Molecular engineering of a therapeutic antibody for Blo t 5–induced allergic asthma
Authors: J.H. Sherlynn Chan, Yen Leong Chua, Hong Yong Peh, Vojislav Jovanovic, Nicholas R.J. Gascoigne, W. S. Fred Wong, Fook Tim Chew, Brendon J. Hanson, David M. Kemeny, Paul A. MacAry
Journal: Journal of Allergy and Clinical Immunology (2016)

Epidemiological studies have established a causal link between sensitization to house dust mite (HDM) allergens and the development of allergic asthma. Despite the coexistence of the 2 major dust mite species in the tropics and dual sensitization profiles in atopic individuals, IgE responses targeting Blomia tropicalis have limited cross-reactivity with Dermatophagoides pteronyssinus.1Kuo I.C. Cheong N. Trakultivakorn M. Lee B.W. Chua K.Y. An extensive study of human IgE cross-reactivity of Blo t 5 and Der p 5.J Allergy Clin Immunol. 2003; 111: 603-609Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Among all known allergens of B tropicalis, Blo t 5 is the principal sensitizing allergen in tropical countries. Blo t 5 has a high allergenic potency with more than 70% of IgE reactivity in subjects with asthma1Kuo I.C. Cheong N. Trakultivakorn M. Lee B.W. Chua K.Y. An extensive study of human IgE cross-reactivity of Blo t 5 and Der p 5.J Allergy Clin Immunol. 2003; 111: 603-609Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 2Arruda L.K. Fernandez-Caldas E. Naspitz C.K. Montealegre F. Vailes L.D. Chapman M.D. Identification of Blomia tropicalis allergen Blo t 5 by cDNA cloning.Int Arch Allergy Immunol. 1995; 107: 456-457Crossref PubMed Scopus (38) Google Scholar, 3Yi F.C. Lee B.W. Cheong N. Chua K.Y. Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.Allergy. 2005; 60: 108-112Crossref PubMed Scopus (20) Google Scholar and 96.7% in children with asthma.4Shek L.P. Chong A.R. Soh S.E. Cheong N. Teo A.S. Yi F.C. et al.Specific profiles of house dust mite sensitization in children with asthma and in children with eczema.Pediatr Allergy Immunol. 2010; 21: e718-e722Crossref PubMed Scopus (22) Google Scholar As such, Blo t 5 is a potential target for therapeutic approaches in B tropicalis–mediated allergic asthma. Previous attempts at neutralizing allergens have been made using allergen-specific polyclonal IgG antibodies. In this study, we sought to explore the potential of using engineered allergen-specific mAbs as a novel treatment modality for HDM-driven allergic asthma. To facilitate this, we first isolated an mAb with high affinity for Blo t 5 (see Fig E1, B, in this article's Online Repository at www.jacionline.org). Epitope mapping studies with western and immuno-dot blot revealed that this mAb binds a linear epitope on an exposed surface of the Blo t 5 protein (Fig E1, C-E). Interestingly, our antibody targets a region of Blo t 5 previously implicated as a conformational IgE-binding epitope.5Naik M.T. Chang C.F. Kuo I.C. Kung C.C. Yi F.C. Chua K.Y. et al.Roles of structure and structural dynamics in the antibody recognition of the allergen proteins: an NMR study on Blomia tropicalis major allergen.Structure. 2008; 16: 125-136Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar Moreover, a dose-dependent inhibition of Blo t 5–specific IgE binding from allergic patients' sera was observed when coincubated with increasing concentrations of anti–Blo t 5 mAb (Fig 1, A). This implies that our mAb may be binding the Blo t 5 antigen in a way that occludes the binding site of Blo t 5–specific IgE in patients' serum by steric hindrance or distorting the Blo t 5 protein conformation such that Blo t 5–specific IgE in patients' sera can no longer bind. We then engendered chimeric variants in which the murine constant region (Fc) of the antibody was replaced with the constant domains of human IgG1, IgG1-LALA, and IgG4-S228P (Fig 1, B). Integrities of the expressed antibodies were verified by SDS-PAGE under reducing and nonreducing conditions. The chimeric antibodies were assembled with a heavy and light chain (Fig 1, C) and were correctly folded into full-length IgGs as depicted by bands at 150 kDa and larger on a nonreducing gel (Fig 1, D). Binding specificities for Blo t 5 were retained despite the molecular manipulations, with comparable affinities in the nM range as the murine IgG1 antibody (Fig 1, E). The chimeric anti–Blo t 5 mAbs were tested for their ability to inhibit allergen-induced human basophil activation. Expression of CD63 was upregulated on stimulation with Blo t extract and anti-IgE (Fig 1, F) and close to 60% of this response was inhibited in the presence of the chimeric antibodies. The extent of inhibition was the strongest and weakest for the chimeric IgG4-S228P and IgG1 variant, respectively (Fig 1, G). In concordance with the widely documented anti-inflammatory properties of IgG4-blocking antibodies elicited by successful allergen-specific immunotherapy (SIT),6Wachholz P.A. Durham S.R. Mechanisms of immunotherapy: IgG revisited.Curr Opin Allergy Clin Immunol. 2004; 4: 313-318Crossref PubMed Scopus (167) Google Scholar our data intimate that engineered IgG4 antibodies can function as antagonists in allergic diseases by impairing basophilic activity. We next examined the in vivo effects of anti–Blo t 5 mAb treatment in a murine model of Blo t 5–induced severe allergic asthma. As opposed to those generated via ovalbumin sensitization, more relevant animal models of HDM-induced allergic asthma are now well-established. We assessed the therapeutic potential of our anti–Blo t 5 mAb in a murine asthma model involving the passive transfer of Blo t 5–specific TH2 cells to naive mice and subsequently challenging the mice intranasally with Blo t 5 protein. The mice exhibited an asthmatic phenotype characterized by pulmonary eosinophilia, increases in serum IgE levels, and mucus hypersecretion.7Huang C.H. Loo E.X. Kuo I.C. Soh G.H. Goh D.L. Lee B.W. et al.Airway inflammation and IgE production induced by dust mite allergen-specific memory/effector Th2 cell line can be effectively attenuated by IL-35.J Immunol. 2011; 187: 462-471Crossref PubMed Scopus (109) Google Scholar The murine anti–Blo t 5 mAb was administered intravenously at 100 μg per mouse on days 1 and 13 (Fig 2, A). Histologic analyses were performed to investigate the impact of mAb treatment on the structural integrity of asthmatic lungs (Fig 2, B and C). Hematoxylin and eosin staining revealed that mAb administration significantly decreased the overall inflammatory status of the airways by ameliorating epithelial thickening and inflammatory cell infiltration. In addition, goblet cell hyperplasia, mucus hypersecretion, and mucus plugging as denoted by the black arrows were majorly alleviated with mAb treatment. Last, the deposition of collagen fibers (denoted by blue staining) is indicative of airway remodeling and fibrosis. Treatment with anti–Blo t 5 mAb attenuated the extent of airway remodeling as highlighted by the reduced collagen deposits around the airway. Overall, anti–-Blo t 5 mAb treatment notably restored the lung architecture in mice with asthma. Treatment with our mAb significantly reduced the total immune cell count and the infiltration of eosinophils into the airways but did not have an impact on neutrophilia (Fig 2, D). Analysis of cytokines in the bronchoalveolar lavage fluid revealed a suppression in eotaxin levels with no changes in other key TH2 cytokines (see Fig E2 in this article's Online Repository at www.jacionline.org). IL-5 was shown to prime eosinophils to chemoattractants, whereas eotaxin was responsible for directing the actual migration to the site of inflammation.8Simson L. Foster P.S. Chemokine and cytokine cooperativity: eosinophil migration in the asthmatic response.Immunol Cell Biol. 2000; 78: 415-422Crossref PubMed Scopus (43) Google Scholar The recruitment of eosinophils to specific tissues and their subsequent activity within these sites might be IL-5–independent.9Adamko D. Odemuyiwa S.O. Moqbel R. The eosinophil as a therapeutic target in asthma: beginning of the end, or end of the beginning?.Curr Opin Pharmacol. 2003; 3: 227-232Crossref PubMed Scopus (29) Google Scholar It is therefore highly plausible that the diminished eosinophilia observed in mAb-treated mice was an effect of reduced chemotaxis to the airways due to lower levels of eotaxin present. Serologic effects of anti–Blo t 5 mAb administration in severely allergic mice with asthma were also analyzed by ELISA. A 5-fold reduction in total IgE levels was observed in the sera from mice treated with anti–Blo t 5 mAb compared with untreated mice, and mice treated with an isotype control antibody. Mice treated with anti–Blo t 5 mAb also had approximately 50% lower Blo t 5–specific IgE levels (Fig 2, E). Furthermore, key lung function parameters indicate an increase in the FEVs (Fig 2, F) and a decrease in respiratory rate (see Fig E3 in this article's Online Repository at www.jacionline.org) when the mice were treated with anti–Blo t 5 mAb. Collectively, the antiallergic effects of anti–Blo t 5 mAb treatment resulted in a major improvement in asthma symptoms (see Video E1 in this article's Online Repository at www.jacionline.org). In summary, we have engineered an allergen-specific mAb targeting Blo t 5, the major allergen of the HDM species B tropicalis, and showed that this mAb is capable of antagonizing Blo t 5–specific IgE binding in patients' sera. More critically, we have demonstrated, for the first time, the protective effects of an allergen-specific mAb treatment in a murine model of severe allergic asthma driven by a clinically relevant and dominant allergen instead of the traditional ovalbumin-induced asthma. It is also important to recognize that murine models with asthma may not reflect the spectrum of immunologic and physiologic changes that underlie the pathology of established asthma in humans. However, airway remodeling, an important hallmark of chronic asthma that is often missing in animal models, was prominent in the mouse model used in this study and anti–Blo t 5 mAb administration was capable of ameliorating this phenomenon. Last, we have used chimeric antibodies to highlight the potential of using allergen-specific IgG4 antibodies in inhibiting basophil activation. Although immunogenic murine components limit the prolonged use of chimeric antibodies in clinical settings, full antibody humanization can potentially reduce the likelihood of this problem arising. This potentially mirrors the approach used when anti–TNF-α is used for chronic inflammatory diseases. Taken together, this work has important implications on the current understanding of allergic asthma and may have an impact on future design of targeted therapeutics for an allergic disease lacking an optimal treatment regime. Female BALB/c mice (8-12 weeks old) used for mAb generation and male C57BL/6 (8-10 weeks old) used for asthma models were maintained under pathogen-free conditions at the Centre for Comparative Medicine. All experiments were performed in accordance with approved institutional guidelines of the Institutional Animal Care and Use Committee of the National University of Singapore, under protocol number 106/10 for hybridoma development and 015/12 for asthma models. Recombinant Blo t 5 protein was expressed in Escherichia coli strain, BL21, and was purified by size exclusion chromatography and anion exchange chromatography (GE Healthcare Life Sciences). Purified Blo t 5 protein was used to immunize female BALB/c mice intraperitoneally with Freud's adjuvant (Sigma-Aldrich, St Louis, Mo), following which the mice were sacrificed for their spleen. Splenocytes were then fused with myeloma cell line NS1 using polyethylene glycol 8000 (PEG8000) (Sigma-Aldrich). Hybridoma clones were screened and identified for anti–Blo t 5 responses 3 weeks postfusion. Responders were subcloned by limiting dilution to obtain monoclones. Once a putative monoclonal hybridoma clone was selected on the basis of superior binding activity as detected by ELISA, it was expanded and cultured in T175 flasks (Thermo Scientific, Rockford, Ill). Anti–Blo t 5 mAb was subsequently purified from the cell culture supernatant by fast protein liquid chromatography with the HiTrap protein G HP column (GE Healthcare Life Sciences). Briefly, varying concentrations of recombinant Blo t 5 protein (125 ng/mL-2 μg/mL) were diluted in 0.1 mol sodium bicarbonate coating buffer and coated onto Maxisorp plates, overnight at room temperature. The plates were washed with PBS-0.05% Tween-20, blocked with 0.2% BSA, and incubated overnight with serial dilutions of anti–Blo t 5 mAb. The plates were washed again and incubated with goat anti-mouse IgG conjugated with horseradish peroxidase (1:5000 vol/vol in 0.2% BSA; Thermo Scientific). Subsequently, the plates were developed with 1-Step Ultra TMB-ELISA Substrate (Thermo Fisher Scientific) and quenched with 1 mol sulphuric acid. OD was acquired using a microplate reader at 450 nm. Calculations of affinity and dissociation constants were performed as described previously. A peptide library of the Blo t 5 protein comprising 15-mer peptides with 10 overlapping amino acids was used. Five micgrogram of each peptide was dotted onto preactivated polyvinylidene difluoride (PVDF) membrane and 1 μg of recombinant Blo t 5 protein was used as positive control. The membrane was air dried and blocked with 5% skimmed milk before incubating with the anti–Blo t 5 mAb (1:500 in 5% skimmed milk). The membrane was then washed thrice with PBS-0.2% Tween-20 for 7 minutes each followed by incubation with goat antimouse IgG conjugated with HRP (1:10,000 vol/vol in 5% skimmed milk; Thermo Scientific). Washing with PBS-0.2% Tween-20 was repeated. Finally, the membrane was developed with Western Lightning Plus Enhanced Chemiluminescence (Perkin Elmer, Waltham, Mass) and the signals on the membrane were captured on light-sensitive film in the dark room. Serum was obtained via cardiac puncture. Total IgE was measured using the Mouse IgE ELISA Set (BD Biosciences, Franklin Lakes, NJ) according to manufacturer's instructions. To measure Blo t 5–specific IgE, sera were preabsorbed with Protein G Sepharose 4 Fast Flow beads (GE Healthcare Life Sciences) before proceeding with the measurement of IgE as described earlier. PBS perfusion was performed and the lungs were fixed by inflating with 0.8 mL of 4% paraformaldehye (Sigma-Aldrich). Lung tissues were further fixed for at least 2 days in 4% paraformaldehyde. Downstream sample processing and staining procedures were performed by the Advanced Molecular Pathology Laboratory situated at the A*Star Institute of Molecular and Cell Biology. Hematoxylin and eosin–stained lung sections were scored on cell infiltration as previously described.E1Myou S. Leff A.R. Myo S. Boetticher E. Tong J. Meliton A.Y. et al.Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT.J Exp Med. 2003; 198: 1573-1582Crossref PubMed Scopus (198) Google Scholar Similarly, periodic acid-Schiff–stained lung sections were quantified for airway mucus production as previously described.E2McMillan S.J. Xanthou G. Lloyd C.M. Manipulation of allergen-induced airway remodeling by treatment with anti-TGF-beta antibody: effect on the Smad signaling pathway.J Immunol. 2005; 174: 5774-5780Crossref PubMed Scopus (229) Google Scholar Total RNA was extracted from 1 × 106 hybridoma cells using the High Pure RNA Isolation kit (Roche, Basel, Switzerland). Extracted RNA was converted to cDNA using Superscript III First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, Calif). Variable regions of the heavy and light chains were PCR amplified with specific primers using the AmpliTaq Gold DNA polymerase (Thermo Fisher Scientific). PCR products were analyzed by DNA gel electrophoresis, bands at corresponding sizes were excised, and DNA was purified using QIAGEN Gel Extraction Kit. Purified PCR products were then cloned into pTT5 plasmid vectors, which possess the human IgG constant regions (Courtesy of A/P Brendon Hanson). The IgG1-LALA variant carries a L234A and L235A double mutation that has been shown to abolish Fc receptor binding,E3Hessell A.J. Hangartner L. Hunter M. Havenith C.E. Beurskens F.J. Bakker J.M. et al.Fc receptor but not complement binding is important in antibody protection against HIV.Nature. 2007; 449: 101-104Crossref PubMed Scopus (719) Google Scholar, E4Hezareh M. Hessell A.J. Jensen R.C. van de Winkel J.G. Parren P.W. Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1.J Virol. 2001; 75: 12161-12168Crossref PubMed Scopus (212) Google Scholar whereas the IgG4-S228P variant carries a S228P mutation that reportedly eliminates the Fab arm exchange phenomenon commonly associated with IgG4 antibodies.E5Labrijn A.F. Buijsse A.O. van den Bremer E.T. Verwilligen A.Y. Bleeker W.K. Thorpe S.J. et al.Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo.Nat Biotechnol. 2009; 27: 767-771Crossref PubMed Scopus (225) Google Scholar, E6Angal S. King D.J. Bodmer M.W. Turner A. Lawson A.D. Roberts G. et al.A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody.Mol Immunol. 1993; 30: 105-108Crossref PubMed Scopus (199) Google Scholar, E7Bloom J.W. Madanat M.S. Marriott D. Wong T. Chan S.Y. Intrachain disulfide bond in the core hinge region of human IgG4.Protein Sci. 1997; 6: 407-415Crossref PubMed Scopus (106) Google Scholar, E8Schuurman J. Perdok G.J. Gorter A.D. Aalberse R.C. The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.Mol Immunol. 2001; 38: 1-8Crossref PubMed Scopus (149) Google Scholar, E9Silva J.P. Vetterlein O. Jose J. Peters S. Kirby H. The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.J Biol Chem. 2015; 290: 5462-5469Crossref PubMed Scopus (42) Google Scholar Constructs were subsequently transfected into suspension HEK-6E cells with polyethylenimine. Cell culture supernatants were harvested 7 days later and chimeric antibodies were purified with Protein A Sepharose Beads (GE Healthcare Life Sciences). The basophil activation test was performed as previously described.E10Sturm E.M. Kranzelbinder B. Heinemann A. Groselj-Strele A. Aberer W. Sturm G.J. CD203c-based basophil activation test in allergy diagnosis: characteristics and differences to CD63 upregulation.Cytometry B Clin Cytom. 2010; 78: 308-318Crossref PubMed Scopus (75) Google Scholar Briefly, whole blood collected from sensitized donors was stained with Pacific-Blue Anti-human HLA-DR, APC Anti-human CD294 (CRTH2), and fluorescein isothiocyanate (FITC) Anti-human CD63 (Biolegend, San Diego, Calif), following which stained whole blood was incubated with PBS, Blo t extract only, or Blo t extract with varying concentrations of anti–Blo t 5 antibody for 20 minutes at 37°C. Activation was terminated with 3.8% EDTA and trythrocytes were lysed with ammonium chloride potassium lysis buffer. Cells were then washed, resuspended in PBS, and analyzed by flow cytometry on Fortessa FACScan. Basophils were identified as the HLA-DR−/CRTH2+ population (Fig E3). A first gate was made around the lymphocyte population in the side scatter versus forward scatter (FSC) dot plot (Fig E3, A). A second gate was placed at the cell singlet population in the FSC-H versus FSC-A dot plot (Fig E3, B), followed by gating around the CRTH2+ population in the HLA-DR versus CRTH2 dot plot (Fig E3, C). Stimulation was then analyzed by the percentage of CD63+ cells in the absence/presence of the Blo t extract. Data were expressed as means ± SEMs. All data analyses were performed with Graphpad Prism 5 software. Comparisons between 2 groups were assessed with 2-tailed unpaired Student t test and results are considered to be statistically significant when the calculated P value is less than .05.Fig E2Cytokine and chemokine levels in the bronchoalveolar lavage fluid. A, Eotaxin levels. B, IL-4 levels. C, IL-5 levels. D, IL-13 levels. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Pulmonary function parameters. A, Respiratory rate. B, Time of expiratory phase. C, Peak expiratory flow (PEF). D, FEV when PEF is reached. E, Dynamic compliance. *P < .05. **P < .01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Basophil gating strategy. A, SSC vs FSC. B, FSC-H vs FSC-A. C, HLA-DR Pacific Blue vs CRTH2 APC. APC, Allophycocyanin; SSC, side scatter.View Large Image Figure ViewerDownload Hi-res image Download (PPT) eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5MDNjNGM1M2YyODc4MGQ4NGUzYjlkNTcyYzhhNzlmNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3ODY2MzY3fQ.ecyMkP5ov7k9-AxqnspfKmMJ6ANMMhEe4ic9G43Qwab2SlSHS8x0n_RlOL8VZQTYAEK7sjHFHx4mwGOnpXd-KxqiOggFqio1hAgYRYnSq4ko8H6t5CQIGbV500Pai7vTDe_Hi3l4zZdh0TiD2PpwbiX6rvtxMeFNIu8dR21Uzi1LEqHKREay7RwfL-mwU-a9M4sVm8eTiYGiue_IHQ0dCcIiqZaeoiDXrbQ-Eg1h3XL6z-t_cwW_wTqNi6FJPEX07NyDt8TRnSFQAHe337DgSOSw3Q9SSJt0ypTt-yjT3j91tWNOwmAAseENdIr2Kz5dWtiq1RCKbRk1AnT7IQEMkA Download .mp4 (8.3 MB) Help with .mp4 files Video E1