PIPE‐cloned human IgE and IgG4 antibodies: New tools for investigating cow's milk allergy and tolerance
Authors: Christina L. Pranger, Judit Fazekas‐Singer, Verena K. Köhler, Isabella Pali‐Schöll, Alessandro Fiocchi, Sophia N. Karagiannis, Olatz Zenarruzabeitia, Francisco Borrego, Erika Jensen‐Jarolim
Journal: Allergy (2020)

Cow's milk (CM) allergy (CMA) is defined as an immune-mediated adverse response to CM proteins. 2% to 3% of children are suffering from CMA, but many develop natural tolerance after 3-4 years.1,S01 Food allergen immunotherapy (FA-AIT) applying increasing antigen doses (oral immunotherapy, OIT) can contribute to improvement of CMA,S02 with variable clinical efficacy,2,S03 but immunologically often resulting in decreased specific IgE levels and increased specific IgG4 levelsS04,S05. IgG4 is (a) anti-inflammatory as it does not activate the complement system; (b) bi-specific due to fab-arm exchange and, thus, has less crosslinking capacity than IgE, but has (c) blocking capacity.3 The interplay of IgE and IgG4 may hence be decisive for the immune balance in CMA. Antibodies of different isotypes with the same variable region could be essential in molecular studies of the mechanism underlying CMA and might shed light onto the function of specific IgG4 antibodies during the acquisition of tolerance. Polymerase Incomplete Prime Extension (PIPE) cloning is a cutting-edge technique to engineer antibodies against various targets and facilitates simple exchange of the constant regions to increase the range of subtypes of antibodies.4-6 In this study, we combined variable regions from the high-affinity IgE antibody "D1"7 (KD = 1.4 × 10−9 M; kon = 1.4 × 106, koff = 1.54 × 10−3)S06 specific for the major CM allergen beta-lactoglobulin (BLG), with the constant domains of IgE or IgG4 antibodies (Figure 1A). Correct antibody assembly was confirmed by SDS-PAGE in comparison with commercial antibodies of the same isotype (Figure 1B). The exclusive specificity of D1 antibodies was proven in ELISA (Figure 1C), in ISAC112 microarray (Figure 1D,E) and in ImmunoCAP (data not shown). Antibodies were furthermore species-specific to bovine BLG when testing milk samples from various animal species and showed a sensitivity comparable to a commercial anti-BLG-ELISA (Figure S1). We next applied our antibodies for the quantification of BLG-specific IgE and IgG4 antibodies in serum of milk allergic patients (n = 9) and sensitised but tolerant patients (n = 8). Allergic children had significantly higher levels of IgE (P < .01), but no differences in the IgG4 levels were seen between the groups (Figure S2), in accordance with previous studies.8 Our data suggest that the PIPE-cloned antibodies may be useful for a precise quantification of allergen-specific IgE and IgG4. This is advantageous, as specific IgE is usually quantified by comparing the reactivity to IgE absorbed to a solid phase, likely leading to an underestimation of specific IgE and semi-quantitative results. Basophil activation test (BAT) is a reliable method to evaluate functional capacities of antibodies, allowing at least an approximation to the real-life situation of an allergic patient. When IgE-stripped basophils from healthy donors were sensitised with D1 IgE antibodies and triggered with BLG, basophil activation levels comparable to primary basophils from milk allergic patients in other studies were achieved.9 Activation of the basophils, measured by the surface expression of the activation marker CD63, was specifically seen after stimulation with BLG, but not with the control milk allergen casein (P < .05; Figure 2A) and this was associated with increased production of the intracellular Th2 cytokines IL-4 (P = .0187) and IL-13 (P = .0048; Figure 2B). Overall, the results propose that our IgE antibodies are functional and may be useful tools to standardise BAT assays to achieve comparability between different laboratories. The blocking capacity of D1 IgG4 antibodies was investigated in an ELISA. As expected, the D1 IgG4, harbouring the same variable region, could decrease D1 IgE binding to plate-bound BLG in a concentration-dependent manner (Figure 2C). In accordance with these results, D1 IgG4 inhibited the activation of IgE-stripped, IL-3 primed, D1 IgE-sensitised and BLG-stimulated basophils from healthy donors in a dose-dependent manner. The addition of 50 nmol/L (P = .0402) or 100 nmol/L D1 IgG4 (P = .0029) significantly decreased the percentage of CD63+ basophils (Figure 2D). In agreement with others, our findings confirmed the importance of the IgG4:IgE-ratio, as no inhibition was achieved with the lowest concentration of D1 IgG4 (10 nmol/L). In line with our D1 IgE/D1 IgG4-model, overlapping epitope-binding of IgG4 and IgE antibodies may play an essential role in acquiring tolerance.8 At the same time, this is also the limitation of our molecular model: the situation in allergic patients with polyclonal antibodies, binding to various epitopes on the allergen, is more complex. However, in a parallel study, with a PIPE-cloned monoclonal IgG4 against the major birch pollen allergen Bet v 1, we were able to demonstrate its blocking capacity using the sera from birch allergic patients.S07 Whether the IgG4 antibodies could be applied as passive immunotherapy in cow milk allergic patients depends on a thorough risk-benefit analysis, but studies in cat allergy promise that this might be possible in the future.S08 Our antibodies have so far proven valuable not only for the quantification of BLG and BLG-specific human serum antibodies, but also for functional studies. With these tools, some questions could be addressed in the future: Are IgG4 antibodies involved in the establishment of persisting tolerance, or in primary tolerance to milk? Contrasting results about the efficacy of oral immunotherapies for CMA may be explained by differences in the methods regarding the duration of the intervention, dosages of the allergen or the composition of the milk. The PIPE-cloned antibodies presented here may add to the comparability of such studies between laboratories. Another application of the D1 antibodies may be in studies on the effect of milk processing on immune responses. Food processing can massively influence the structure of the allergens and therefore alter their allergenicity.S09,S10 Especially during heat processing, important BLG epitopes are lost, but IgE binding is significantly increased after pasteurisation.S11 Overall, the generated antibodies may shed light on milk allergenicity, bring critical insight into the development of CMA and help developing prevention strategies. We would like to thank Gerlinde Hofstetter for her excellent help in the laboratory and Mira Matz for her assistance with antibody purification. We acknowledge Iñigo Terrén, Joana Vitallé and Ane Orrantia for their practical assistance and interesting discussions at the Biocruces Bizkaia Health Research Institute, Barakaldo, Spain. EJJ is founder and shareholder of Biomedical Int. R+D GmbH, Vienna, Austria, and business partner of Bencard Allergie GmbH, Germany as well as AllergyTherapeutics Ltd., UK, but has no COI in relation to the presented work. EJJ also holds patents in lipocalins including BLG for allergen immunotherapy. SNK is founder and shareholder of Epsilogen Ltd. and holds patents on IgE antibodies for cancer therapy. AF is conducting research supported by Danone SA and Hipp GmbH. He has been in the advisory boards of Abbott SA and Hipp GmbH over the last two years. The other authors have no potential conflicts of interest to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.