and IL‐1β. Administration of LPS (at a dose that elicited levels of proinflammatory cytokines comparable to those by the anti‐TLR4 mAb) plus OVA induced no or less‐marked activation of OVA‐specific T‐cells and failed to suppress tumour growth in mice. In conclusion, the agonistic anti‐TLR4 mAb induces potent CD8 T‐cell‐dependent antitumour immunity and an inflammatory response of lesser magnitude than does LPS. The agonistic anti‐TLR4 mAb has potential as an adjuvant for use in vaccines against cancer.

show abstract

“…43 Data were analysed using FlowJo software (Tree Star, Ashland, OR). 43 Data were analysed using FlowJo software (Tree Star, Ashland, OR).…”

Section: Cell Staining and Flow Cytometrymentioning

confidence: 99%

An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…The TLR4-MD2 dimerization occurs to initiate a downstream signaling cascade (60). The LPS induction enhances macrophage activation markers like CD14, MHC-II and CD86 expressions and results in the internalization of cell surface TLR4 (51,52,(61)(62)(63)(64). As reported previously, activation of TLR4 leads to phosphorylation of NF-kB (55) and thus has a direct correlation with inflammation (58, 59).…”

Section: Discussionmentioning

confidence: 73%

TLR4 is one of the receptors for Chikungunya virus envelope protein E2 and regulates virus induced pro-inflammatory responses in host macrophages

Mahish

Chatterjee

et al. 2023

Front. Immunol.

View full text Add to dashboard Cite

Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.

show abstract

“…Hu 15C1 is a potent anti-TLR4 mAb, which, owing to its efficacy, has reached clinical trials and is being evaluated in RA patients [ 25 ]. The epitope of mAb Hu 15C1 has been mapped by alanine mutagenesis and has been found to be located in the ectodomain of TLR4 near the dimerization interface [ 27 ]. This epitope has also been investigated in another study [ 26 ], in which a new derivative antibody, C2E3, was designed.…”

Section: Resultsmentioning

confidence: 99%

“…These observed impacts including steric clashes, hydrogen bond network disruption or abrogation of antibody binding, can lead to gain or loss in function of the receptor [ 33 , 34 ]. Moreover, Tsukamoto et al, assessed the functional role of TLR4 activation by a dual-luciferase NF-κB reporter assay, and demonstrated that the overexpression of TLR4 wt induced NF-κB activation was reduced upon transfection of the mutated plasmid in which LLR13 residues (TLR4 residing epitope) were mutated [ 27 ]. From these studies, we can assume that the mutation-driven structure change makes the antibody unable to recognize the epitope, and these residues play a functional role in TLR4 activation as they are essential for dimerization.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

Ahmad

Batool

Kim

et al. 2021

IJMS

View full text Add to dashboard Cite

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.

show abstract

An inhibitory epitope of human Toll‐like receptor 4 resides on leucine‐rich repeat 13 and is recognized by a monoclonal antibody

Cited by 8 publications

References 30 publications

An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

An agonistic anti‐Toll‐like receptor 4 monoclonal antibody as an effective adjuvant for cancer immunotherapy

TLR4 is one of the receptors for Chikungunya virus envelope protein E2 and regulates virus induced pro-inflammatory responses in host macrophages

Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

Contact Info

Product

Resources

About