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• W2109201764 abstract "The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcγ receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit. The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcγ receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit. In mammals, the innate immune system has evolved to principally distinguish between self and non-self and to sense a large spectrum of invading microbes. Recognition of pathogenic microorganisms evokes a proinflammatory response that is essential for the survival of the host organism and does not necessarily require previous exposure to the pathogen in question. Induction of an effective innate immune response requires recognition of conserved microbial ligands such as lipopolysaccharide (LPS), 3The abbreviations used are: LPS, lipopolysaccharide; Mal, MyD88 adaptor-like protein; TRIF, TIR-containing adaptor protein; TRAM, TRIF-related adaptor molecule; iDC, immature dendritic cell; DAP12, DNAX-activating protein 12; TREM, triggering receptor expressed on myeloid cells; IL, interleukin; HUVEC, human umbilical vein endothelial cell; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; WT, wild type; mAb, monoclonal antibody; TLR, Toll-like receptor; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; cfu, colony-forming units; h, human. lipoproteins, lipoteichoic acids, flagellin, and CpG DNA. These ligands are collectively referred to as pattern-associated molecular patterns (1Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6247) Google Scholar). Several receptor types have been shown to participate in the recognition of microbial ligands and are collectively known as pattern recognition receptors. One family that has been the focus of intense research over the past 10 years is the Toll-like receptor (TLR) family, originally identified based on their homology to the Drosophila Toll protein (2Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4458) Google Scholar) and known to play a key role in the recognition of pathogens across a broad range of species. This family contains at least 10 mammalian homologs collectively recognizing a wide range of pattern-associated molecular patterns (3Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1270) Google Scholar). The TLRs have a common structure characterized by a leucine-rich repeat extracellular region and a cytoplasmic region essential for downstream signaling, which is homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. This region is known as the Toll/IL-1R (TIR) domain and is the region sharing homology with Drosophila Toll (2Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4458) Google Scholar, 4Gay N.J. Keith F.J. Nature. 1991; 351: 355-356Crossref PubMed Scopus (458) Google Scholar). TLR4 is arguably the best characterized receptor of the mammalian TLR family and has been shown to recognize the major structural component of the cell wall of Gram-negative bacteria, LPS (5Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6478) Google Scholar, 6Qureshi S.T. Lariviere L. Leveque G. Clermont S. Moore K.J. Gros P. Malo D. J. Exp. Med. 1999; 189: 615-625Crossref PubMed Scopus (1356) Google Scholar, 7Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1626) Google Scholar). Exposure of mammals to LPS as a result of bacterial infection leads to rapid cell activation via TLR4. The signaling pathways known to be involved to date exploit several adaptor proteins, including myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like protein (Mal), TIR-containing adaptor protein (TRIF), and TRIF-related adaptor molecule (TRAM) (8Kawai T. Akira S. Semin. Immunol. 2007; 19: 24-32Crossref PubMed Scopus (1270) Google Scholar). It appears that the LPS response can be divided into an MyD88/Mal-dependent “early phase” NF-κB activation (characterized by the production of many proinflammatory cytokines such as TNF-α and IL-6) and a TRIF/TRAM-dependent phase characterized by “late phase” NF-κB activation and the interferon regulatory factor 3 (IRF3) activation (characterized by the production of interferon-β) (9Palsson-McDermott E.M. O'Neill L.A. Immunology. 2004; 113: 153-162Crossref PubMed Scopus (967) Google Scholar). In certain cases, overactivation of the innate immune system via pattern recognition receptors such as TLR4, resulting in the systemic release of high levels of proinflammatory cytokines, is thought to trigger the majority of symptoms associated with acute inflammatory disorders such as septic shock (10Annane D. Bellissant E. Cavaillon J.M. Lancet. 2005; 365: 63-78Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar, 11Cohen J. Nature. 2002; 420: 885-891Crossref PubMed Scopus (2179) Google Scholar, 12Hotchkiss R.S. Karl I.E. N. Engl. J. Med. 2003; 348: 138-150Crossref PubMed Scopus (3244) Google Scholar). In order for TLR4 to respond effectively to LPS and initiate signal transduction, contribution from the co-receptors myeloid differentiation protein 2 (MD-2) and CD14 is essential (13Nagai Y. Akashi S. Nagafuku M. Ogata M. Iwakura Y. Akira S. Kitamura T. Kosugi A. Kimoto M. Miyake K. Nat. Immunol. 2002; 3: 667-672Crossref PubMed Scopus (858) Google Scholar, 14Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3420) Google Scholar, 15Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1760) Google Scholar). TLR4, MD-2, and CD14 are expressed on many immune cells, including monocytes, macrophages, and dendritic cells. Innate immune recognition of LPS is initiated by its interaction with the serum protein LPS-binding protein (16Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1381) Google Scholar), followed by a rapid transfer to membrane-bound or -soluble CD14. This sequence of events is required for LPS recognition by the TLR4-MD-2 complex and signal transduction (17da Silva C.J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). MD-2 is a secreted glycoprotein that serves as an extracellular adaptor protein for TLR4 activation and itself is key for ligand recognition by TLR4. MD-2 must be bound to TLR4 on the cell surface for receptor activation to occur. LPS has been shown to directly bind MD-2, subsequently inducing an interaction with the leucine-rich repeats of TLR4 leading to TLR4 aggregation and downstream signal transduction (18Visintin A. Latz E. Monks B.G. Espevik T. Golenbock D.T. J. Biol. Chem. 2003; 278: 48313-48320Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). LPS stimulation of TLR4-MD-2 has been described to induce a micro-domain on the cell surface containing an “activation cluster” comprising several cell surface molecules, including CD14, Fcγ receptors (CD16, CD32, CD64), CD55, CD11b/CD18, CD36, and CD81 (19Pfeiffer A. Bottcher A. Orso E. Kapinsky M. Nagy P. Bodnar A. Spreitzer I. Liebisch G. Drobnik W. Gempel K. Horn M. Holmer S. Hartung T. Multhoff G. Schutz G. Schindler H. Ulmer A.J. Heine H. Stelter F. Schutt C. Rothe G. Szollosi J. Damjanovich S. Schmitz G. Eur. J. Immunol. 2001; 31: 3153-3164Crossref PubMed Scopus (272) Google Scholar). In this study we describe the generation of a TLR4 mAb antagonist. The mAb is highly specific for human TLR4 in the presence or absence of MD-2 and potently blocks the effects of LPS stimulation on a variety of isolated cell types and cells in human whole blood. 15C1 was found to bind to a functionally important region of the extracellular region of TLR4 known to be important for LPS recognition and receptor activation. Furthermore, we show that the potency of this mAb is enhanced on cells bearing the regulatory Fcγ receptor CD32A, and we demonstrate a functional contribution of the Fc portion of the mAb. These results suggest a cross-talk between the TLR4 and CD32A signaling pathways. We propose 15C1 as a potent novel inhibitor of TLR4 with potential therapeutic applications in acute inflammatory disorders such as septic shock driven by Gram-negative bacterial infection, and we identify the importance of addressing the role of CD32A to obtain maximal clinical efficacy. Reagents—Anti-human TLR2 mAb T2.5 (mouse IgG1 κ), anti-human TLR4 mAb HTA125 (mouse IgG2a κ), and anti-human CD14 mAb 28C5 (mouse IgG1 κ) were described previously (15Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1760) Google Scholar, 20Meng G. Rutz M. Schiemann M. Metzger J. Grabiec A. Schwandner R. Luppa P.B. Ebel F. Busch D.H. Bauer S. Wagner H. Kirschning C.J. J. Clin. Investig. 2004; 113: 1473-1481Crossref PubMed Scopus (187) Google Scholar, 21Pugin J. Schurer-Maly C.C. Leturcq D. Moriarty A. Ulevitch R.J. Tobias P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2744-2748Crossref PubMed Scopus (731) Google Scholar). T2.5 and HTA125 were purchased from eBioscience. Anti-human TLR4 mAb 15C1 (mouse IgG1 κ) and the anti-human MD-2 mAb 18H10 (mouse IgG2b κ) were generated as described previously (22Pugin J. Stern-Voeffray S. Daubeuf B. Matthay M.A. Elson G. Dunn-Siegrist I. Blood. 2004; 104: 4071-4079Crossref PubMed Scopus (84) Google Scholar). Isotype controls were purchased from BD Biosciences; anti-human CD32 (AT10) was from Serotec, and anti-human CD32 (IV.3) was from StemCell. All mAbs were tested on human whole blood and found to have no effect on cell viability using TruCOUNT™ (BD Biosciences). Ultrapure Escherichia coli K12LCD25 LPS and Pam3CSK4 were purchased from InvivoGen. The E. coli (wild type) strain was isolated in a 70-year-old male patient who developed sepsis of urinary origin caused by these bacteria (a gift from Dr. P. Rohner, University Hospital of Geneva, Switzerland). This strain was sensitive to all antibiotics tested, except for first generation cephalosporins. Bacteria were heat-inactivated as described previously (23Elson G. Dunn-Siegrist I. Daubeuf B. Pugin J. Blood. 2007; 109: 1574-1583Crossref PubMed Scopus (162) Google Scholar). Human umbilical vein endothelial cells (HUVEC) were from Dr. F. Mach, University Medical Center, Geneva, Switzerland. The HEK 293, CHO, and Sp2/0 cell lines were obtained from the American Type Culture Collection (ATCC). PEAK™ cells were purchased from Edge Biosystems. A stably transfected cell line expressing human TLR4-MD-2 was generated as described previously (22Pugin J. Stern-Voeffray S. Daubeuf B. Matthay M.A. Elson G. Dunn-Siegrist I. Blood. 2004; 104: 4071-4079Crossref PubMed Scopus (84) Google Scholar). Generation of Immature Dendritic Cells (iDC)—Peripheral blood mononuclear cells were isolated by Ficoll-Paque™ Plus (Amersham Biosciences) using a buffy coat. Monocytes were then passed on MACS Magnetic Microbeads (Miltenyi Biotec) using a positive selection with anti-human CD14. Purity of the monocytes was confirmed (over 90%) using a FACSCalibur® flow cytometer on the basis of forward and side scatter. The cells were then cultivated in RPMI 1640 medium, 1 mm pyruvate, 1× nonessential amino acids, 0.5 mm β-mercaptoethanol, 10% fetal calf serum, 60 ng/ml granulocyte-macrophage colony-stimulating factor, 200 ng/ml hIL-4 for 8 days, changing the medium every 2 days. Maturation was monitored by FACS using the CD80/CD86 co-stimulatory molecules as well as CD83 (mAbs purchased from Pharmingen) and anti-human CD14-(R)-phycoerythrin (DAKO). The CD32A phenotype for iDC used in this study was CD80/CD86/CD83hi (in comparison to the level on blood-derived monocytes prior to maturation) and CD14neg and heterozygous for the Arg/His polymorphism at amino acid position 131, as determined by PCR (described below). Generation of Mouse-Human Hybrid TLR4 Mutant—To generate MHHH (i.e. containing mouse (M) TLR4 residues at position 23–291), human (H) TLR4, cloned into the mammalian expression vector pCDNA3.1(–)hygro (Invitrogen), was modified by introducing a novel HpaI site and destroying an existing HpaI site by site-directed mutagenesis (QuikChange™ kit, Stratagene). The N-terminal region of mouse TLR4 was PCR-amplified and used to replace the corresponding human DNA fragment in the HpaI-mutated human TLR4 vector by cloning at the unique NotI and HpaI restriction sites. HHHM (i.e. containing mouse TLR4 residues at position 487–629) was generated by PCR-amplifying the C-terminal region of mouse TLR4, which was used to replace the corresponding human DNA fragment in the human TLR4 vector by cloning at the unique EcoRV and XhoI restriction sites. MMHH (i.e. containing mouse TLR4 residues at position 23–371) was generated by modifying the MHHH construct (site-directed mutagenesis) to introduce a unique AgeI restriction site into the TLR4 sequence. In parallel, an internal region of mouse TLR4 was PCR-amplified. This mouse DNA fragment replaced the corresponding human DNA fragment in the AgeI-mutated MHHH vector by cloning at the unique HpaI and AgeI restriction sites. Finally to generate MHMH (i.e. containing mouse residues at positions 23–291 and 371–487), an internal region of mouse TLR4 was PCR-amplified and used to replace the corresponding human DNA fragment in the AgeI-mutated MHHH vector by cloning at the unique AgeI and EcoRV restriction sites. To generate MMHHa and MMHHb, PCR fragments were generated where mouse sequences were introduced in the non-hybridizing region of extended oligonucleotides. These fragments were cloned between the unique HpaI/AgeI sites of MHHH. To generate site-specific TLR4 alanine mutants, site-directed mutagenesis with oligonucleotides was performed using the QuikChange™ kit following manufacturer's guidelines (Stratagene). Cloning and Expression of Recombinant Antibody 15C1—Total RNA extraction from the 15C1 hybridoma cell line and cDNA synthesis was performed using standard techniques. The heavy (H) and light (L) chain variable regions coding for 15C1 mAb were determined using the PCR-based method described previously (24Jones S.T. Bendig M.M. Bio/Technology. 1991; 9: 88-89Crossref PubMed Scopus (3) Google Scholar). The 15C1 L chain variable region was subcloned into the Lonza GS expression vectors containing the human or mouse κ constant region. The 15C1 H chain variable region was subcloned into the Lonza GS expression vectors containing the human γ-4, mouse γ-1, or mouse D265A γ-1 constant region. The genomic versions of the mouse γ-1 and κ constant regions were obtained directly from 15C1 hybridomas cells by nested PCR. The D265A mouse γ-1 mutant was obtained by site-directed mutagenesis using the QuikChange mutagenesis PCR protocol from Stratagene. The full-length 15C1 human IgG4 κ, mouse IgG1 κ, or mouse D265A IgG1 κ antibody was produced by transient transfection of PEAK™ cells. Cells were co-transfected with 0.75 μg of combinations of expression vectors encoding the appropriate full-length L and H chains using FuGENE 6™ transfection reagent (Roche Applied Science). Conditioned supernatants were collected 72 h post-transfection, and recombinant antibody was purified by protein G affinity column chromatography (GE Healthcare). Transient Transfection of TLR4 and MD-2 in PEAK™ Cells—Cells were transfected with human or human/mouse hybrid TLR4 and/or human MD-2. PEAK™ cells were co-transfected with 0.75 μg of combinations of expression vectors encoding human TLR4-myc (N-terminal c-Myc tag), mouse MD-2-FLAG™ (C-terminal FLAG tag) human MD-2-FLAG™ (C-terminal FLAG tag) and chimeras of human and mouse TLR4-myc (N-terminal c-Myc tag, generated in house) using the Fugene6™ transfection reagent (Roche Applied Science). Cells were analyzed 48 h post-transfection. Flow Cytometry—For HUVEC and iDC, 5 × 105 cells/ml were incubated in phosphate-buffered saline, 1% bovine serum albumin, and 10 μg/ml of the appropriate antibody. Cells were washed and incubated in the same buffer with allophycocyanin-conjugated goat anti-mouse IgG antibody (1:250 dilution; Molecular Probes). Cells were analyzed using a FACSCalibur® flow cytometer (BD Biosciences) in the FL-4 channel. For iDC, monoclonal mouse anti-human CD14-(R)-phycoerythrin (DAKO) was used, and cells were analyzed in the FL-2 channel. For circulating leukocytes, the appropriate antibodies (10 μg/ml) were added to human whole blood. Following two washes, cells were incubated with secondary antibody (allophycocyanin-conjugated anti-mouse IgG diluted 1:250) containing 100 μg/ml human IgG (Sigma) to prevent Fc-mediated interactions. Red blood cells were lysed, and the remaining cells were washed twice. Cells were analyzed as above. Different leukocyte populations were distinguished on the basis of forward and side light scatter. HEK 293, HUVEC, and iDC Assays—Cells were plated in 96-well plates at 6 × 104 cells/well. HUVEC cells were cultured on gelatin-coated plates (Sigma). The medium was removed on the day of the experiment, and 30 μl of medium containing 5% heat-inactivated fetal calf serum (human serum for HUVEC) was added. mAbs were diluted in 30 μl of basal medium to the appropriate concentration and added to the cells for 1 h at 37 °C. LPS was diluted in 30 μl of medium, added to the cells, and left to incubate for 21 h at 37 °C. IL-6 (iDC) or IL-8 (HEK 293, HUVEC) secretion in the culture supernatant was monitored by ELISA (Endogen). LPS concentrations were as follows: 100 ng/ml for HUVEC, 30 ng/ml for HEK 293 cells, and 2 ng/ml for iDC. Whole Blood Assays—Fresh heparinized blood from healthy volunteers was obtained by venipuncture and diluted 1:2 with RPMI 1640 medium. Blood was plated at 60 μl/well in a 96-well plate and incubated for 15 min at 37 °C, and in some experiments anti-human CD32 (5 μg/ml final) was added in 60 μl of blood for 1 h. mAbs (15C1 or isotype control) were diluted in RPMI 1640 medium (30 μl final volume) and added to the blood. 1 h later, 30 μl of either E. coli K12 LPS (4 ng/ml final), Pam3CSK4 (100 ng/ml final), or heat-inactivated E. coli WT (107 cfu/ml final) was added to the blood and incubated for 6 h (24 h for heat-inactivated E. coli). These concentrations corresponded to the maximal response observed with these agonists. IL-6 levels were measured by ELISA. In some experiments, TNF-α, IL-8, and IP-10 were measured in parallel by ELISA (R & D Systems). All blood samples used in this study were obtained anonymously, and there is no reference to the donors within. Institutional review board approval for informed consent therefore was not required by our institution. Screening of Healthy Individuals for Their CD32A Genotype at the Histidine or Arginine Polymorphism CD32A (Amino Acid 131)—To genotype the FcγRIIa R131H polymorphism, the method was partly obtained from that described by Carlsson et al. (25Carlsson L.E. Santoso S. Baurichter G. Kroll H. Papenberg S. Eichler P. Westerdaal N.A. Kiefel V. van de Winkel J.G. Greinacher A. Blood. 1998; 92: 1526-1531Crossref PubMed Google Scholar). Briefly, genomic DNA was isolated from sodium citrate-anticoagulated peripheral blood (Vacutainer Systems, BD Biosciences) using QIAamp DNA blood kit (Qiagen). The FcγRIIa-specific PCR amplification was carried out using the HotStar Taq DNA polymerase kit (Qiagen). One hundred and fifty nanograms of genomic DNA was added to 50-μl reaction mixes, containing 1× PCR buffer, 100 μg/ml bovine serum albumin, 2.75 mm MgCl2, 0.25 mm of each dNTP, 0.4 mm each of P63 (5′-CAA GCC TCT GGT CAA GGT C) and P52 (5′-GAA GAG CTG CCC ATG CTG) primers, and 5 units of HotStar Taq (Qiagen). PCR conditions were as follows: 1 cycle at 95 °C for 15 min, 55 °C for 5 min, and 72 °C for 5 min. This was followed by 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, ending with an extension step at 72 °C for 10 min. To analyze the polymorphic region, PCR products amplified by primers P63 and P52 were purified by GeneElute PCR Clean-Up kit (Sigma) and sequenced directly with a modified version of primer P13 (5′-AGG CTT CCA CCC CAC TCC TC). DNA sequencing was performed by Fasteris S.A. (Plan-lesOuates, Switzerland). All average results are presented as means ± S.D. Two-way analysis of variance computation combined with the Bonferroni test was used to analyze data using Prism version 5 (GraphPad). A probability level of 0.05 was considered significant. Generation of a Panel of Anti-TLR4-MD-2 mAbs—Sera from BALB/c mice immunized with transfected CHO cells overexpressing TLR4-MD-2 were screened by FACS for TLR4-MD-2-specific titers. Positive mice were “hyper-boosted” intravenously with a soluble TLR4-MD-2 fusion protein and isolated splenocytes subjected to PEG-mediated fusion with the SP2/0 myeloma cell line as described previously (22Pugin J. Stern-Voeffray S. Daubeuf B. Matthay M.A. Elson G. Dunn-Siegrist I. Blood. 2004; 104: 4071-4079Crossref PubMed Scopus (84) Google Scholar). Supernatants from resulting hybridoma clones were screened for TLR4-MD-2 specific mAb binding on mock-transfected or TLR4-MD-2-transfected CHO cells. The HTA125 anti-TLR4 mAb was used as a positive control to confirm specificity. Supernatants from clones showing specific activity were evaluated for their ability to neutralize LPS-mediated IL-8 production in TLR4-MD-2-transfected HEK 293 cells (data not shown). Twelve clones showing neutralizing activity were expanded, and antibody was purified from supernatants by protein G chromatography. Specificity for the TLR4-MD-2 complex was determined using TLR4-MD-2-transfected PEAK™ cells as described previously (22Pugin J. Stern-Voeffray S. Daubeuf B. Matthay M.A. Elson G. Dunn-Siegrist I. Blood. 2004; 104: 4071-4079Crossref PubMed Scopus (84) Google Scholar). Neutralizing activity of these 12 antibodies was further evaluated in LPS-stimulated human whole blood using IL-6 production as a readout. 7 of the 12 antibodies were found to inhibit LPS in this assay (data not shown). Table 1 summarizes the characteristics of the TLR4-/MD-2 mAbs generated in this study.TABLE 1Mouse monoclonal antibodies generated to the human TLR4/MD-2 complexSpecificityNo. of hybridoma clones binding hTLR4-MD-2 CHONo. of hybridoma clones neutralizing hTLR4-MD-2 HEK 293aData are based on an inhibition of greater than 50% of LPS-induced IL-8 production.No. of hybridoma clones neutralizing whole bloodbData are based on an inhibition of greater than 50% of LPS-induced IL-6 production.TLR464MD-210641TLR4-MD-222a Data are based on an inhibition of greater than 50% of LPS-induced IL-8 production.b Data are based on an inhibition of greater than 50% of LPS-induced IL-6 production. Open table in a new tab 15C1 Binds TLR4 on Transfected and Primary Cells in Vitro—We chose to further characterize mAb 15C1 due to its highly potent inhibition of LPS in whole blood. 15C1 produces a mouse IgG1 κ immunoglobulin. 15C1 binds the TLR4-MD-2 complex on transfected CHO cells in a dose-dependent manner (Fig. 1A) and binds neither the mouse nor rabbit TLR4-MD-2 complexes (data not shown). FACS analysis of transiently transfected PEAK™ cells revealed that like the commercially available TLR4 mAb HTA125, 15C1 binds to TLR4 either in the presence or absence of MD-2 (Fig. 1B). 15C1 also binds to monocytes, granulocytes, iDC, and HUVEC (Fig. 1C). The Epitope for 15C1 Lies in a Functionally Important Region of the TLR4 Extracellular Domain—Human-mouse hybrids were used to determine the region of 15C1 binding to TLR4. As 15C1 does not cross-react with mouse TLR4, expression constructs encoding human-mouse hybrid versions of TLR4 were generated to determine the precise region of human TLR4 containing the epitope recognized by 15C1. The extracellular region of TLR4 was nominally divided into four regions. We then constructed four human-mouse hybrids, MHHH, MMHH, MHMH, and MHHM (Fig. 2A). Each construct was co-transfected into PEAK™ cells along with human MD-2. 15C1 binding was not detected on cells expressing the MMHH construct (Fig. 2B). As cells expressing the MHHH construct were positive for 15C1 binding, we conclude that the epitope is at least partially contained within the 2nd region of human TLR4, corresponding to amino acids 289–375. MHMH was also negative for 15C1 binding. We were unable to draw conclusions from this observation, as MHMH was poorly expressed on the cell surface and was also negative for MD-2 binding, probably suggesting that the hybrid protein was misfolded or conformationally unrepresentative of TLR4. Investigation of the binding of other neutralizing and non-neutralizing mAbs generated in this study using the human-mouse TLR4 hybrids is summarized in Table 2. With one exception, all mAbs showing TLR4-neutralizing activity in human whole blood had epitopes at least partially located in the 2nd region of TLR4 (from the N to the C terminus). All four of a panel of non-neutralizing anti-TLR4 mAbs tested were located in the N-terminal region of TLR4.TABLE 2Epitope localization of anti-TLR4 mAbs on human TLR4 based on binding detected by flow cytometryNo. of hybridoma clones binding to hybrid TLR4 constructsMHHHMMHHMHMHMHHMNeutralizingaData are based on an inhibition of greater than 50% of LPS-induced IL-6 production in human whole blood.0510Non-neutralizing4000a Data are based on an inhibition of greater than 50% of LPS-induced IL-6 production in human whole blood. Open table in a new tab MMHHa and MMHHb were constructed to further define the epitope for 15C1 binding (Fig. 2A). 15C1 bound MMHHa but failed to bind MMHHb, suggesting that the epitope was located in the C-terminal region of the TLR4 fragment spanning amino acids 289–375. To determine amino acids involved in 15C1 binding to TLR4, alanine scanning of this region was performed. The human-mouse alignment for this region is shown in Fig. 2C. Twenty “blocks” of 2–4 amino acids were identified where differences were identified. Wherever complete amino acid mismatches occurred within these blocks, the human amino acid was converted to an alanine by site-directed mutagenesis (for example, block one sequence was converted from LDY to ADA). 15C1 binding to these mutants co-transfected with MD-2 in PEAK™ cells was analyzed by FACS (Fig. 2D). c-Myc binding was determined to assess the expression of these mutants on the cell surface (the TLR4 mutants expressed a c-Myc tag at the N terminus), and 18H10 binding was determined to assess MD-2 binding to the mutants. 15C1 binding is expressed as a ratio between the mean fluorescence intensity of 15C1 and that of c-Myc. 15C1 binding was significantly compromised with mutants 10 (amino acids 328–329), 15 (amino acids 349–351), and 20 (amino acids 369–371), confirming the observed lack of binding with human-mouse hybrid TLR4 mutant MMHHb and suggesting that amino acids located within these blocks are essential for the interaction between 15C1 and human TLR4. 15C1 Potently Inhibits LPS-dependent TLR4 Induction on a Panel of Human Cells—mAb 15C1 was tested at a range of concentrations for inhibition of LPS-induced IL-8 production in TLR4-MD-2 transfected HEK 293 cells along with an irrelevant control mAb and the commercially available anti-human TLR4 mAb HTA 125. In contrast to control mAb and HTA 125, 15C1 showed a dose-dependent inhibition of LPS activity (Fig. 3" @default.
• W2109201764 created "2016-06-24" @default.
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• W2109201764 date "2007-11-01" @default.
• W2109201764 modified "2023-10-10" @default.
• W2109201764 title "Pivotal Involvement of Fcγ Receptor IIA in the Neutralization of Lipopolysaccharide Signaling via a Potent Novel Anti-TLR4 Monoclonal Antibody 15C1" @default.
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