Manufacture of monoclonal antibodies
(mAb) and related biologics is performed
in eukaryotic cell lines to allow proper
glycosylation, which is often critical for
function. The first mAbs were produced
in the mouse-derived cell lines SP2/0 and
NS0, which have the genetic makeup to
produce proteins containing nonhuman
glycan structures, such as galactose-α-1,3-
galactose (α-Gal) and N-glycolylneuramic
acid1, that can adversely affect safety and
half-life. The best example is Erbitux
(cetuximab), produced in SP2/0 cells,
clinical use of which has been associated
with α-Gal–specific, IgE-mediated
anaphylactic responses2. In addition,
patients with IgE antibodies to α-Gal may
experience allergic reactions to red meat3,4.
The quantity of α-Gal required to induce
hypersensitivity reactions is not known;
however, it is clear that binding of IgE
antibodies against α-Gal is an important
trigger in this pathology.
Chinese hamster ovary (CHO) cells are widely used for the manufacture of biotherapeutics, in part because of their ability to produce proteins with desirable properties, including 'human-like' glycosylation profiles. For biotherapeutics production, control of glycosylation is critical because it has a profound effect on protein function, including half-life and efficacy. Additionally, specific glycan structures may adversely affect their safety profile. For example, the terminal galactose-α-1,3-galactose (α-Gal) antigen can react with circulating anti α-Gal antibodies present in most individuals. It is now understood that murine cell lines, such as SP2 or NSO, typical manufacturing cell lines for biotherapeutics, contain the necessary biosynthetic machinery to produce proteins containing α-Gal epitopes. Furthermore, the majority of adverse clinical events associated with an induced IgE-mediated anaphylaxis response in patients treated with the commercial antibody Erbitux (cetuximab) manufactured in a murine myeloma cell line have been attributed to the presence of the α-Gal moiety. Even so, it is generally accepted that CHO cells lack the biosynthetic machinery to synthesize glycoproteins with α-Gal antigens. Contrary to this assumption, we report here the identification of the CHO ortholog of N-acetyllactosaminide 3-α-galactosyltransferase-1, which is responsible for the synthesis of the α-Gal epitope. We find that the enzyme product of this CHO gene is active and that glycosylated protein products produced in CHO contain the signature α-Gal antigen because of the action of this enzyme. Furthermore, characterizing the commercial therapeutic protein abatacept (Orencia) manufactured in CHO cell lines, we also identified the presence of α-Gal. Finally, we find that the presence of the α-Gal epitope likely arises during clonal selection because different subclonal populations from the same parental cell line differ in their expression of this gene. Although the specific levels of α-Gal required to trigger anaphylaxis reactions are not known and are likely product specific, the fact that humans contain high levels of circulating anti-α-Gal antibodies suggests that minimizing (or at least controlling) the levels of these epitopes during biotherapeutics development may be beneficial to patients. Furthermore, the approaches described here to monitor α-Gal levels may prove useful in industry for the surveillance and control of α-Gal levels during protein manufacture.
Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.
Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.
Transgenic tobacco plants expressing a cyanobacterial fructose-1,6/sedoheptulose-1,7-bisphosphatase targeted to chloroplasts show enhanced photosynthetic efficiency and growth characteristics under atmospheric conditions (360 p.p.m. CO2). Compared with wild-type tobacco, final dry matter and photosynthetic CO2 fixation of the transgenic plants were 1.5-fold and 1.24-fold higher, respectively. Transgenic tobacco also showed a 1.2-fold increase in initial activity of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) compared with wild-type plants. Levels of intermediates in the Calvin cycle and the accumulation of carbohydrates were also higher than those in wild-type plants. This is the first report in which expression of a single plastid-targeted enzyme has been shown to improve carbon fixation and growth in transgenic plants.
Accurate simulation of intracellular biochemical networks is essential to furthering our understanding of biological system behavior. The number of protein complexes and of chemical interactions among them has traditionally posed significant problems for simulation algorithms. Here we describe an approach to the exact stochastic simulation of biochemical networks that emphasizes the contribution of protein complexes to these systems. This simulation approach starts from a description of monomeric proteins and specifications for binding, unbinding and other reactions. This manageable specification is reasonably intuitive for biologists. Rather than requiring the inclusion of all possible complexes and reactions from the outset, our approach incorporates new complexes and reactions only when needed as the simulation proceeds. As a result, the simulation generates much smaller reaction networks, which can be exported to other simulators for further analysis. We apply this approach to the automatic generation of reaction systems for the study of signal transduction networks.
A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.