Abstract
Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.
Highlights
Polyclonal antibodies have been used as a therapeutic agent for many years in treating a variety of diseases, for infectious diseases
In a series of our study, we hypothesized that there could be some interspecies-incompatibilities between human and bovine that may generally hamper high production of fully human IgG (hIgG) in the bovine environment
To construct KcHACΔ vector, part of the hIGHM constant region gene (CH1 through TM2 domains) was bovinized so that in such a chimeric IgM structure [which was named as cIgM (CH1) for chimeric human IgM bovinized at CH1 through TM2 domain], the pre-BCR/BCR could interact more effectively with bovine surrogate light chain, orthodox light chain and Igα/Ig-β molecules for better pre-BCR/BCR signaling (Fig 1)
Summary
Polyclonal antibodies (pAbs) have been used as a therapeutic agent for many years in treating a variety of diseases, for infectious diseases. Current IgG products, such as human intravenous immunoglobulin (IVIG), monoclonal antibodies, and animal derived polyclonal antibodies, have known limitations. Animal-derived pAbs could be an alternative to human polyclonal antibody products, they typically have relatively high toxicity since these animal-derived antibody products are highly immunogenic that can cause a variety of severe adverse effects such as allergic reactions. To avoid serious side effects, animal antibodies are usually processed into smaller F(ab) or F(ab’) fragments which significantly reduces their half-life and potency, and typically restricts their availability to a limited number of administrations. Disadvantage for monoclonal antibodies is that they are directed against a single epitope that may be subject to rapid mutational escape and the cost of manufacturing mAb products are very high
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