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Antigen-specific human polyclonal antibodies from hyperimmunized cattle

Nature Biotechnology volume 27, pages 173–181 (2009)Cite this article

Abstract

Antigen-specific human polyclonal antibodies (hpAbs), produced by hyperimmunization, could be useful for treating many human diseases. However, yields from available transgenic mice and transchromosomic (Tc) cattle carrying human immunoglobulin loci are too low for therapeutic applications. We report a Tc bovine system that produces large yields of hpAbs. Tc cattle were generated by transferring a human artificial chromosome vector carrying the entire unrearranged, human immunoglobulin heavy (hIGH) and κ-light (hIGK) chain loci to bovine fibroblasts in which two endogenous bovine IgH chain loci were inactivated. Plasma from the oldest animal contained >2 g/l of hIgG, paired with either human κ-light chain (up to ∼650 μg/ml, fully human) or with bovine κ- or λ-light chain (chimeric), with a normal hIgG subclass distribution. Hyperimmunization with anthrax protective antigen triggered a hIgG-mediated humoral immune response comprising a high proportion of antigen-specific hIgG. Purified, fully human and chimeric hIgGs were highly active in an in vitro toxin neutralization assay and protective in an in vivo mouse challenge assay.

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Figure 1: Generation and analysis of κHAC/IGHM−/− and κHAC/IGHM−/−IGHML1−/− cattle.
Figure 2: Characterization of antigen-specific hIgG produced in κHAC/IGHM−/−IGHML1−/− calf 468.
Figure 3: Glycosylation analysis of antigen-specific hIgG produced in κHAC/IGHM−/−IGHML1−/− calf 468.
Figure 4: In vivo mouse protection assay of PA-specific hIgG produced in κHAC/IGHM−/−IGHML1−/− calf 468.

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Acknowledgements

We thank the teams at Transova Genetics for their efforts in embryo transfer and at the Research Development Center for their assistance in fetal and sample collection, calf delivery and care. We thank the teams at Hematech Inc. for their assistance in gene targeting, cell culture, embryonic cloning and any other technical procedures. We also thank Ralph Kubo, Sandra Rickert, Mareto Hosono and Tomoyuki Tahara for their useful comments on the immunological study.

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Authors and Affiliations

  1. Hematech, Inc, 4401 S. Technology Dr., Sioux Falls, 57106, South Dakota, USA

    Yoshimi Kuroiwa, Poothappillai Kasinathan, Thillainayagen Sathiyaseelan, Jin-an Jiao, Hiroaki Matsushita, Janaki Sathiyaseelan, Hua Wu, Jenny Mellquist, Melissa Hammitt, Julie Koster & James M Robl

  2. Kyowa Hakko Kirin Co., Ltd., 1-6-1 Ohtemachi, Chiyoda-ku, Tokyo, 100-8185, Japan

    Yoshimi Kuroiwa, Satoru Kamoda, Katsumi Tachibana & Isao Ishida

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  1. Yoshimi Kuroiwa
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  2. Poothappillai Kasinathan
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Contributions

Y.K. and J.M.R. led the work and wrote the manuscript. P.K. and J.K. led animal cloning. T.S. and H.W. led immunological analyses and immunization. J.J. led purification and protein chemistry. H.M. and J.S. carried out gene targeting experiments. J.M. conducted the mouse challenge assay. M.H. performed flow cytometry analysis. S.K. and K.T. implemented sugar chain analyses. I.I. initiated the work.

Corresponding authors

Correspondence to Yoshimi Kuroiwa or James M Robl.

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Competing interests

Work described in this paper was funded in part by Kyowa Hakko Kirin Co., Ltd. (Kyowa Kirin) and in part by a Small Business Innovation Research Phase II grant (no. 5R44A1054086-03) from the National Institute of Allergy and Infectious Diseases/National Institutes of Health to Hematech, Inc. During the project, Hematech was acquired by Kyowa Kirin and is now a wholly-owned subsidiary of Kyowa Kirin. As a result, all authors are currently either directly or indirectly employed by Kyowa Kirin.

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Kuroiwa, Y., Kasinathan, P., Sathiyaseelan, T. et al. Antigen-specific human polyclonal antibodies from hyperimmunized cattle. Nat Biotechnol 27, 173–181 (2009). https://doi.org/10.1038/nbt.1521

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