Identification and characterization of FcγRs in Göttingen minipigs – implications for preclinical assessment of therapeutic antibodies

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Purpose – Antibodies of the human (hu) Immunoglobulin G (IgG) isotype are used as therapeutics for patients with cancer, rheumatoid arthritis, asthma, and other diseases. Often, these therapeutic huIgG antibodies mediate effects by binding to human Fc gamma receptors (FcγRs) expressed on various cells of the patient’s immune system. Three classes of huFcγRs comprising a total of six receptors are known in humans, namely FcγRIa (CD64), FcγRIIa/b/c (CD32a/b/c), and FcγRIIIa/b (CD16a/b). FcγR-mediated effector functions range from desired depletion of tumor cells via antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis, to unwanted toxic effects by exaggerated cytokine release, thrombosis, and infusion reactions. These functions depend on the FcγR, the binding strength, and the involved immune cells. Prior to human use, the safety and efficacy of therapeutics have to be demonstrated in animal studies where human antibodies interact with the immune system of the selected species. The Gottingen minipig is highly suitable for such mandatory preclinical studies. However, the relevance of such studies for assessing the safety and efficacy of therapeutic antibodies is limited due to unknown characteristics of porcine (po)FcγRs. Therefore, this thesis aims to characterize the poFcγRs, focusing on the expression on immune cells of the minipig and the binding to huIgG. Methods – To study the set of poFcγRs in minipigs, we performed a detailed genome analysis of the locus coding for most FcγRs by polymerase chain reaction (PCR) and manual assembly of existing sequences. We used single cell ribonucleic acid (RNA) sequencing to determine the transcription, and flow cytometry to show the expression of different poFcγRs on various cells within blood, lymph node, and spleen. Cloning and expression of all poFcγRs as soluble proteins enabled the binding assessment of monomeric, as well as immune complexed huIgG1 therapeutic antibodies to poFcγRs by surface plasmon resonance (SPR; Biacore). Furthermore, we investigated the binding of monomeric antibodies and immune complexes to FcγR-expressing cell lines and immune cells of the minipig by flow cytometry. Results – We used genome analysis to identify the missing poFcγRIIa and to map the gene coding for the known poFcγRIIIa, which had not been annotated to date. The genomic organization of poFcγRs resembles that of most mammals except humans, who have two additional genes coding for huFcγRIIc and IIIb. In general, the distribution of FcγRs on immune cells and the binding properties to free- and immune-complexed huIgG1, both prerequisites for effector functions mediated by huIgG1, are similar in minipigs and humans. However, we observed several key differences which may affect the use of minipigs in preclinical studies with therapeutic huIgG1 antibodies. Firstly, the binding of huIgG1 to FcγRIIa, which is expressed on blood platelets, was stronger in minipigs (poFcγRIIa) compared to humans (huFcγRIIa). Despite this, the minipig could be a valuable model to study IgG-mediated platelet activation, aggregation, and thrombosis. Secondly, for the inhibitory poFcγRIIb, we observed stronger binding versus huFcγRIIb. In humans, FcγRIIb regulates the immune response and is expressed on B cells, dendritic cells, and tissue monocytes. In contrast, we reported expression of poFcγRIIb on blood monocytes in minipigs. We suggest that anti-inflammatory effects with therapeutic huIgG1 antibodies could be stronger in minipigs than in humans due to the divergent expression and the stronger binding to the inhibitory poFcγRIIb. Lastly, we observed a lack of binding of huIgG1 to poFcγRIIIa. In humans, cytotoxic huIgG1 antibodies mediate ADCC via binding to huFcγRIIIa expressed on natural killer (NK) cells and on a subset of monocytes in the blood. The lacking binding of huIgG1 to poFcγRIIIa excludes NK-mediated ADCC and additionally restricts functions of monocytes, thus limiting studies with certain huIgG1 therapeutics. However, we reported binding of endogenous poIgG1 enabling effector functions in tumor vaccination or infection studies. Conclusion – The results compiled in this thesis generally recommend the use of minipigs for the assessment of therapeutic huIgG1 antibodies. However, the limitations of this animal model regarding differential binding of huIgG1 to poFcγRs and their expression pattern on immune cells in comparison to the human have to be considered. Therefore, functional studies are recommended to further assess the translatability of FcγR-mediated effector functions with various therapeutic antibodies from the minipig to the human. Nevertheless, this work delivers a foundation for species selection and allows the interpretation of results from preclinical safety and efficacy studies with Gottingen minipigs.