Transgenic goats producing an improved version of cetuximab in milk

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

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Transgenic goats producing an improved version of cetuximab in milk

10.1101/2020.06.05.137463 ◽

2020 ◽

Author(s):

Götz Laible ◽

Sally Cole ◽

Brigid Brophy ◽

Paul Maclean ◽

Li How Chen ◽

...

Keyword(s):

Mammalian Cells ◽

Large Scale ◽

Growth Factor Receptor ◽

Cost Effective ◽

Scale Production ◽

Cancer Treatments ◽

Large Scale Production ◽

Immunogenic Epitope ◽

Dependent Cell ◽

Transgenic Goats

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

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Production of human lactoferrin in animal milk1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o11-088 ◽

2012 ◽

Vol 90 (3) ◽

pp. 513-519 ◽

Cited By ~ 15

Author(s):

I.L. Goldman ◽

S.G. Georgieva ◽

Ya.G. Gurskiy ◽

A.N. Krasnov ◽

A.V. Deykin ◽

...

Keyword(s):

Peer Review Process ◽

Micrococcus Luteus ◽

Chemical Properties ◽

Human Lactoferrin ◽

Natural Ligands ◽

Terminal Amino ◽

Transgenic Goats ◽

Genetic Constructs ◽

Physical And Chemical ◽

Circular Dichroic

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.

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Health and Reproductive Profiles of Malaria Antigen-Producing Transgenic Goats Derived by Somatic Cell Nuclear Transfer

Cloning and Stem Cells ◽

10.1089/clo.2005.7.107 ◽

2005 ◽

Vol 7 (2) ◽

pp. 107-118 ◽

Cited By ~ 30

Author(s):

E. Behboodi ◽

S.L. Ayres ◽

E. Memili ◽

M. O'coin ◽

L.H. Chen ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Malaria Antigen ◽

Transgenic Goats

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The Effects of GH Transgenic Goats on the Microflora of the Intestine, Feces and Surrounding Soil

PLoS ONE ◽

10.1371/journal.pone.0139822 ◽

2015 ◽

Vol 10 (10) ◽

pp. e0139822

Author(s):

Zekun Bao ◽

Xue Gao ◽

Qiang Zhang ◽

Jian Lin ◽

Weiwei Hu ◽

...

Keyword(s):

Transgenic Goats ◽

Surrounding Soil

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335 GENETIC ENGINEERING OF GOATS FOR THE PRODUCTION OF A BIOSIMILAR ANTIBODY IN MILK

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab335 ◽

2013 ◽

Vol 25 (1) ◽

pp. 315

Author(s):

G. Laible ◽

S.-A. E. Cole ◽

B. K. Brophy ◽

M. J. Wright ◽

M. C. Berg ◽

...

Keyword(s):

Copy Number ◽

Simultaneous Detection ◽

Number Line ◽

Easy Access ◽

Regulatory Sequences ◽

Position Effects ◽

Copy Numbers ◽

Cell Clones ◽

Fetal Fibroblasts ◽

Transgenic Goats

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.

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