Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery

Abstract

If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.

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Figure 1: Engineering of the mouse Igh locus.
Figure 2: Human and mouse variable region usage in transgenic mice.
Figure 3: Normal B-cell maturation and antibody production in HK and HL mice.
Figure 4: Sequence analysis of antibody transcripts in HK mice.
Figure 5: Analysis of V-region sequences of ovalbumin-specific mAbs isolated from prototype mice with two or three heavy-chain BACs and an inversion.
Figure 6: Binding and function of mAbs isolated from HK mice.

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Acknowledgements

We thank A. Sandham, J. Clube and D. Casson for frequent critical input and discussions on this work and P. Alexander, A. Muncey and A. Lloyd for administrative support. Kymab was funded by investments from the Wellcome Trust, London.

Author information

A. Bradley, G.A.F., E.-C.L. and M.J.O. planned and coordinated the project and wrote the manuscript; Q.L. and W.W. devised transgenic strategies and performed experiments; H.L., D.S., S.O., H.A. and A.C. performed molecular biology and created modified and compound BACs; Q.L., D.S., H.H., J.P., L. Bayliss and A. Beasley performed all the ES cell manipulations and culturing; H.L., R.B., A.C. and T.B.-G. performed molecular biology and genotyped all ES cell lines and mice; S.C. microinjected ES cells into blastocysts and maintained the mouse colonies; D.S. and B.F. analyzed mouse immune cells by FACS; A.W. and H.L. sequenced antibody transcripts of full repertoires; N.E. performed bioinformatic and statistical analysis; S.H. planned mAb discovery experiments; V.G., I.K., J.C. and A.D. conducted and coordinated all antibody discovery and characterization; A.L., H.A., A.M., N.W., D.S. and L.T. performed functional assays on mAbs; M.K., L. Bonoli, B.F. and R.S. cloned antibody heavy and light chains; Q.L., V.W., S.W. and A.F.-D. created hybridomas for mAb discovery; and A.S. performed peripheral immune cell analysis.

Correspondence to Allan Bradley.

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Lee, E., Liang, Q., Ali, H. et al. Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery. Nat Biotechnol 32, 356–363 (2014). https://doi.org/10.1038/nbt.2825

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