AllergoOncology: Generating a canine anticancer IgE against the epidermal growth factor receptor
Authors: Judit Fazekas-Singer, Josef Singer, Kristina M. Ilieva, Miroslawa Matz, Ina Herrmann, Edzard Spillner, Sophia N. Karagiannis, Erika Jensen-Jarolim
Journal: Journal of Allergy and Clinical Immunology (2018)

Cancer immunotherapy with antibodies has revolutionized clinical oncology, giving hope for patients with cancer. Among these, cetuximab, a mouse-human chimeric antibody, targeting the human epidermal growth factor receptor (EGFR),1Ferris R.L. Jaffee E.M. Ferrone S. Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape.J Clin Oncol. 2010; 28: 4390-4399Crossref PubMed Scopus (249) Google Scholar inhibits growth signaling and mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP)2Pozzi C. Cuomo A. Spadoni I. Magni E. Silvola A. Conte A. et al.The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death.Nat Med. 2016; 22: 624-631Crossref PubMed Scopus (169) Google Scholar, E5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar of tumor cells. Harnessing TH2 immune responses against cancer is a subject in the emerging field of "AllergoOncology," taking advantage of the high capacity of IgE to trigger immune reactions against cancer.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar Unlike Fcγ-receptors, all Fcε-receptors are activating.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar Because fewer than 1 in 10 anticancer drugs translate from murine preclinical studies to human clinical trials,4Fulkerson C.M. Dhawan D. Ratliff T.L. Hahn N.M. Knapp D.W. Naturally occurring canine invasive urinary bladder cancer: a complementary animal model to improve the success rate in human clinical trials of new cancer drugs.Int J Genomics. 2017; 2017: 6589529Crossref PubMed Scopus (29) Google Scholar we focused on dogs (Canis lupus familiaris) with spontaneously occurring cancer as potential efficacy models. Mammary carcinomas and osteosarcomas in dogs and humans frequently overexpress EGFR.4Fulkerson C.M. Dhawan D. Ratliff T.L. Hahn N.M. Knapp D.W. Naturally occurring canine invasive urinary bladder cancer: a complementary animal model to improve the success rate in human clinical trials of new cancer drugs.Int J Genomics. 2017; 2017: 6589529Crossref PubMed Scopus (29) Google Scholar, 5Mantovani F.B. Morrison J.A. Mutsaers A.J. Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.BMC Veter Res. 2016; 12: 82Crossref PubMed Scopus (18) Google Scholar Also, human and canine immune systems are highly similar,3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar rendering canine clinical trials a promising alternative to rodent experiments. We previously developed a caninized cetuximab IgG, "can225IgG,"6Singer J. Fazekas J. Wang W. Weichselbaumer M. Matz M. Mader A. et al.Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.Mol Cancer Therapeut. 2014; 13: 1777-1790Crossref PubMed Scopus (23) Google Scholar which here we engineered into cetuximab IgE, "can225IgE-λ," retaining the exact epitope specificity, but with the canine λ- and ε-constant domains. Can225IgE-λ was cloned using a ligation-free cloning techniqueE13Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (12) Google Scholar and expressed in Expi293F cells on the basis of our previously published method7Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (2) Google Scholar (Fig 1, A; see this article's Methods section in the Online Repository at www.jacionline.org). The affinity-purified can225IgE-λ had an apparent molecular mass of 235 kDa (Fig 1, B) and was correctly assembled (Fig 1, C and D) with low amounts of free chains. We confirmed antibody specificity to recombinant human EGFR by Western blot (Fig 1, E), to native EGFR on human A431 breast cancer cells (Fig 1, F), to P114 canine mammary carcinoma (Fig 1, G) and D17 canine osteosarcoma (Fig 1, H) cells, but no binding to EGFR-negative CHO K1 (control) cells (Fig 1, I). Next, we assessed FcεRI expression on effector cell lines. Both human promonocytic U937 and canine monocytic/macrophage-like DH82 cells showed moderate to high FcεRI expression (Fig 1, J and K). Furthermore, canine MPT-1 mast cells showed relatively lower FcεRI expression (Fig 1, L) compared with RBL-SX38 rat basophilic leukemia cells transfected with human tetrameric FcεRI (Fig 1, M). Because no cross-reactive antibody for canine FcεRII (CD23) was available, we evaluated CD23 expression on canine effector cell lines by RT-PCR. We observed a more than 1000-fold higher relative CD23 expression by MPT-1, compared with DH82 cells (Fig 1, N). The can225IgE-λ antibody bound to Fcε receptors on U937 cells (Fig 2, A), DH82 cells (Fig 2, B), and MPT-1 (Fig 2, C) cells. Having shown tumor target specificity and recognition of immune effector cells by can225IgE-λ, we next used a tumor cell killing assayE4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar to quantify immune-mediated tumor cell killing by can225IgE-λ via flow cytometry. Tumor cells killed by cytotoxicity (ADCC) and phagocytosis (ADCP) were counted separately (for gating strategy, see Fig E1, A, in this article's Online Repository at www.jacionline.org). We first used human A431 as cancer target cells, U937 as effector cells, and human and dog cetuximab IgGE5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar and IgE versions. Both cetuximab IgG and IgE showed high tumor cell killing potential, mostly via ADCC (Fig 2, D) mediated by effector cells. Canine IgE shows a 33 times weaker binding strength to the human FcεRI compared with human IgE. Accordingly, can225IgE-λ–triggered tumor killing with human effector cells was significantly lower than that triggered by human cetuximab-IgE (Fig 2, E). None of the antibodies had a significant effect on the ADCP of tumor cells (see Tables E1 and E3 in this article's Online Repository at www.jacionline.org). We furthermore evaluated whether can225IgE-λ could activate canine effector cells (Fig 2, F and G; Table E1). In a time-dependent manner (Fig E1, K), can225IgE-λ triggered significantly higher levels of ADCC by canine DH82 macrophages compared with can225IgG (Fig 2, F). Conversely, and in agreement with previous studies,8Karagiannis P. Singer J. Hunt J. Gan S.K. Rudman S.M. Mechtcheriakova D. et al.Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.Cancer Immunol Immunother. 2009; 58: 915-930Crossref PubMed Scopus (83) Google Scholar, E7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar can225IgG was significantly superior to IgE with regard to triggering tumor cell ADCP (see Table E4 in this article's Online Repository at www.jacionline.org). In addition, we investigated the effects of an approach using 50:50 can225IgG and can225IgE-λ at the same total antibody amount as samples treated with individual antibodies. Combinational treatment performed comparable tumor cell ADCC to that by can225IgE-λ alone, but significantly better ADCC than that by can225IgG alone. ADCP was lower than that triggered by can225IgG, but higher than in can225IgE-λ monotherapy (Fig 2, F). Overall, can225IgE-λ mediated higher levels of total tumor cell killing compared with those mediated by can225IgG alone. Moreover, combining can225IgG and can225IgE-λ was significantly superior to each isotype alone (Fig 2, G; see Table E2 and Table E4 in this article's Online Repository at www.jacionline.org). Similarly, we evaluated the cytotoxic killing potency of can225IgE-λ by MPT-1 canine mast cells against A431 and measured specific tumor cell killing (Fig 2, H). Although ADCP assessment by flow cytometry was not possible in this experimental setting due to cell clumping, we could indeed confirm mast cell–mediated ADCC by fluorescence microscopy (Fig 2, I). Such effector functions may be due to can225IgE-λ specifically binding to IgE receptors (FcεRI on RBL-SX38 cells, Fig 2, J) and canine CD23, expressed on transfected CHO K1 cells (Fig 2, K). Therefore, we established an in vitro system for quantification and side-by-side comparison of anticancer ADCC/ADCP triggered by cetuximab IgG, IgE, and their canine counterparts. Unlike in humans where κ:λ light chains are expressed in a ratio of 2:1, the average κ:λ ratio in dogs is 1:9.E11Arun S.S. Breuer W. Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.Zentralblatt fur Veterinarmedizin Reihe A. 1996; 43: 573-576Crossref PubMed Scopus (86) Google Scholar Hence, we engineered can225IgE-λ. Because canine IgE is able to bind human FcεRI, we could demonstrate dose-dependent binding of can225IgE-λ on human U937 (Fig 2, A) in addition to the expected binding to canine FcεRI on DH82 and MPT-1 cells (Fig 2, B and C). Furthermore, we showed that DH82 macrophages express low levels of CD23 (Fig 1, N), but high levels of functional FcεRI (Fig 1, K), which is likely responsible for triggering IgE-mediated tumor cell killing (Fig 2, F and G), comparable to that mediated by human U937 monocytic effector cells. Using the canine DH82 macrophages, we were able to generate a species-relevant in vitro model system with which we confirmed the tumoricidal potencies of our canine antibodies, can225IgG or can225IgE-λ, alone, or combined. It has been observed that human IgG triggers considerable levels of ADCP, whereas IgE mainly elicits ADCC by human monocytes/macrophages.8Karagiannis P. Singer J. Hunt J. Gan S.K. Rudman S.M. Mechtcheriakova D. et al.Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.Cancer Immunol Immunother. 2009; 58: 915-930Crossref PubMed Scopus (83) Google Scholar, E7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar We observed the same in the dog system and we also report significantly higher total tumor cell killing induced by IgE. We furthermore anticipated that IgE could act complementary to IgG, through using different Fc-receptors on effector cells. Coincubation with can225IgG and can225IgE-λ exerted higher tumor cell death, than did IgG or IgE treatment alone (Fig 2, F). Finally, we also demonstrate that can225IgE-λ is able to trigger specific ADCC against EGFR+ tumor cells by canine MPT-1 mast cells (Fig 2, H and I). Additional studies are needed to further evaluate these promising results with other IgG or IgE effector cells.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar, 9Singer J. Jensen-Jarolim E. IgE-based immunotherapy of cancer: challenges and chances.Allergy. 2014; 69: 137-149Crossref PubMed Scopus (25) Google Scholar This study presents the first in vitro functionally active canine anticancer IgE antibody for future clinical studies in dog cancer patients that may offer new options in cancer immunotherapy.9Singer J. Jensen-Jarolim E. IgE-based immunotherapy of cancer: challenges and chances.Allergy. 2014; 69: 137-149Crossref PubMed Scopus (25) Google Scholar In contrast to previous reports,E1Peng Z. Estelle F. Simons R. Becker A.B. Differential binding properties of protein A and protein G for dog immunoglobulins.J Immunol Method. 1991; 145: 255-258Crossref PubMed Scopus (22) Google Scholar we did not succeed in purifying can225IgE-λ by Protein A affinity columns. This might be possibly due to 2 IgE isoforms in dogs, IgE1 having a high affinity toward Staphylococcal Protein A and IgE2 not at all binding to protein A.E2Peng Z. Arthur G. Rector E.S. Kierek-Jaszczuk D. Simons F.R. Becker A.B. Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity.J Allergy Clin Immunol. 1997; 100: 87-95Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The EGFR-negative CHO K1 cell line was used as control target cells. These cells were not recognized by can225IgE-λ (Fig 1, I). Consequently, CHO K1 cells were also not attacked by the human monocytic U937 cells (Fig E1, C and D) or the canine monocytic/macrophage-like DH82 cells (Fig E1, E and F). DH82 cells appeared as single, round cells in culture and showed only 1 distinct population in flow cytometry scatter plots (Fig E1, B); 77.4% of DH82 cells were positive for CD16 (ΔMFI, 713), 99.9% for CD14 (ΔMFI, 12922), and 72.2% for CD80 (ΔMFI, 2797) (Fig E1, G-I). DH82 cells transform over time from a monocyte- to a macrophage-like phenotype, associated with changes in CD14 and CD80 surface marker expression.E3Heinrich F. Contioso V.B. Stein V.M. Carlson R. Tipold A. Ulrich R. et al.Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).Veter Immunol Immunopathol. 2015; 163: 86-92Crossref PubMed Scopus (18) Google Scholar However, both CD14 and CD80 expression levels, as well as morphology and the uniform population in flow-cytometric scatter plots, identified a monocytic phenotype,E3Heinrich F. Contioso V.B. Stein V.M. Carlson R. Tipold A. Ulrich R. et al.Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).Veter Immunol Immunopathol. 2015; 163: 86-92Crossref PubMed Scopus (18) Google Scholar which was advantageous because the 3-color flow-cytometric assay had been established for monocytes.E4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar For proof-of-concept studies in the field of comparative immuno-oncology and AllergoOncology, the recombinant canine IgE antibody can225IgE-λ was developed against EGFR, which has 95% amino acid sequence homology and 92% identity among humans and dogs.E5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar The antibody was expressed in a transient expression system and characterized with respect to correct assembly, EGFR specificity, and binding to IgE receptors FcεRI and CD23. In addition, the functionality of can225IgE-λ was evaluated in a flow-cytometric ADCC and ADCP assay using human and canine monocyte-like cells, as well as canine mast cells as effectors against EGFR-overexpressing tumor and EGFR-negative control cells, as described below. Human monocytic cell line U937 (CRL-1593.2), human epidermoid carcinoma cell line A431 (CRL-1555), canine osteosarcoma cell line D17 (CCL183), canine monocyte/macrophage-like cell line DH82 (CRL-10389), and Chinese hamster ovary cell line CHO K1 (CCL-61) were purchased from the American Type Culture Collection (Manassas, Va). Canine mammary carcinoma cell line P114 was a kind gift of G. Rutteman, University of Utrecht (Utrecht, The Netherlands). Canine mast cell line MPT-1E6Amagai Y. Tanaka A. Ohmori K. Matsuda H. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.Biochemic Biophys Res Commun. 2008; 366: 857-861Crossref PubMed Scopus (11) Google Scholar was a kind gift of Prof. H. Matsuda, Tokyo University of Agriculture and Technology (Tokyo, Japan). U937 was cultured in RPMI 1640 GlutaMAX medium (Gibco, Thermo Fisher Scientific, Waltham, Mass) supplemented with 10% (v/v) FBS (Gibco, Thermo Fisher Scientific) and 100 U/mL penicillin and 100 μg/mL Streptomycin (Gibco, Thermo Fisher Scientific). A431 was grown in high glucose (4.5 g/L) Dulbecco modified Eagle medium (DMEM) GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. D17 was grown in Minimum Essential Medium (MEM) GlutaMAX (Gibco, Thermo Fisher Scientific), augmented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. P114 was cultured in DMEM/F-12 GlutaMAX (Gibco, Thermo Fisher Scientific) media supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. DH82 was cultured in low glucose (1 g/L) DMEM GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. MPT-1 was grown in alpha-MEM GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. CHO K1 was grown in Ham's F-12 Nutrient Mix GlutaMAX (Gibco, Thermo Fisher Scientific), augmented with 0% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were maintained in a humidified incubator at 37°C under a 5% CO2 atmosphere. Cetuximab (Erbitux) was purchased from Merck (Darmstadt, Germany). Rituximab (MabThera) was used as human IgG isotype control and was purchased from Roche (Basel, Switzerland). Cetuximab IgEE7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar was provided by Dr E. Spillner, Aarhus University (Aarhus, Denmark). MOv18-IgEE8Gould H.J. Mackay G.A. Karagiannis S.N. O'Toole C.M. Marsh P.J. Daniel B.E. et al.Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.Eur J Immunol. 1999; 29: 3527-3537Crossref PubMed Scopus (88) Google Scholar—used as human IgE isotype control—was provided by Dr S.N. Karagiannis and Prof. H.J. Gould, King's College London (London, UK). Purified canine IgG (cat# IR-DG-GF, Innovative Research, Novi, Mich) was used as canine IgG isotype control and purified canine IgE (cat# P115, Bethyl Laboratories, Montgomery, Tex) as canine IgE isotype control. Can225IgG was produced as described by Singer et al.E9Singer J. Fazekas J. Wang W. Weichselbaumer M. Matz M. Mader A. et al.Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.Mol Cancer Therapeut. 2014; 13: 1777-1790Crossref PubMed Scopus (40) Google Scholar Recombinant human EGFR (extracellular domain) was purchased from ACROBiosystems (cat# EGR-H5222). Recombinant human epidermal growth factor receptor 2 (extracellular domain) was produced in-house by Lec-1 cells,E10Cho H.S. Mason K. Ramyar K.X. Stanley A.M. Gabelli S.B. Denney Jr., D.W. et al.Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.Nature. 2003; 421: 756-760Crossref PubMed Scopus (1222) Google Scholar which were a kind gift of D. Leahy from Johns Hopkins University School of Medicine (Baltimore, Md). Cetuximab heavy-chain variable region sequence was fused with the canine immunoglobulin epsilon constant sequence (NCBI GenBank: AAA56797.1). Cetuximab light-chain variable region sequence was fused with the canine lambda (because of its higher prevalence in dogs than in humans).E11Arun S.S. Breuer W. Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.Zentralblatt fur Veterinarmedizin Reihe A. 1996; 43: 573-576Crossref PubMed Scopus (86) Google Scholar The light-chain constant sequence (NCBI Ref Seq: XP_013963655.1) can be found among the coding sequences in Table E5. The final sequences were synthesized by GeneArt in Strings DNA format (Thermo Fisher Scientific) and cloned into pCR-Blunt vector using Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific). Coding sequences were recloned into pVitro1-hygro (Invivogen, San Diego, Calif) using a ligation-free polymerase incomplete primer extension cloning method adapted from Dodev et al.E12Dodev T.S. Karagiannis P. Gilbert A.E. Josephs D.H. Bowen H. James L.K. et al.A tool kit for rapid cloning and expression of recombinant antibodies.Sci Rep. 2014; 4: 5885Crossref PubMed Scopus (62) Google Scholar All primers used for polymerase incomplete primer extension cloning are listed in Table E6. The pVitro-hygro-can225IgE-λ construct was then transformed into Escherichia coli Top10 (Thermo Fisher Scientific). Correct clones were verified using Sanger sequencing. Can225IgE-λ was produced according to Ilieva et alE13Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (12) Google Scholar in human Expi293F cells using the pVitro1 vector (Fig 1, A) and purified with a custom affinity column using the ÄKTAFPLC system from GE Healthcare Life Sciences (Chicago, Ill). Custom anti-dog IgE column was made by crosslinking 1 mg goat anti-dog IgE (cat# NB7343, Novus Biologicals, Littleton, Colo) to HiTrap Protein G HP column (GE Healthcare Life Sciences) in the presence of 50-fold molar excess disuccinimidylsuberate (Sigma-Aldrich, St Louis, Mo). PBS, pH 7.4, was used as binding and washing buffer. Bound can225IgE-λ was then eluted with 0.1 M glycine (pH 2.5) followed by immediate pH neutralization using 1 M tris-HCl, pH 8.0. PAGE was run using tris-glycine–based 4% to 20% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, Calif), Laemmli loading buffer, and molecular weight markers Spectra Multicolor High Range Protein Ladder (Fig 1, B-D) and PageRuler Plus Prestained Protein Ladder (Fig 2, A) from Thermo Fisher. The gels were loaded with 0.5 μg can225IgE-λ/canine IgE isotype or 1 μg recombinant human EGFR/recombinant human epidermal growth factor receptor 2/BSA and the proteins were subsequently detected either by silver staining or by immunoblot. Blocking and antibody dilutions were performed with 5% (w/v) skimmed milk powder dissolved in tris buffered saline, pH 7.2, + 0.1% (v/v) tween-20. Canine IgE was detected using horseradish peroxidase–labeled goat anti-dog IgE (cat# 7346, Novus Biologicals) at 1:5000 for 1 hour, or by 0.5 μg/mL can225IgE-λ for 2 hours at room temperature. Blots were developed with Clarity Western enhanced chemiluminescence substrate (Bio-Rad) using the VersaDoc Imaging System (Bio-Rad). Light chains were detected using 0.5 μg/mL anti-dog light-chain antibody (cat# A40-124A, Bethyl Laboratories) followed by alkaline phosphatase-labeled donkey anti-goat IgG detection antibody (cat# A16008, Thermo Fisher) at 1:2000, and subsequently visualized using 1-Step NBT/BCIP substrate solution (Thermo Fisher). The apparent molecular mass of can225IgE-λ on silver-stained PAGE was determined using GelAnalyzer 2010a software (Istvan Lazar and Dr Istvan Lazar). Spectra Multicolor High Range Protein Ladder (Thermo Fisher Scientific) was used as a reference. A431, D17, P114, and CHO K1 cells were detached using Accutase solution (Sigma-Aldrich). A total of 3 × 105 cells/tube were stained with 10 μg/mL can225IgE-λ (or isotype control) for 30 minutes on ice followed by 10 μg/mL fluorescein isothiocyanate (FITC)-labeled goat anti-dog IgE (cat# NB7345, Novus Biologicals) for 30 minutes on ice in a light-protected environment. The cells were analyzed on a 3-laser FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). DH82 and MPT-1 (3 × 105 per test) were stained with 10 μg/mL phycoerythrin-labeled anti-mouse FcεRI (clone MAR-1) (cat# 12-5898-83, eBioscience, Santa Clara, Calif) or appropriate isotype controls. U937 and RBL-SX38 cells (3 × 105 per test) were stained with 10 μg/mL anti-human FcεRI (clone CRA-1) (cat# 14-5899-82, eBioscience, Santa Clara, Calif), or mouse IgG2b isotype control, detected by 10 μg/mL FITC-labeled goat anti-mouse IgG (cat# F0479, DAKO, Agilent Technologies, Santa Clara, Calif). U937, DH82, and MPT-1 cells (3 × 105 per test) were incubated with can225IgE-λ at different concentrations (1, 2.5, 5, and 10 μg/mL) for 30 minutes at 37°C. Bound can225IgE-λ was detected with 10 μg/mL FITC-labeled goat anti-dog IgE. RBL-SX38 or canine CD23-transfected CHO K1 cells (3 × 105 per test) were incubated with 10 μg/mL can225IgE-λ for 30 minutes. Following a washing step, bound can225IgE-λ was detected with 10 μg/mL FITC-labeled goat anti-dog IgE. DH82 cells (passages 16-27, 3 × 105 per test) were stained with APC-labeled anti-human CD14 clone Tük4 (cat# MHCD1405, Thermo Fisher)/FITC-labeled anti-human CD16 (clone LNK16) (cat# MA1-19611, Thermo Fisher)/phycoerythrin-labeled anti-mouse CD80 (clone 16-10A1) (cat# 12-0801-82, eBioscience), or the respective controls. Cells were incubated with antibodies at concentrations according to the manufacturer's instructions for 30 minutes and washed before acquisition. Total RNA of 5 × 106 DH82 and MPT-1 was isolated using the RNeasy Mini Kit (cat# 74104, Qiagen, Venlo, The Netherlands) and transcribed using the SuperScript II reverse transcriptase (cat# 18064022, Thermo Fisher) along with oligo(dT)12-18 primers (cat# 18418012, Thermo Fisher) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green PCR Master Mix (cat# 4309155, Applied Biosystems, Thermo Fisher). Primers for canine CD23: forward: CCCAGAGCTTGAACGAGAGAA, reverse: TCCTCGCCGAAGTAGTAGCAC, as described by Amagai et al.E6Amagai Y. Tanaka A. Ohmori K. Matsuda H. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.Biochemic Biophys Res Commun. 2008; 366: 857-861Crossref PubMed Scopus (11) Google Scholar Primers for beta-actin: forward: ATTGCCGACAGGATGCAGAA, reverse: GCTGATCCACATCTGCTGAA. RT-PCR analysis was performed using 3 biological replicates (and 3 technical replicates of each biological replicate). Flow-cytometric ADCC/ADCP assays were performed using the method described by Bracher et al.E4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar A431 tumor cells were prestained with 5 μM of the green fluorescent dye carboxyfluoresceinsuccinimidyl ester for 10 minutes, 18 hours before the experiment. The tumor cells (70000/test) were incubated with 5 μg/mL antibody solution in assay buffer (RPMI 1640 GlutaMAX +2% (v/v) FBS) at 37°C for 30 minutes. Next, 210,000 U937 cells/tube were coincubated with the tumor cells at 37°C for 2.5 hours. Following the incubation, U937 were labeled with APC-conjugated anti-human CD89 antibody (cat# 354106, BioLegend, San Diego, Calif). Dead cells were detected using the blue fluorescent DNA dye 4′,6-diamidino-2-phenylindole (cat# D1306, Thermo Fisher), few seconds before acquisition on the FACSCanto II. When using DH82 or MPT-1 as effector cells, both tumor and effector cells were stained with cell-tracking dyes 18 hours in advance to the treatment antibodies and coincubation: Tumor cells were prestained with carboxyfluoresceinsuccinimidyl ester (as described earlier), whereas DH82 and MPT-1 were prestained with 1 μM CellTrace Far Red (Thermo Fisher) for 10 minutes. Because effector cells were already prelabeled in this case, no CD89 staining was needed to tag the cells after the coincubation of the tumor and effector cells. All assays were performed at least 3 times using 3 technical replicates per condition. Data were analyzed using FlowJo V10.0.7 (Flow Jo LLC, Ashland, Ore). Samples were prepared as described in the 3-color flow-cytometric ADCC and ADCP assay section using MPT-1 canine mast cells and A431 tumor cells. Fifteen microliter of the nonfixed cell suspension was mounted on a glass slide immediately after 4′,6-diamidino-2-phenylindole staining, and fluorescence microscopy pictures were recorded 20 minutes within mounting using an Axioplan 2 fluorescent microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 20× air objective. CHO K1 cells were transfected with pcDNA3.3-TOPO (Thermo Fisher) containing the coding sequence of canine CD23 (back-translated from the amino acid sequence disclosed under GenBank accession number AAN20273.1) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions. Forty-eight hours after transfection, 1 mg/mL G418 (cat# CP11.3, Carl Roth) was added and selection pressure was maintained over 3 weeks before proceeding to IgE-binding assays. Statistical analyses were carried out using GraphPad Prism v5.00 for Windows (GraphPad Software, Inc, La Jolla, Calif). Fig 2, A-C, shows mean values + SEM, and Fig 2, D-H, and Fig E1, C-F, depict mean + SD. To calculate the statistical significance of induced killing between the different treatment groups (Table E3 and Table E4), we used 2-way ANOVA with Bonferroni posttest (phagocytosis and cytotoxicity). To compare the total killing induced by the antibodies (Fig 2, E and G, and Fig E1, D and F), we used 1-way ANOVA followed by Tukey multiple comparison test. Statistical significances are defined as *P < .05, **P < .01, and ***P < .001.Table E1ADCC and ADCP triggered by different antibody treatmentsU937 as effector cellsKilling mechanismPBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgE-λCytotoxicity20% ± 6.0%18% ± 5.0%56% ± 8.6%18% ± 5.2%68% ± 4.7%18% ± 4.0%55 % ± 14%Phagocytosis4.0% ± 2.3%3.3% ± 2.4%5.3% ± 4.3%4.4% ± 4.0%4.2% ± 3.3%3.7% ± 2.9%3.6% ± 3.4%DH82 as effector cellsKilling mechanismPBSDog IgG ISOcan225IgGDog IgE ISOcan225IgE-λDog IgG + dog IgE ISOcan225IgG + can225IgE-λCytotoxicity15% ± 2.3%13% ± 1.7%40% ± 4.9%14% ± 2.4%64% ± 3.4%15% ± 2.8%63% ± 5.1%Phagocytosis18% ± 2.3%19% ± 2.9%28% ± 2.6%19% ± 2.5%11% ± 1.3%19% ± 1.1%18% ± 2.5%Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6.ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Open table in a new tab Table E2Total tumor cell killing triggered by different antibody treatmentsU937 as effector cellsTreatmentPBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgE-λTotal killing24% ± 6.0%21% ± 5.2%61% ± 12%22% ± 6.1%72% ± 7.5%21% ± 5.0%58% ± 16%DH82 as effector cellsTreatmentPBSDog IgG ISOcan225IgGDog IgE ISOcan225IgEDog IgG + dog IgE ISOcan225IgG + can225IgE-λTotal killing32% ± 2.9%32% ± 3.6%67% ± 3.8%32% ± 1.6%74% ± 2.6%34% ± 1.9%82% ± 3.1%Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6.ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Open table in a new tab Table E3Detailed statistical analysis of antibody treatments in ADCC and ADCP assays using the human U937 as effector cellsPhagocytosis ↓Cytotoxicity →PBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgEPBSns***P < .001.ns***P < .001.ns***P < .05.Human IgG ISOns***P < .001.ns***P < .001.ns***P < .001.Cetuximab IgGnsns***P < .001.***P < .001.∗P < .05.nsHuman IgE ISOnsnsns***P < .001.ns***P < .001.Cetuximab IgEnsnsnsns***P < .001.***P < .001.Dog IgE ISOnsnsnsnsns***P < .001.can225IgEnsnsnsnsnsnsStatistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05).∗ P < .05.∗∗∗ P < .001. Open table in a new tab Table E4Detailed statistical analysis of antibody treatments in ADCC and ADCP assays using the canine DH82 as effector cellsPhagocytosis ↓Cytotoxicity →PBSDog IgG ISOcan225IgGDog IgE ISOcan225IgEDog IgG + dog IgE ISOcan225IgG + can225IgEPBSns***P < .001.ns***P < .001.ns***P < .001.Dog IgG ISOns***P < .001.ns***P < .001.ns***P < .001.can225IgG***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.Dog IgE ISOnsns***P < .001.∗P < .05.ns***P < .001.can225IgE***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.nsDog IgG + dog IgE ISOnsns***P < .001.ns***P < .001.***P < .001.can225IgG +can225IgEnsns***P < .001.ns***P < .001.nsStatistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05).∗ P < .05.∗∗∗ P < .001. Open table in a new tab Table E5Amino acid and coding DNA sequence of can225IgE-λSequence descriptionSequenceCan225IgE-λ EPSILON chain amino acid sequence Legend:Bold, dog immunoglobulin heavy-chain variable region leader (ACI25521.1)Underlined, 225 variable regionRegular, dog immunoglobulin epsilon constant regionMESVLGWVFLVAILQGVQGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAATSQDLSVFPLASCCKDNIASTSVTLGCLVTGYLPMSTTVTWDTGSLNKNVTTFPTTFHETYGLHSIVSQVTASGKWAKQRFTCSVAHAESTAINKTFSACALNFIPPTVKLFHSSCNPVGDTHTTIQLLCLISGYVPGDMEVIWLVDGQKATNIFPYTAPGTKEGNVTSTHSELNITQGEWVSQKTYTCQVTYQGFTFKDEARKCSESDPRGVTSYLSPPSPLDLYVHKAPKITCLVVDLATMEGMNLTWYRESKEPVNPGPLNKKDHFNGTITVTSTLPVNTNDWIEGETYYCRVTHPHLPKDIVRSIAKAPGKRAPPDVYLFLPPEEEQGTKDRVTLTCLIQNFFPADISVQWLRNDSPIQTDQYTTTGPHKVSGSRPAFFIFSRLEVSRVDWEQKNKFTCQVVHEALSGSRILQKWVSKTPGKCan225IgE-λ EPSILON chainDNA sequence(HEK293-optimized)Legend:Bold, STOP codonATGGAAAGCGTGCTGGGCTGGGTGTTCCTGGTGGCCATTCTGCAGGGCGTGCAGGGACAGGTGCAGCTGAAGCAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACCAACTACGGCGTGCACTGGGTGCGCCAGAGCCCTGGAAAAGGCCTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCCTTCACCAGCAGACTGTCCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTACTGCGCTCGGGCCCTGACCTACTATGACTACGAGTTCGCCTACTGGGGCCAGGGCACACTCGTGACAGTGTCTGCCGCCACCAGCCAGGACCTGAGCGTGTTCCCTCTGGCCAGCTGCTGCAAGGACAATATCGCCAGCACCAGCGTGACCCTGGGCTGCCTCGTGACCGGCTACCTGCCTATGAGCACCACAGTGACCTGGGACACCGGCAGCCTGAACAAGAACGTGACCACCTTCCCCACCACCTTTCACGAGACATACGGCCTGCACAGCATCGTGTCTCAAGTGACCGCCAGCGGCAAGTGGGCCAAGCAGAGATTCACATGCAGCGTGGCCCACGCCGAGAGCACCGCCATTAACAAGACCTTCAGCGCCTGCGCCCTGAACTTCATCCCTCCCACCGTGAAGCTGTTCCACAGCAGCTGCAACCCCGTGGGCGATACCCACACCACCATTCAGCTGCTGTGCCTGATCAGCGGCTACGTGCCCGGCGACATGGAAGTGATCTGGCTGGTGGATGGCCAGAAGGCTACCAACATCTTCCCCTACACCGCCCCTGGCACCAAAGAGGGCAATGTGACCAGCACCCACTCCGAGCTGAACATCACCCAGGGCGAGTGGGTGTCACAGAAAACCTACACCTGTCAAGTGACATACCAGGGCTTCACCTTCAAGGACGAGGCCCGGAAGTGCAGCGAGAGCGATCCTAGAGGCGTGACCTCCTACCTGAGCCCCCCTAGCCCTCTGGACCTGTACGTGCACAAGGCCCCCAAGATCACCTGTCTGGTGGTGGACCTGGCCACAATGGAAGGCATGAACCTGACCTGGTACAGGGAAAGCAAAGAACCCGTGAACCCAGGCCCTCTGAACAAAAAGGACCACTTCAACGGCACCATCACCGTGACAAGCACCCTGCCCGTGAACACCAACGACTGGATCGAGGGCGAGACTTACTACTGCAGAGTGACCCACCCTCATCTGCCCAAGGACATCGTGCGGTCTATCGCCAAGGCCCCAGGCAAAAGGGCCCCTCCCGATGTGTACCTGTTCCTGCCTCCCGAGGAAGAACAGGGCACCAAGGACAGAGTGACACTGACCTGCCTGATCCAGAACTTCTTCCCCGCCGACATCTCCGTGCAGTGGCTGAGAAACGACAGCCCCATCCAGACCGACCAGTACACCACCACCGGCCCTCACAAGGTGTCCGGAAGCAGACCCGCCTTCTTCATCTTCAGCAGGCTGGAAGTGTCCCGGGTGGACTGGGAGCAGAAGAACAAGTTCACATGCCAGGTGGTGCACGAGGCCCTGAGCGGCTCTCGGATCCTGCAGAAATGGGTGTCCAAGACCCCCGGCAAGTGACan225IgE-λ LAMBDA chain amino acid sequence Legend:Bold, dog immunoglobulin lambda light-chain variable region leader (XP_013963655.1)Underlined, 225 light-chain variable regionRegular, dog immunoglobulin lambda constant regionMTSTMAWSSFLLTLLAHFTGSWADILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECSCan225IgE-λLAMBDA light-chainDNA sequence(HEK293-optimized)Legend:Bold, STOP codonATGACCAGCACAATGGCCTGGTCCAGCTTCCTGCTGACCCTGCTGGCCCACTTTACAGGCAGCTGGGCCGACATTCTGCTGACACAGAGCCCCGTGATCCTGAGCGTGTCCCCTGGCGAGAGAGTGTCCTTCAGCTGCAGAGCCAGCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACGGCAGCCCCAGACTGCTGATTAAGTACGCCAGCGAGTCCATCAGCGGCATCCCCAGCAGATTTTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAACAGCGTGGAAAGCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCACCAAGCTGGAACTGAAGAGAACCGTGGCCCAGCCCAAGGCCAGCCCTAGCGTGACACTGTTCCCTCCAAGCAGCGAGGAACTGGGCGCCAACAAGGCCACACTCGTGTGCCTGATCAGCGACTTCTACCCCAGCGGCGTGACCGTGGCCTGGAAGGCTGATGGCTCTCCTGTGACCCAGGGCGTGGAAACCACCAAGCCCAGCAAGCAGTCCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGATAAGTGGAAGTCCCACAGCAGCTTCAGCTGTCTCGTGACCCACGAGGGCAGCACCGTGGAAAAGAAAGTGGCCCCTGCCGAGTGCAGCTGATAG Open table in a new tab Table E6PIPE primers used for ligation-free cloning can225IgE-λ heavy- and light- chain sequences into pVitro-1 hygroPrimer nameSequencecan225IgE-λ epsilon forwardTACAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGcan225IgE-λ epsilon reverseTCCGGATTGCTTTGAATTAGCGGTGGCTTTCACAACACCTcan225IgE-λ lambda forwardAGGGATCCCGTACGCCTAGGAGCAGGTTTCCCCAATGACAcan225IgE-λ lambda reverseACCGGTTGCTTTGAATTAGCGGTGGTTTTCACAACACCTAPIPE, Polymerase incomplete primer extension. Open table in a new tab Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6. ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6. ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Statistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05). Statistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05). PIPE, Polymerase incomplete primer extension.