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

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).

本研究以在转染色体（transchromosomic, Tc）牛中制备完全人源多克隆抗体（human polyclonal antibodies, hpAbs）或人免疫球蛋白G（human immunoglobulin G, hIgGs）为目标。我们此前曾报道，当同时纯合敲除牛免疫球蛋白μ重链基因bIGHM与bIGHML1（即bIGHM−/−、bIGHML1−/−，双敲除（double knockouts, DKO））时，携带包含完整未重排人免疫球蛋白（immunoglobulin, Ig）重链（hIGH）、κ链（hIGK）与λ链（hIGL）生殖系基因座的人人工染色体（human artificial chromosome, HAC）的转染色体牛，可产生生理水平的hIgGs。然而，由于内源性牛免疫球蛋白轻链基因座仍保持完整，轻链既可由人人工染色体上的hIGK与hIGL基因组位点表达，也可由内源性牛κ链（bIGK）和λ链（bIGL）基因组位点表达，由此会同时产生两类抗体：完全人源hIgGs（免疫球蛋白重链与轻链均为人源：hIgG/hIgκ或hIgG/hIgλ），以及嵌合型hIgGs（免疫球蛋白重链为人源，而轻链为牛源：hIgG/bIgκ或hIgG/bIgλ）。为提升转染色体牛中完全人源hIgGs的产量，本研究利用Cre/loxP介导的位点特异性染色体重组技术，敲除了完整的牛IGL连接（joining, J）区与恒定（constant, C）基因簇（bIGLJ1-IGLC1至bIGLJ5-IGLC5），并成功制备了三重敲除（bIGHM−/−、bIGHML1−/−及bIGL−/−，TKO）转染色体牛。我们进一步证实，牛IGL基因簇的敲除可显著提升三重敲除转染色体牛血清中完全人源hIgGs的产量，其占比可达51.3%（hIgG/hIgκ与hIgG/hIgλ之和）。