335 Genetic Engineering of Goats for the Production of a Biosimilar Antibody in Milk

Abstract

Dairy animals provide an attractive production platform for biosimilar antibodies due to the high protein production capacity of the mammary gland and easy access to milk. Goats are well suited for this approach as they offer a relatively short gestation time and good milk yield and are fully validated for the production of recombinant therapeutics. To generate transgenic goats capable of producing a biosimilar version of cetuximab, a monoclonal antibody for epidermal growth factor receptor and approved for the treatment of specific cancers, we co-transfected primary female fetal fibroblasts with expression constructs for cetuximab’s heavy (HC) and light (LC) chains under the control of the goat β-casein regulatory sequences. Beta-globin insulators were added to both transgenes to minimize position effects, and an antibiotic selection marker was placed downstream of the HC transgene sequences to allow for the isolation of stable transgenic cell clones. Selected cell clones were screened by PCR for the presence of both transgenes. Positive cell clones were analysed by Southern blot with a β-casein-specific probe. This allowed for the simultaneous detection of both transgenes, and the endogenous β-casein gene served as a standard to determine transgene copy numbers. The cell clones showed a broad range of copy numbers, from single copy insertions to >100 copies for the HC and LC transgenes. Interestingly, most of the cell clones had more LC than HC transgene copies. Ten cell clones were selected to generate transgenic founders using somatic cell nuclear transfer. We were able to produce 43 live kids from 9 cell lines following transfer of between 26 and 153 one- and two-cell embryos per line into recipients (range of 4 to 15 embryos per recipient). The one cell clone that we used unsuccessfully had the lowest number of transferred embryos (11). The efficiency for the production of live kids per transferred embryos was, on average, 5.1% (range of 1.0 to 9.7%). Kids from 5 lines were hormonally induced into lactation at the age of 10 weeks. Two lines with high copy numbers (≥30) produced either no or only a few drops of milk, whereas the lines with ≤25 transgene copies gave up to several milliliters of milk per day. Western analyses confirmed cetuximab production levels of 15 g L–1 in 2 of the lines with ≤25 transgene copies and ~45 g L–1 in a high copy number line; one low copy number line showed good HC but very low LC expression. Our data demonstrate that cetuximab can be produced in significant quantities in transgenic goats. Future work is aimed at determining production levels under natural lactation conditions and characterising glycosylation patterns to fully understand the pharmacodynamic properties of the antibody. Supported by GTC, the NZ Ministry of Science and Innovation and AgResearch.

Cetuximab produced from a goat mammary gland expression system is equally efficacious as innovator cetuximab in model systems of cancer

“…no a-gal linkages) as is seen with the innovator and mammalian cell culture derived cetuximab product. Additionally, we have previously reported on the potential for increased efficacy from the transgenic goat milk derived cetuximab based on its increased antibody dependant cellular toxicity (ADCC) profile (10). We now report that, compared to the commercially available cetuximab, the goat derived cetuximab is equally suited as a antibody for attaching a toxin to create an ADC form of cetuximab.…”

“…High costs of monoclonal anti-cancer antibody therapies restricts their overall routine use in the clinic. We have previously described a transgenic animal production system for the monoclonal antibody cetuximab in which high levels of the therapeutic proteins are expressed in the mammary gland of lactating goats (10). In this study, we investigated antitumour effects of goat-produced cetuximab by a range of biological assays both in vitro and in vivo.…”

“…Cetuximab was expressed in the milk of lactating goats and purified as previously described (10). Commercially sourced cetuximab was obtained from Onelink (NZ) Ltd.…”

“…Clinical grade cetuximab (Erbitux), is produced by expressing it in expensive mammalian cell bioreactors, driving interest in alternative production systems. We have recently reported the development of a system for expressing cetuximab in the mammary gland of lactating transgenic goats and purifying this version of cetuximab (gCetuximab) from the resulting milk (10). Our earlier report described the gCetuximab and provided evidence that it may be a "Biobetter"as; (a) it has unique characteristics and benefits that increase its safety profile (i.e.…”

“…Our earlier report described the gCetuximab and provided evidence that it may be a "Biobetter"as; (a) it has unique characteristics and benefits that increase its safety profile (i.e. no a-gal linkage or chance for immunogenicity), and (b) it has an increased antibody dependent cellcular cytotoxity (ADCC) profile (10). This report describes a range of studies that demonstrate that the biological properties of the cetuximab produced in the transgenic milk system are equivalent to those of the commercial cell culture product.…”