Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

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

doi:10.1371/journal.pone.0090383

Hiroaki Matsushita, Yoshimi Kuroiwa + Show 7 more

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https://doi.org/10.1371/journal.pone.0090383

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Abstract

Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/−, bIGHML1−/−; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM−/−, bIGHML1−/− and bIGL−/−; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

Highlights

Human polyclonal antibodies prepared from plasma donated from the general population or convalescing human donors have been widely used to treat several human diseases, such as autoimmunity, immunodeficiency and infection [1]
These Tc cattle were engineered by homozygous knockout of both of the bovine immunoglobulin mu heavy-chain genes, bIGHM and bIGHML1 and by reconstituting B cell function with a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain, kappa-chain, and lambda-chain germline loci
As the bovine endogenous immunoglobulin lightchain loci, the lambda locus and the kappa locus, are intact in these Tc cattle, the light-chains are expressed both from hIGK and hIGL loci on the HAC and from the endogenous bIGL and bIGK loci, resulting in a mixture of hIgGs composing fully hIgGs and chimeric hIgGs

Summary

Introduction

Human polyclonal antibodies (hpAbs or hIgGs) prepared from plasma donated from the general population or convalescing human donors have been widely used to treat several human diseases, such as autoimmunity, immunodeficiency and infection [1]. We previously reported our success in creating double knockout (DKO) transchromosomic (Tc) cattle that produce physiological levels of hIgGs (Sano A et al, 2013). These Tc cattle were engineered by homozygous knockout of both of the bovine immunoglobulin mu heavy-chain genes, bIGHM and bIGHML1 (bIGHM2/2, bIGHML12/2; DKO) and by reconstituting B cell function with a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci. As bIgl is the predominantly expressed light-chain isotype in bovine B cells, we further reasoned that inactivation of bovine lambda light-chain genes, in addition to the KO of the two bIGHM and bIGHML1 loci, would dramatically improve fully hIgG production in the resulting Tc cattle

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