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Polymorphisms of Neonatal Fc Receptor in Cynomolgus and Rhesus Macaques
Drug Metab. Pharmacokinet. 29 (5): 427–430 (2014).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Note Polymorphisms of Neonatal Fc Receptor in Cynomolgus and Rhesus Macaques Yasuhiro U NO * , Masahiro U TOH and Kazuhide I WASAKI Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., Kainan, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and ¢ 2 -microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed. Keywords: cynomolgus monkey; genetic polymorphism; mRNA expression; neonatal Fc receptor (FcRn)
in the acidic environment of the endosome, but not at physiological pH.2,3) Although sequences and overall structures of FcRn are conserved well among species, their binding specificities to various types of IgG actually differ.2) Several amino acid residues of FcRn are involved in an interaction with IgG, and differences in these residues are considered to be responsible for species differences in the binding affinity of FcRn between humans and rodents.3) Hence, mutations of the amino acid residues critical for function, if any, might cause variable binding affinity of FcRn with IgG, and serum half-life of therapeutic antibodies. Indeed, a non-synonymous mutation of B2M appears to result in reduced serum levels of IgG and albumin in familial hypercatabolic hypoproteinemia patients.4) Cynomolgus macaques (Macaca fascicularis) are frequently used in the evaluation of therapeutic antibodies. Cynomolgus macaques are bred mainly in Asian countries such as China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN). Functional mutations of FCGRT and B2M, if any, would cause variations in serum half-life of therapeutic antibodies tested in cynomolgus macaques. However, the existence of polymorphisms of FCGRT and B2M has not been investigated in cynomolgus macaques.
Introduction Neonatal Fc receptor (FcRn) is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of monomeric IgG, and thus, FcRn plays a major role in regulating serum IgG levels.1,2) FcRn also binds to albumin and affects the serum half-life of albumin. FcRn is a heterodimer of ¢2microglobulin (¢2m) and a transmembrane-anchored heavy chain (MHC class I-like protein), composed of 99 and 342 amino acid residues, encoded by B2M and FCGRT, containing three and six exons, respectively. FcRn is involved in the transfer of IgG from mother to fetus in the placenta and to neonates in the small intestine. In rodents, FcRn plays an essential role in the neonatal period for transporting maternal IgG in ingested milk across the small intestine.2) In contrast, FcRn transports maternal antibody to the fetus across placenta in humans. Therefore, the major site of FcRn function appears to be different between rodents and humans. Moreover, in adult humans, FcRn is widely expressed in various tissues, including brain, lung, kidney, liver, and intestine.1,2) FcRn binds to IgG in a pH-dependent manner: FcRn binds IgG
Received March 28, 2014; Accepted April 28, 2014 J-STAGE Advance Published Date: May 6, 2014, doi:10.2133/dmpk.DMPK-14-NT-033 *To whom correspondence should be addressed: Yasuhiro UNO, D.V.M., Ph.D., Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami-Akasaka, Kainan 642-0017, Japan. Tel.©81-73-483-8881, Fax. ©81-73-483-7377, E-mail: [email protected] 427
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In this study, 71 cynomolgus and 24 rhesus macaques were screened for genetic variants of FCGRT and B2M, and the distribution of these variants was compared in cynomolgus macaques (MacfaCHN, MacfaCAM, and MacfaIDN) and rhesus macaques. Moreover, quantitative polymerase chain reaction (qPCR) was conducted to measure expression of FCGRT and B2M in 10 tissue types. Materials and Methods Materials: Oligonucleotides were synthesized by Greiner (Tokyo, Japan). All other reagents were purchased from SigmaAldrich (St. Louis, MO) or Wako (Osaka, Japan), unless otherwise specified. Extraction of genomic DNA and total RNA: Whole blood samples were collected from 71 cynomolgus macaques (24 MacfaCHN, 24 MacfaCAM, and 23 MacfaIDN, 4–5 years of age, weighing 3–5 kg) and 24 rhesus macaques (from China, 7 years of age, weighing 3–5 kg). Genomic DNA was extracted from whole blood samples using the PUREGENE DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. Tissue samples (brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus) were collected from six cynomolgus macaques (three males and three females of MacfaCHN, 4–5 years of age, weighing 3–5 kg), from which total RNA was extracted, as previously described5) and used for qPCR analysis. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. Re-sequencing: To identify genetic variants, genome samples of 71 cynomolgus macaques and 24 rhesus macaques were subjected to direct sequencing of exons 2–6 in FCGRT and exons 1–3 in B2M. Exon 1 of FCGRT was not included in the analysis due to difficulty of amplification under the conditions currently employed. PCR was carried out in a 20-µL reaction containing 1 ng of genomic DNA, 5 pmol of forward and reverse primers, and 1 unit of ExTaq HS polymerase (Takara, Ohtsu, Japan). The thermal cycler conditions were an initial denaturation at 95°C for 2 min, and 35 cycles of 20 s at 95°C, 30 s at 60°C, and 1 min at 72°C, followed by a final extension step of 10 min at 72°C; however, for exons 2–3 of FCGRT annealing was conducted at 62°C. Sequencing was performed using ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), followed by electrophoresis with the ABI PRISM 3730 DNA Analyzer (Applied Biosystems). The primers used for amplification of FCGRT were mmFCGRT (5int2a) 5A-ATCTTCCCTCTCCATAATAGATTCTTC-3A and mmFCGRT (3int4a) 5A-CAAGAGTGAAACTTCGTCTCAACA-3A for exons 2–3, mmFCGRT (5int4a) 5A-CCTCCTCTTTTACCACCACATTATC-3A and mmFCGRT (3int5a) 5A-ACAAGGCAGTCAGATTCAAGGA3A for exon 4, mmFCGRT (5int5a) 5A-GCTGTGGCTGGTTCTTACATC-3A and mmFCGRT (3int7a) 5A-GTGTTATATTATCGAGGTGGAAAAGAG-3A for exons 5–6; and those of B2M were mmB2M (5flk1a) 5A-CTCTAACCTAGCACTGCGTCTCT-3A and mmB2M (3int1a) 5A-CTTATCAACGCCCTAAACTTTGTC-3A for exon 1, and mmB2M (5int1a) 5A-GATACCAAGTAATGGTTTATTCCTCAA-3A and mmB2M (3int3a) 5A-CCTTCTTCACACCACAGGAGAA-3A for exons 2–3. The primers used for sequencing of FCGRT were mfFCGRT (3btj1) 5A-CTGCTTGAGGTCGAAATTCAT-3A for exon 2, mfFCGRT (5btj1) 5A-GGATCAAGGAGAAGCTCTTTCTG-3A for exon 3; mmFCGRT (3int5a) for exon 4,
mmFCGRT (5int5a) for exon 5, and mmFCGRT (3int7a) for exon 6; and those of B2M were mmB2M (5flk1a) for exon 1, mmB2M (5int1a) for exon 2, and mmB2M (3int3a) for exon 3. Sequence data were analyzed using Sequencher (Gene Codes, Ann Arbor, MI) and DNASIS Pro (Hitachi Software, Tokyo, Japan). Genetic variants were determined by comparing the sequence of cynomolgus FCGRT cDNA (AF485818) or cynomolgus B2M cDNA (AF485817). Measurement of gene expression: Gene expression of FCGRT and B2M was quantified by qPCR in brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus, as described previously5) using gene-specific primers. Each tissue RNA originated from six cynomolgus macaques. Briefly, reverse transcription reactions were carried out as described above using random primers (Invitrogen, Carlsbad, CA), and a twenty-fifth of the reaction was used for the subsequent PCR. PCR amplification was conducted in a total volume of 20 µL using SYBR Green PCR Master Mix (Applied Biosystems) with the ABI PRISM 7500 sequence detection system (Applied Biosystems), according to the manufacturer’s protocols. The primers, used at the final concentrations of 300 nM, were mfFCGRT (5btj1) and mfFCGRT (3btj1) for FCGRT, and mfB2M (5btj1) 5A-CTGGCGCTACTCTCTCTTTCTG-3A and mfB2M (3btj1) 5A-CACATAGCAATTCAGGAAATTTGG-3A for B2M. To determine the relative expression level, the raw data were normalized with the 18S ribosomal RNA level based on three independent amplifications. Results and Discussion Re-sequencing of FCGRT and B2M was carried out using genome samples from 71 cynomolgus macaques (24 MacfaCHN, 24 MacfaCAM, and 23 MacfaIDN) and 24 rhesus macaques. For FCGRT, a total of 21 variants were identified, including 4 non-synonymous variants, c.77G>A, c.763G>A, c.937G>T, and c.1064C>T (Table 1). Among these 21 variants, 15 (71%) were unique to cynomolgus macaques; 3, 2, and 5 were found only in MacfaCHN, MacfaCAM, and MacfaIDN, respectively, whereas 2 (9.5%) were unique to rhesus macaques. Four non-synonymous variants were shared by both cynomolgus and rhesus macaques (Table 1). Similarly, uneven distribution of alleles has been described between cynomolgus and rhesus macaques or between MacfaCHN and MacfaIDN, for the genes such as P450 genes.6,7) In the variants unique to cynomolgus macaque, 5 were found in both MacfaCHN and MacfaCAM; none of the variants found in MacfaIDN was shared by MacfaCHN or MacfaCAM, indicating genetic similarities of MacfaCHN and MacfaCAM as previously described,8) most likely due to the fact that the founder animals of MacfaCHN originated from Indochina, including Cambodia.9) In contrast, for B2M, only 5 variants were found, including 2 nonsynonymous variants, c.8C>G and c.335C>A (Table 1). Among these 5 variants, 4 were found only in cynomolgus macaques, and the remaining 1 variant was found in both cynomolgus and rhesus macaques. Several amino acid residues are important for FcRn function. It should be noted that amino acid numbers of FcRn described in the present paper were counted from the beginning of the signal peptide. For example, E138, E139, D153, W154, and L158 in the ¡2-domain of a transmembrane-anchored heavy chain of FcRn participate in an interaction of FcRn with IgG in humans,3) and the differences in these amino acid residues would likely account for the species differences in binding affinity of FcRn with IgG
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Polymorphisms of Neonatal Fc Receptor in Macaques Table 1. Genetic variants identified in cynomolgus and rhesus FCGRT and B2M
Variant
Exon
Nucleotide changea
FCGRT: c.77G>A c.78C>T c.81C>T c.120C>T c.135T>A c.378C>T c.543C>T c.558G>T c.576G>A c.633C>T c.723C>T c.763G>A c.843G>A c.849G>A c.891G>A c.933G>C c.937G>T c.1023G>A c.1041G>A c.1064C>T c.1071T>C
2 2 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 6 6 6 6
AGAAA(G>A)CCACC GAAAG(C>T)CACCT AGCCA(C>T)CTCTC TCGCC(C>T)GCCCC ACGCC(T>A)GCCTT AACAC(C>T)TCGGT TTCTC(C>T)TGCCC CGGCT(G>T)CGGGA GAGAG(G>A)GGCCG CGACC(C>T)GGCAA ATGGC(C>T)GCTGG GTGAC(G>A)GCTCC GCGGG(G>A)CTGGC CTGGC(G>A)CAGCC TCCTC(G>A)GTGCT CTCAC(G>C)GCAGC CGGCA(G>T)CGGCT ACCGG(G>A)TCCCT ACCCC(G>A)GGGGA TGATT(C>T)GAAGG AAGGA(T>C)ATAAA
B2M: c.8C>G c.192G>A c.261C>T c.327G>A c.335C>A
1 2 2 2 2
GTCTC(C>G)CTCAG GGAGA(G>A)AAAAT TACTA(C>T)ACTGA TCAGG(G>A)CCCAG CAGGA(C>A)AGTTA
Amino acid change
S26N
G255S
A313S
S355L
P3R
T112K
MacfaCHN
Allele frequency Cynomolgus MacfaCAM MacfaIDN
Rhesus
0/48 19/48 0/48 48/48 1/48 0/48 30/48 0/48 21/48 17/42 0/42 1/42 0/42 1/42 0/46 0/46 3/46 0/48 11/48 1/48 1/48
0/46 20/46 0/46 46/46 5/46 0/46 22/46 1/46 22/46 15/40 1/40 0/40 0/40 2/40 0/44 0/44 2/44 0/42 5/42 0/42 3/42
3/48 5/48 2/48 6/48 0/48 0/48 0/48 0/48 48/48 0/46 0/46 0/46 0/46 0/46 1/46 1/46 0/46 3/46 0/46 0/46 0/46
0/42 19/42 0/42 42/42 0/42 3/48 48/48 0/48 11/48 0/46 0/46 0/46 1/46 0/46 0/32 1/32 0/32 0/36 0/36 0/36 0/36
9/40 0/26 0/26 2/26 2/26
4/34 0/20 0/20 0/20 2/20
7/44 4/40 1/40 0/40 0/40
22/38 0/36 0/36 0/36 0/36
a
Nucleotide changes were detected by comparison with the cDNA sequence of cynomolgus FCGRT (AF485818) or B2M (AF485817), which were tentatively designated as a wild-type sequence in this study.
between humans and rodents.2) These amino acid residues are conserved in cynomolgus macaques. The variants found in the present study were located in the ¡1- (S26N) or ¡3- (G255S, A313S, and S355L) domain, which are not relevant to FcRn function. Moreover, the variants found in this study did not change the amino acid residue H189, important for binding with albumin.1) A previous study provided the evidence that mutation of ¢2m (A11P) apparently results in hypercatabolic hypoproteinemia due to hypercatabolic albumin and IgG deficiencies in humans,4) which have also been demonstrated in mice lacking FcRn.10) Moreover, I1 of ¢2m is involved in IgG binding.2,3) However, the nonsynonymous variants (P3R and T112K) found in macaque B2M did not change this amino acid residue. Overall, the results indicated that the cynomolgus macaques analyzed in the present study did not possess mutations corresponding to those functionally important in human FcRn. Efforts have been made to identify functional FCGRT alleles in humans. Thirty-three genetic variations were found in FCGRT, but the two non-synonymous variations characterized were similar to the wild type in their intracellular localization and antibody recycling efficiencies.11) The variable number of tandem repeat (VNTR) found in human FCGRT promoter influenced the transcriptional activity of FCGRT in monocytes, but has not shown association with risk for glomerular nephritis or with transfer of IgG from mother to fetus.12) In the present study, the genetic variants found in cynomolgus and rhesus FCGRT and B2M did not appear to influence the FcRn function. Functionally relevant mutation is yet to be identified in human or macaque FCGRT.
Fig. 1. Tissue expression patterns of cynomolgus FCGRT and B2M The expression level of FCGRT and B2M was measured by qPCR analysis, as described in Materials and Methods, in the brain, lung, heart, liver, kidney, jejunum, adrenal gland, testis, ovary, and uterus. The expression level of each gene was normalized to the 18S rRNA level and is shown as the mean « S.D. from three independent amplifications. The value of the most abundant expression was adjusted to 1, to which all other expression levels were adjusted accordingly.
FcRn is ubiquitously expressed in human tissues.1,2) To analyze tissue expression patterns of FcRn in cynomolgus macaques, expression of FCGRT and B2M was measured by qPCR in the brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Both of these genes showed universal expression patterns in these 10 tissue types (Fig. 1). Therefore, like in humans, the genes for FcRn appear to be expressed in various tissue types for cynomolgus macaques. In contrast, expression patterns are different in rodents. For example, FcRn is expressed in
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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epithelial cells of the fetal and adult human intestines, but not abundantly expressed in the adult rodent intestines.2) In the present study, FCGRT and B2M were expressed in adult intestines (jejunum) (Fig. 1), similar to the previous report,13) partly indicating similarities in tissue expression patterns of FcRn between cynomolgus macaques and humans. In conclusion, our screening of cynomolgus macaques (MacfaCHN, MacfaCAM, and MacfaIDN) and rhesus macaques successfully identified 21 and 5 variants in FCGRT and B2M, including 4 and 2 non-synonymous variants, respectively. None of these variants appeared to result in mutations of amino acid residues important for FcRn function, and thus these cynomolgus (and rhesus) macaques might not show inter-animal variability in FcRn function, or serum-half life of therapeutic antibodies. Moreover, expression of FCGRT and B2M was detected in cynomolgus macaque tissues. These results, together with the differences in FcRn in binding affinity and tissue expression pattern between humans and rodents, suggest that cynomolgus macaques would be an ideal animal species for studies of therapeutic antibodies. Acknowledgments: We greatly thank Mr. Lance Bell for his advice on English writing. References 1) 2) 3)
4)
Kuo, T. T. and Aveson, V. G.: Neonatal Fc receptor and IgG-based therapeutics. MAbs, 3: 422–430 (2011). Roopenian, D. C. and Akilesh, S.: FcRn: the neonatal Fc receptor comes of age. Nat. Rev. Immunol., 7: 715–725 (2007). Andersen, J. T. and Sandlie, I.: The versatile MHC class I-related FcRn protects IgG and albumin from degradation: implications for development of new diagnostics and therapeutics. Drug Metab. Pharmacokinet., 24: 318–332 (2009). Wani, M. A., Haynes, L. D., Kim, J., Bronson, C. L., Chaudhury, C., Mohanty, S., Waldmann, T. A., Robinson, J. M. and Anderson, C. L.:
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Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant ¢2-microglobulin gene. Proc. Natl. Acad. Sci. USA, 103: 5084–5089 (2006). Uno, Y., Fujino, H., Kito, G., Kamataki, T. and Nagata, R.: CYP2C76, a novel cytochrome P450 in cynomolgus monkey, is a major CYP2C in liver, metabolizing tolbutamide and testosterone. Mol. Pharmacol., 70: 477–486 (2006). Uno, Y., Matsushita, A., Osada, N., Uehara, S., Kohara, S., Nagata, R., Fukuzaki, K., Utoh, M., Murayama, N., et al.: Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques. Drug Metab. Dispos., 38: 209–214 (2010). Uno, Y., Sakuraba, H., Uehara, S., Kumano, T., Matsuno, K., Nakamura, C., Kito, G., Kamataki, T. and Nagata, R.: A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism. Drug Metab. Dispos., 37: 14–17 (2009). Ise, R., Nakanishi, Y., Kohara, S., Yamashita, H., Yoshikawa, T., Iwasaki, K., Nagata, R., Fukuzaki, K., Utoh, M., et al.: Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes. Drug Metab. Pharmacokinet., 27: 307–316 (2012). Zhiyong, F. and Yanling, S.: Chinese primate status and primate captive breeding for biomedical research in China. In Institute for Laboratory Animal Research (eds.): International perspectives: the future of nonhuman primate resources, Washington, D. C., National Academies Press, 2002, pp. 36–45. Chaudhury, C., Mehnaz, S., Robinson, J. M., Hayton, W. L., Pearl, D. K., Roopenian, D. C. and Anderson, C. L.: The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J. Exp. Med., 197: 315–322 (2003). Ishii-Watabe, A., Saito, Y., Suzuki, T., Tada, M., Ukaji, M., Maekawa, K., Kurose, K., Kaniwa, N., Sawada, J., et al.: Genetic polymorphisms of FCGRT encoding FcRn in a Japanese population and their functional analysis. Drug Metab. Pharmacokinet., 25: 578–587 (2010). Roopenian, D. C. and Sun, V. Z.: Clinical ramifications of the MHC family Fc receptor FcRn. J. Clin. Immunol., 30: 790–797 (2010). Hornby, P. J., Cooper, P. R., Kliwinski, C., Ragwan, E., Mabus, J. R., Harman, B., Thompson, S., Kauffman, A. L., Yan, Z., et al.: Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis. Pharm. Res., 31: 908–922 (2014).
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
Note Polymorphisms of Neonatal Fc Receptor in Cynomolgus and Rhesus Macaques Yasuhiro U NO * , Masahiro U TOH and Kazuhide I WASAKI Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., Kainan, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and ¢ 2 -microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed. Keywords: cynomolgus monkey; genetic polymorphism; mRNA expression; neonatal Fc receptor (FcRn)
in the acidic environment of the endosome, but not at physiological pH.2,3) Although sequences and overall structures of FcRn are conserved well among species, their binding specificities to various types of IgG actually differ.2) Several amino acid residues of FcRn are involved in an interaction with IgG, and differences in these residues are considered to be responsible for species differences in the binding affinity of FcRn between humans and rodents.3) Hence, mutations of the amino acid residues critical for function, if any, might cause variable binding affinity of FcRn with IgG, and serum half-life of therapeutic antibodies. Indeed, a non-synonymous mutation of B2M appears to result in reduced serum levels of IgG and albumin in familial hypercatabolic hypoproteinemia patients.4) Cynomolgus macaques (Macaca fascicularis) are frequently used in the evaluation of therapeutic antibodies. Cynomolgus macaques are bred mainly in Asian countries such as China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN). Functional mutations of FCGRT and B2M, if any, would cause variations in serum half-life of therapeutic antibodies tested in cynomolgus macaques. However, the existence of polymorphisms of FCGRT and B2M has not been investigated in cynomolgus macaques.
Introduction Neonatal Fc receptor (FcRn) is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of monomeric IgG, and thus, FcRn plays a major role in regulating serum IgG levels.1,2) FcRn also binds to albumin and affects the serum half-life of albumin. FcRn is a heterodimer of ¢2microglobulin (¢2m) and a transmembrane-anchored heavy chain (MHC class I-like protein), composed of 99 and 342 amino acid residues, encoded by B2M and FCGRT, containing three and six exons, respectively. FcRn is involved in the transfer of IgG from mother to fetus in the placenta and to neonates in the small intestine. In rodents, FcRn plays an essential role in the neonatal period for transporting maternal IgG in ingested milk across the small intestine.2) In contrast, FcRn transports maternal antibody to the fetus across placenta in humans. Therefore, the major site of FcRn function appears to be different between rodents and humans. Moreover, in adult humans, FcRn is widely expressed in various tissues, including brain, lung, kidney, liver, and intestine.1,2) FcRn binds to IgG in a pH-dependent manner: FcRn binds IgG
Received March 28, 2014; Accepted April 28, 2014 J-STAGE Advance Published Date: May 6, 2014, doi:10.2133/dmpk.DMPK-14-NT-033 *To whom correspondence should be addressed: Yasuhiro UNO, D.V.M., Ph.D., Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minami-Akasaka, Kainan 642-0017, Japan. Tel.©81-73-483-8881, Fax. ©81-73-483-7377, E-mail: [email protected] 427
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In this study, 71 cynomolgus and 24 rhesus macaques were screened for genetic variants of FCGRT and B2M, and the distribution of these variants was compared in cynomolgus macaques (MacfaCHN, MacfaCAM, and MacfaIDN) and rhesus macaques. Moreover, quantitative polymerase chain reaction (qPCR) was conducted to measure expression of FCGRT and B2M in 10 tissue types. Materials and Methods Materials: Oligonucleotides were synthesized by Greiner (Tokyo, Japan). All other reagents were purchased from SigmaAldrich (St. Louis, MO) or Wako (Osaka, Japan), unless otherwise specified. Extraction of genomic DNA and total RNA: Whole blood samples were collected from 71 cynomolgus macaques (24 MacfaCHN, 24 MacfaCAM, and 23 MacfaIDN, 4–5 years of age, weighing 3–5 kg) and 24 rhesus macaques (from China, 7 years of age, weighing 3–5 kg). Genomic DNA was extracted from whole blood samples using the PUREGENE DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. Tissue samples (brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus) were collected from six cynomolgus macaques (three males and three females of MacfaCHN, 4–5 years of age, weighing 3–5 kg), from which total RNA was extracted, as previously described5) and used for qPCR analysis. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. Re-sequencing: To identify genetic variants, genome samples of 71 cynomolgus macaques and 24 rhesus macaques were subjected to direct sequencing of exons 2–6 in FCGRT and exons 1–3 in B2M. Exon 1 of FCGRT was not included in the analysis due to difficulty of amplification under the conditions currently employed. PCR was carried out in a 20-µL reaction containing 1 ng of genomic DNA, 5 pmol of forward and reverse primers, and 1 unit of ExTaq HS polymerase (Takara, Ohtsu, Japan). The thermal cycler conditions were an initial denaturation at 95°C for 2 min, and 35 cycles of 20 s at 95°C, 30 s at 60°C, and 1 min at 72°C, followed by a final extension step of 10 min at 72°C; however, for exons 2–3 of FCGRT annealing was conducted at 62°C. Sequencing was performed using ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), followed by electrophoresis with the ABI PRISM 3730 DNA Analyzer (Applied Biosystems). The primers used for amplification of FCGRT were mmFCGRT (5int2a) 5A-ATCTTCCCTCTCCATAATAGATTCTTC-3A and mmFCGRT (3int4a) 5A-CAAGAGTGAAACTTCGTCTCAACA-3A for exons 2–3, mmFCGRT (5int4a) 5A-CCTCCTCTTTTACCACCACATTATC-3A and mmFCGRT (3int5a) 5A-ACAAGGCAGTCAGATTCAAGGA3A for exon 4, mmFCGRT (5int5a) 5A-GCTGTGGCTGGTTCTTACATC-3A and mmFCGRT (3int7a) 5A-GTGTTATATTATCGAGGTGGAAAAGAG-3A for exons 5–6; and those of B2M were mmB2M (5flk1a) 5A-CTCTAACCTAGCACTGCGTCTCT-3A and mmB2M (3int1a) 5A-CTTATCAACGCCCTAAACTTTGTC-3A for exon 1, and mmB2M (5int1a) 5A-GATACCAAGTAATGGTTTATTCCTCAA-3A and mmB2M (3int3a) 5A-CCTTCTTCACACCACAGGAGAA-3A for exons 2–3. The primers used for sequencing of FCGRT were mfFCGRT (3btj1) 5A-CTGCTTGAGGTCGAAATTCAT-3A for exon 2, mfFCGRT (5btj1) 5A-GGATCAAGGAGAAGCTCTTTCTG-3A for exon 3; mmFCGRT (3int5a) for exon 4,
mmFCGRT (5int5a) for exon 5, and mmFCGRT (3int7a) for exon 6; and those of B2M were mmB2M (5flk1a) for exon 1, mmB2M (5int1a) for exon 2, and mmB2M (3int3a) for exon 3. Sequence data were analyzed using Sequencher (Gene Codes, Ann Arbor, MI) and DNASIS Pro (Hitachi Software, Tokyo, Japan). Genetic variants were determined by comparing the sequence of cynomolgus FCGRT cDNA (AF485818) or cynomolgus B2M cDNA (AF485817). Measurement of gene expression: Gene expression of FCGRT and B2M was quantified by qPCR in brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus, as described previously5) using gene-specific primers. Each tissue RNA originated from six cynomolgus macaques. Briefly, reverse transcription reactions were carried out as described above using random primers (Invitrogen, Carlsbad, CA), and a twenty-fifth of the reaction was used for the subsequent PCR. PCR amplification was conducted in a total volume of 20 µL using SYBR Green PCR Master Mix (Applied Biosystems) with the ABI PRISM 7500 sequence detection system (Applied Biosystems), according to the manufacturer’s protocols. The primers, used at the final concentrations of 300 nM, were mfFCGRT (5btj1) and mfFCGRT (3btj1) for FCGRT, and mfB2M (5btj1) 5A-CTGGCGCTACTCTCTCTTTCTG-3A and mfB2M (3btj1) 5A-CACATAGCAATTCAGGAAATTTGG-3A for B2M. To determine the relative expression level, the raw data were normalized with the 18S ribosomal RNA level based on three independent amplifications. Results and Discussion Re-sequencing of FCGRT and B2M was carried out using genome samples from 71 cynomolgus macaques (24 MacfaCHN, 24 MacfaCAM, and 23 MacfaIDN) and 24 rhesus macaques. For FCGRT, a total of 21 variants were identified, including 4 non-synonymous variants, c.77G>A, c.763G>A, c.937G>T, and c.1064C>T (Table 1). Among these 21 variants, 15 (71%) were unique to cynomolgus macaques; 3, 2, and 5 were found only in MacfaCHN, MacfaCAM, and MacfaIDN, respectively, whereas 2 (9.5%) were unique to rhesus macaques. Four non-synonymous variants were shared by both cynomolgus and rhesus macaques (Table 1). Similarly, uneven distribution of alleles has been described between cynomolgus and rhesus macaques or between MacfaCHN and MacfaIDN, for the genes such as P450 genes.6,7) In the variants unique to cynomolgus macaque, 5 were found in both MacfaCHN and MacfaCAM; none of the variants found in MacfaIDN was shared by MacfaCHN or MacfaCAM, indicating genetic similarities of MacfaCHN and MacfaCAM as previously described,8) most likely due to the fact that the founder animals of MacfaCHN originated from Indochina, including Cambodia.9) In contrast, for B2M, only 5 variants were found, including 2 nonsynonymous variants, c.8C>G and c.335C>A (Table 1). Among these 5 variants, 4 were found only in cynomolgus macaques, and the remaining 1 variant was found in both cynomolgus and rhesus macaques. Several amino acid residues are important for FcRn function. It should be noted that amino acid numbers of FcRn described in the present paper were counted from the beginning of the signal peptide. For example, E138, E139, D153, W154, and L158 in the ¡2-domain of a transmembrane-anchored heavy chain of FcRn participate in an interaction of FcRn with IgG in humans,3) and the differences in these amino acid residues would likely account for the species differences in binding affinity of FcRn with IgG
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Polymorphisms of Neonatal Fc Receptor in Macaques Table 1. Genetic variants identified in cynomolgus and rhesus FCGRT and B2M
Variant
Exon
Nucleotide changea
FCGRT: c.77G>A c.78C>T c.81C>T c.120C>T c.135T>A c.378C>T c.543C>T c.558G>T c.576G>A c.633C>T c.723C>T c.763G>A c.843G>A c.849G>A c.891G>A c.933G>C c.937G>T c.1023G>A c.1041G>A c.1064C>T c.1071T>C
2 2 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 6 6 6 6
AGAAA(G>A)CCACC GAAAG(C>T)CACCT AGCCA(C>T)CTCTC TCGCC(C>T)GCCCC ACGCC(T>A)GCCTT AACAC(C>T)TCGGT TTCTC(C>T)TGCCC CGGCT(G>T)CGGGA GAGAG(G>A)GGCCG CGACC(C>T)GGCAA ATGGC(C>T)GCTGG GTGAC(G>A)GCTCC GCGGG(G>A)CTGGC CTGGC(G>A)CAGCC TCCTC(G>A)GTGCT CTCAC(G>C)GCAGC CGGCA(G>T)CGGCT ACCGG(G>A)TCCCT ACCCC(G>A)GGGGA TGATT(C>T)GAAGG AAGGA(T>C)ATAAA
B2M: c.8C>G c.192G>A c.261C>T c.327G>A c.335C>A
1 2 2 2 2
GTCTC(C>G)CTCAG GGAGA(G>A)AAAAT TACTA(C>T)ACTGA TCAGG(G>A)CCCAG CAGGA(C>A)AGTTA
Amino acid change
S26N
G255S
A313S
S355L
P3R
T112K
MacfaCHN
Allele frequency Cynomolgus MacfaCAM MacfaIDN
Rhesus
0/48 19/48 0/48 48/48 1/48 0/48 30/48 0/48 21/48 17/42 0/42 1/42 0/42 1/42 0/46 0/46 3/46 0/48 11/48 1/48 1/48
0/46 20/46 0/46 46/46 5/46 0/46 22/46 1/46 22/46 15/40 1/40 0/40 0/40 2/40 0/44 0/44 2/44 0/42 5/42 0/42 3/42
3/48 5/48 2/48 6/48 0/48 0/48 0/48 0/48 48/48 0/46 0/46 0/46 0/46 0/46 1/46 1/46 0/46 3/46 0/46 0/46 0/46
0/42 19/42 0/42 42/42 0/42 3/48 48/48 0/48 11/48 0/46 0/46 0/46 1/46 0/46 0/32 1/32 0/32 0/36 0/36 0/36 0/36
9/40 0/26 0/26 2/26 2/26
4/34 0/20 0/20 0/20 2/20
7/44 4/40 1/40 0/40 0/40
22/38 0/36 0/36 0/36 0/36
a
Nucleotide changes were detected by comparison with the cDNA sequence of cynomolgus FCGRT (AF485818) or B2M (AF485817), which were tentatively designated as a wild-type sequence in this study.
between humans and rodents.2) These amino acid residues are conserved in cynomolgus macaques. The variants found in the present study were located in the ¡1- (S26N) or ¡3- (G255S, A313S, and S355L) domain, which are not relevant to FcRn function. Moreover, the variants found in this study did not change the amino acid residue H189, important for binding with albumin.1) A previous study provided the evidence that mutation of ¢2m (A11P) apparently results in hypercatabolic hypoproteinemia due to hypercatabolic albumin and IgG deficiencies in humans,4) which have also been demonstrated in mice lacking FcRn.10) Moreover, I1 of ¢2m is involved in IgG binding.2,3) However, the nonsynonymous variants (P3R and T112K) found in macaque B2M did not change this amino acid residue. Overall, the results indicated that the cynomolgus macaques analyzed in the present study did not possess mutations corresponding to those functionally important in human FcRn. Efforts have been made to identify functional FCGRT alleles in humans. Thirty-three genetic variations were found in FCGRT, but the two non-synonymous variations characterized were similar to the wild type in their intracellular localization and antibody recycling efficiencies.11) The variable number of tandem repeat (VNTR) found in human FCGRT promoter influenced the transcriptional activity of FCGRT in monocytes, but has not shown association with risk for glomerular nephritis or with transfer of IgG from mother to fetus.12) In the present study, the genetic variants found in cynomolgus and rhesus FCGRT and B2M did not appear to influence the FcRn function. Functionally relevant mutation is yet to be identified in human or macaque FCGRT.
Fig. 1. Tissue expression patterns of cynomolgus FCGRT and B2M The expression level of FCGRT and B2M was measured by qPCR analysis, as described in Materials and Methods, in the brain, lung, heart, liver, kidney, jejunum, adrenal gland, testis, ovary, and uterus. The expression level of each gene was normalized to the 18S rRNA level and is shown as the mean « S.D. from three independent amplifications. The value of the most abundant expression was adjusted to 1, to which all other expression levels were adjusted accordingly.
FcRn is ubiquitously expressed in human tissues.1,2) To analyze tissue expression patterns of FcRn in cynomolgus macaques, expression of FCGRT and B2M was measured by qPCR in the brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Both of these genes showed universal expression patterns in these 10 tissue types (Fig. 1). Therefore, like in humans, the genes for FcRn appear to be expressed in various tissue types for cynomolgus macaques. In contrast, expression patterns are different in rodents. For example, FcRn is expressed in
Copyright © 2014 by the Japanese Society for the Study of Xenobiotics (JSSX)
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epithelial cells of the fetal and adult human intestines, but not abundantly expressed in the adult rodent intestines.2) In the present study, FCGRT and B2M were expressed in adult intestines (jejunum) (Fig. 1), similar to the previous report,13) partly indicating similarities in tissue expression patterns of FcRn between cynomolgus macaques and humans. In conclusion, our screening of cynomolgus macaques (MacfaCHN, MacfaCAM, and MacfaIDN) and rhesus macaques successfully identified 21 and 5 variants in FCGRT and B2M, including 4 and 2 non-synonymous variants, respectively. None of these variants appeared to result in mutations of amino acid residues important for FcRn function, and thus these cynomolgus (and rhesus) macaques might not show inter-animal variability in FcRn function, or serum-half life of therapeutic antibodies. Moreover, expression of FCGRT and B2M was detected in cynomolgus macaque tissues. These results, together with the differences in FcRn in binding affinity and tissue expression pattern between humans and rodents, suggest that cynomolgus macaques would be an ideal animal species for studies of therapeutic antibodies. Acknowledgments: We greatly thank Mr. Lance Bell for his advice on English writing. References 1) 2) 3)
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Familial hypercatabolic hypoproteinemia caused by deficiency of the neonatal Fc receptor, FcRn, due to a mutant ¢2-microglobulin gene. Proc. Natl. Acad. Sci. USA, 103: 5084–5089 (2006). Uno, Y., Fujino, H., Kito, G., Kamataki, T. and Nagata, R.: CYP2C76, a novel cytochrome P450 in cynomolgus monkey, is a major CYP2C in liver, metabolizing tolbutamide and testosterone. Mol. Pharmacol., 70: 477–486 (2006). Uno, Y., Matsushita, A., Osada, N., Uehara, S., Kohara, S., Nagata, R., Fukuzaki, K., Utoh, M., Murayama, N., et al.: Genetic variants of CYP3A4 and CYP3A5 in cynomolgus and rhesus macaques. Drug Metab. Dispos., 38: 209–214 (2010). Uno, Y., Sakuraba, H., Uehara, S., Kumano, T., Matsuno, K., Nakamura, C., Kito, G., Kamataki, T. and Nagata, R.: A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism. Drug Metab. Dispos., 37: 14–17 (2009). Ise, R., Nakanishi, Y., Kohara, S., Yamashita, H., Yoshikawa, T., Iwasaki, K., Nagata, R., Fukuzaki, K., Utoh, M., et al.: Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes. Drug Metab. Pharmacokinet., 27: 307–316 (2012). Zhiyong, F. and Yanling, S.: Chinese primate status and primate captive breeding for biomedical research in China. In Institute for Laboratory Animal Research (eds.): International perspectives: the future of nonhuman primate resources, Washington, D. C., National Academies Press, 2002, pp. 36–45. Chaudhury, C., Mehnaz, S., Robinson, J. M., Hayton, W. L., Pearl, D. K., Roopenian, D. C. and Anderson, C. L.: The major histocompatibility complex-related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J. Exp. Med., 197: 315–322 (2003). Ishii-Watabe, A., Saito, Y., Suzuki, T., Tada, M., Ukaji, M., Maekawa, K., Kurose, K., Kaniwa, N., Sawada, J., et al.: Genetic polymorphisms of FCGRT encoding FcRn in a Japanese population and their functional analysis. Drug Metab. Pharmacokinet., 25: 578–587 (2010). Roopenian, D. C. and Sun, V. Z.: Clinical ramifications of the MHC family Fc receptor FcRn. J. Clin. Immunol., 30: 790–797 (2010). Hornby, P. J., Cooper, P. R., Kliwinski, C., Ragwan, E., Mabus, J. R., Harman, B., Thompson, S., Kauffman, A. L., Yan, Z., et al.: Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis. Pharm. Res., 31: 908–922 (2014).
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