At protein level, Asn was found at position 95, as well as at position 97 in the only published sequence of rb2m [31], whereas NCBI reference sequence analysis (“type”:”entrez-protein”,”attrs”:”text”:”XP_008267300.1″,”term_id”:”655862972″,”term_text”:”XP_008267300.1″XP_008267300.1) and our cDNA clone (the sequence was deposited into GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”KT151665″,”term_id”:”960500104″,”term_text”:”KT151665″KT151665) predicted Asp at both positions. The sequence comparisons show high identity between human (GenBank: “type”:”entrez-protein”,”attrs”:”text”:”ARO48560″,”term_id”:”1189402594″,”term_text”:”ARO48560″ARO48560) and rabbit (Genbank: “type”:”entrez-protein”,”attrs”:”text”:”AEN74950″,”term_id”:”345122362″,”term_text”:”AEN74950″AEN74950) [28] variants of FcRn -chain (72%), human (GenBank: “type”:”entrez-protein”,”attrs”:”text”:”CAG33347″,”term_id”:”48146249″,”term_text”:”CAG33347″CAG33347) and rabbit (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”KT151665″,”term_id”:”960500104″,”term_text”:”KT151665″KT151665) 2m (74%), as well as human (hIgG1 GenBank: “type”:”entrez-protein”,”attrs”:”text”:”P01857″,”term_id”:”2500440404″,”term_text”:”P01857″P01857, hIgG2 GenBank: “type”:”entrez-protein”,”attrs”:”text”:”P01859″,”term_id”:”2500440430″,”term_text”:”P01859″P01859, hIgG3 GenBank: “type”:”entrez-protein”,”attrs”:”text”:”P01860″,”term_id”:”2500440431″,”term_text”:”P01860″P01860, hIgG4 GenBank: “type”:”entrez-protein”,”attrs”:”text”:”P01861″,”term_id”:”2500440406″,”term_text”:”P01861″P01861) and rabbit IgG (GenBank: “type”:”entrez-protein”,”attrs”:”text”:”P01870″,”term_id”:”121088″,”term_text”:”P01870″P01870) isotypes (69C71%) querying the NCBI Reference Sequences database with Blastp. rabbit FcRn binds rabbit IgG and human IgG1 with nearly identical affinity, whereas it has stronger interactions with the other human IgG isotypes. The comparable affinity of rabbit IgG and human IgG1 for rabbit FcRn was confirmed by FcRn-mediated recycling assay. These data verify that rabbit is an appropriate animal model for analyzing the pharmacokinetics of human therapeutic monoclonal antibodies. Introduction The neonatal Fc receptor, FcRn is usually a heterodimer consisting of an MHC-I like -chain and 2-microglobulin (2m) [1]. FcRn plays an important role in the transcytosis of maternal IgG to the fetus and in maintaining IgG and albumin homeostasis in adult [2], as well as in antigen presentation by professional Ag presenting cells in case of Ag-IgG immune complexes [3C5]. FcRn functions in pH-dependent manner, as it binds IgG at slightly acidic pH (pH 5.5C6.0) whereas this conversation is negligible at around neutral pH (pH 7.2C7.4) [6C8]. The 2 2:1 FcRn:IgG binding stoichiometry, i.e. two FcRn Monotropein molecules bind one IgG at impartial sites was first proposed by FcRn:Fc co-crystal structures [9C11] and was further confirmed by gel filtration studies in solution [12C14], and recently, by surface plasmon resonance measurements [15]. However, FcRn can also form 1:1 complexes with the Fc region of IgG when assayed under non-equilibrium conditions [16]. In rabbit, it was found decades ago that this transfer of maternal IgG and at a lower extent, albumin occurs across the rabbit fetal yolk sac membrane (YSM) from the maternal uterine lumen to the fetus [17]. Furthermore, human IgG (hIgG) injected into the maternal circulation was also transported well to the rabbit fetus [17] indicating that rabbit FcRn (rbFcRn) binds efficiently hIgG. Low level antibody transfer could be observed during early gestation, prior to gestation day (GD) 8 due to the incomplete tight junctions of the bilaminar yolk sac membrane [18], however, no or only limited antibody transport could be detected during the period of yolk sac inversion (GD 9C13). Once inversion is usually completed (around GD 15), then IgG transport starts up Monotropein through FcRn-mediated transcytosis, and the rate constantly increases with the progression of gestation [19C21]. Accordingly, the available data regarding the placental transfer of human therapeutic monoclonal antibodies (mAb IgGs) and Fc-containing biopharmaceuticals, similar to endogenous maternal IgG, indicate low fetal exposures until inverted yolk sac placenta is usually evolved, and near maternal level is usually reached at the end of gestation (GD 29C31) [19, 21C23]. The blood clearance of rabbit IgG (rbIgG) and hIgG was also investigated in rabbits and it Monotropein was found that the half-life of rbIgG and a hIgG preparation was quite comparable, around 6 and 5 days, respectively, which indicates that this FcRn-mediated salvage mechanism in rabbits works for hIgG, as Monotropein well [24C26]. The comparable half-lives of rbIgG and hIgG also suggest that rbFcRn binds similarly these IgGs, as IgG half-life depends on its binding affinity to FcRn [27]. Since then, it was clearly exhibited that FcRn is usually highly expressed in the apical plasma membrane of the brush borders endodermal cells of rabbit fetal yolk sac membrane (YSM) and in the placental capillary endothelial cells indicating that maternal IgG transport through the placenta is usually fulfilled by FcRn [28]. In addition, based on the highly conserved FcRn-IgG contact residues the pH-dependent IgG binding of FcRn was exhibited in IgG-binding assay by Monotropein Western blot using rbIgG and yolk sac lysates of rabbit fetuses [28]. Despite being an important animal model in pharmacological studies, like investigating placental transfer of therapeutic mAbs and Fc-containing biopharmaceuticals [19], the interactions of rbFcRn with rbIgG and hIgG isotypes at molecular level have not been analyzed. Therefore, we generated and purified rbFcRn and analyzed first its pH-dependent binding of rbIgG and hIgG isotypes by surface plasmon resonance assay, which provided detailed kinetic data of the conversation. Moreover, these data were further validated by FcRn-mediated recycling assay using rabbit macrophages. Materials and methods Amplification of soluble rbFcRn -chain and rabbit 2-microglobulin (rb2m) cDNAs Total RNA purification CD40 from 50 g rabbit spleen was carried out using RNeasy Plus Kit.