PDF - 94K, Supplementary Methods.

PDF - 210K, Can225IgG heavy and light chain amino acid sequences.

PDF - 446K, Figure S1. Purification of can225IgG from cell culture supernatant is efficient with Protein G, but not with Protein A. Figure S2. Elution of can225IgG from Protein G columns at a low pH does not affect integrity of the antibody. Figure S3. Binding of can225IgG to the cell lines CF33, CF41, TLM-1 and BT474 in FACS. Figure S4. Can225IgG shows a slightly lower binding affinity towards canine EGFR, compared with human EGFR. Figure S5. Staining of EGFR+ cell line and canine mammary carcinoma section by can225IgG.

Abstract Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. Mol Cancer Ther; 13(7); 1777–90. ©2014 AACR.

To facilitate comparative oncology trials we compared the biological and molecular homologies of canine (dog; Canis lupus familiaris) and human tumor-associated antigens ErbB-1 and -2. Further, we investigated whether they could serve as targets for anti-ErbB-1 (cetuximab) and anti-ErbB-2 antibodies (trastuzumab), which are highly relevant in human clinical oncology. Immunohistochemistry of canine mammary cancer showed ErbB-1 overexpression in 3/10 patients and ErbB-2 in 4/10. We report 91% amino acid homology for ErbB-1 and 92% for ErbB-2 between canine and human molecules. Modeling of canine on human ErbB-1 revealed that the cetuximab epitope only differs by 4 amino acids: Lys443 is replaced by Arg, Ser468 by Asn, Gly471 by Asp, and Asn473 by Lys in canines. The trastuzumab binding site is identical in human and canine ErbB-2 apart from a single amino acid change (Pro557 to Ser). Binding of cetuximab and trastuzumab to canine mammary carcinoma cells CF33, CF41, Sh1b and P114 was confirmed by flow cytometry. Both antibodies significantly inhibited canine tumor cell proliferation partly due to growth arrest in G(0)/G(1) phase. We explain the lower efficiency on the tested canine than on human SKBR3 and A431 cells, by a 2-log lower expression level of the canine ErbB-1 and -2 molecules. Our results indicate significant homology of human and canine Erb-1 and -2 tumor associated antigens. The fact that the canine homologues express the cetuximab and trastuzumab epitopes may facilitate antibody-based immunotherapy in dogs. Importantly, the striking similarities of ErbB-1 and -2 molecules open up avenues towards comparative strategies for targeted drug development.

In human medicine, passive immunotherapy is a well-established tool to fight cancer. Cetuximab and trastuzumab are famous examples of effective monoclonal antibody therapies for the treatment of ErbB-1 (EGFR) overexpressing colon tumors and for ErbB-2 (HER2/neu) overexpressing metastatic breast cancer. Mammary carcinomas of dogs are the most important cause of tumor-related death in females. It is known that these tumors also express tumor markers similar to human breast cancer and that regarding the idea of comparative oncology cancer in pet dogs are a valuable model for speeding up drug development. Therefore, we aimed to investigate more closely canine ErbB-1 and ErbB-2 and the applicability of cetuximab and trastuzumab in the dog. Screenings with FDA-approved immunohistochemical diagnostic tests of canine mammary tumor samples showed 3/10 ErbB-1 and 4/10 ErbB-2 overexpression, which seems similar to the expression pattern in human breast cancer patients. Sequence analyses revealed amino acid sequence identities of 91% between human and canine ErbB-1 and 92% for ErbB-2. Modeling of those proteins showed that the human cetuximab epitope differs in only four amino acids compared to the canine counterpart and that the trastuzumab binding site is identical in human and canine ErbB-2 except for only one amino acid. Using flow cytometry analyses cetuximab and trastuzumab binding could be confirmed in four canine mammary carcinoma cell lines. Furthermore, cell viability assays showed that incubation of canine cell lines with both antibodies inhibited tumor cell proliferation, even though there are lower numbers of ErbB molecules on canine than on human cells, which was confirmed by a flow cytometry-based assay. This comparative oncology study indicates that ErbB-1 and ErbB-2 are highly conserved molecules with significant concordance in terms of structure and expression pattern between human and canine cancer. Importantly, the canine homologous molecules are susceptible to cetuximab and trastuzumab targeting, which may open up new avenues towards antibody-based immunotherapies in companion dogs and improvements in anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C46.

Comparative oncology aims at speeding up developments for both, human and companion animal cancer patients. Following this line, carcinoembryonic antigen (CEA, CEACAM5) could be a therapeutic target not only for human but also for canine (Canis lupus familiaris; dog) patients. CEACAM5 interacts with CEA-receptor (CEAR) in the cytoplasm of human cancer cells. Our aim was, therefore, to phylogenetically verify the antigenic relationship of CEACAM molecules and CEAR in human and canine cancer.Anti-human CEACAM5 antibody Col-1, previously being applied for cancer diagnosis in dogs, immunohistochemically reacted to 23 out of 30 canine mammary cancer samples. In immunoblot analyses Col-1 specifically detected human CEACAM5 at 180 kDa in human colon cancer cells HT29, and the canine antigen at 60, 120, or 180 kDa in CF33 and CF41 mammary carcinoma cells as well as in spontaneous mammary tumors. While according to phylogenicity canine CEACAM1 molecules should be most closely related to human CEACAM5, Col-1 did not react with canine CEACAM1, -23, -24, -25, -28 or -30 transfected to canine TLM-1 cells. By flow cytometry the Col-1 target molecule was localized intracellularly in canine CF33 and CF41 cells, in contrast to membranous and cytoplasmic expression of human CEACAM5 in HT29. Col-1 incubation had neither effect on canine nor human cancer cell proliferation. Yet, Col-1 treatment decreased AKT-phosphorylation in canine CF33 cells possibly suggestive of anti-apoptotic function, whereas Col-1 increased AKT-phosphorylation in human HT29 cells. We report further a 99% amino acid similarity of human and canine CEA receptor (CEAR) within the phylogenetic tree. CEAR could be detected in four canine cancer cell lines by immunoblot and intracellularly in 10 out of 10 mammary cancer specimens from dog by immunohistochemistry. Whether the specific canine Col-1 target molecule may as functional analogue to human CEACAM5 act as ligand to canine CEAR, remains to be defined. This study demonstrates the limitations of comparative oncology due to the complex functional evolution of the different CEACAM molecules in humans versus dogs. In contrast, CEAR may be a comprehensive interspecies target for novel cancer therapeutics.