Physiological Level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

Akiko Sano,Zhongde Wang,Eddie J. Sullivan,Hiroaki Matsushita,Jin-An Jiao,Poothappillai Kasinathan,Hua Wu,Yoshimi Kuroiwa,Jason Glenn Knott

doi:10.1371/journal.pone.0078119

Akiko Sano, Zhongde Wang + Show 7 more

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https://doi.org/10.1371/journal.pone.0078119

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Abstract

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

Highlights

Immunotherapy with human polyclonal antibodies or immunoglobulins prepared from the plasma of normal and convalescing human donors have been shown to be an effective treatment for several human diseases [1]
For deleting the nonimmunoglobulin genes on hChr14, intact hChr14 was transferred to chicken DT40 cells by using Micro-cell Mediated Chromosome Transfer (MMCT) for chromosomal modification as described previously [17]
To prepare the correctly shortened hChr14 as one of the building blocks for the construction of the cKSL-HACΔ, it was further modified by integrating a loxP sequence and a GFP reporter cassette at the RNR2 locus as described in Materials and Methods and previously [12]

Summary

Introduction

Immunotherapy with human polyclonal antibodies (hpAbs) or immunoglobulins (hIgs) prepared from the plasma of normal and convalescing human donors have been shown to be an effective treatment for several human diseases [1]. To address the medical demand for hpAbs, different alternatives have been explored to substitute or even replace human plasma-derived hpAbs. For example, IgG or Fabs (fragments of antigen binding), prepared from the plasma of immunized horses or sheep has been widely used to treat severe envenomation resulting from snake and spider bites [7,8]. An in vitro antibody production system based on recombinant DNA and mammalian cell culture technologies has been in the development by Symphogen A/S [11] This approach tries to mimic polyclonal nature of humoral immune response by producing mixtures of monoclonal antibodies (mAbs) that recognize multiple epitopes of an antigen. As pre-defined antigens are needed to identify the mAbs and a lengthy process is needed to engineer cell lines expressing the recombinant mAbs, this system may not be useful for treatment of diseases in which antigens are not well characterized, such as in autoimmunity, nor in dealing with sudden outbreaks of infectious diseases such as the 2002 SARS epidemic [5]

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