Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells
Fang et al. Biological Procedures Online
Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells
Xiaotian T. Fang 1
Dag Sehlin 1
Lars Lannfelt 1
Stina Syvänen 1
Greta Hultqvist 0 1
0 Department of Pharmaceutical biosciences, Uppsala University, Biomedical center , Husargatan 3, Box 591, 75124 Uppsala , Sweden
1 Department of Public Health and Caring Sciences, Uppsala University, Rudbeck laboratory , Dag Hammarskjöldsväg 20, 751 85 Uppsala , Sweden
Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels. Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). Results: The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo. Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.
Bispecific antibodies; Expi293; HEK293; PEI; Transient protein expression
Background
Immunotherapy is today a very fast advancing fields
within drug discovery. Efforts are being made to develop
antibodies that can be used to treat a large number of
different diseases. With the recent advances in
multifunctional antibody design the possibilities have increased
even further. For efficient testing of potential drug
candidates, it is essential to have a fast and inexpensive
system to express many different antibodies. However,
antibodies are difficult to expresses due to their large size.
Furthermore, the two heavy and two light chains need
to be paired correctly. Multispecific antibodies with
additional binding domains, e.g. tetravalent bispecific
antibodies (Fig. 1), further complicate the expression.
For many applications, post-translational modifications
are essential and hence an expression system as similar
to the human body as possible is desired. Traditionally,
stable cell lines expressing antibodies were a necessity to
generate sufficient amounts of the antibody, even for
validation experiments. However, there has been a lot of
progress in the last decade in transient expression of
recombinant proteins. For instance, the robust and easily
transfected HEK293 cell line has been adapted to growth
in suspension [
1
]. Furthermore, it has been discovered
that addition of cell cycle inhibiting substances after the
transfection step will increase the protein yield [
2, 3
].
Promoters such as CMW and SV40 and other regulatory
elements have been improved [4]. Alternative
transfection techniques have been discovered and upgraded. It
was shown that polyethylenimine (PEI) can be used to
transfect HEK293 cells [
5
]. Most of the protocols for the
production of recombinant proteins at high yields are
based on the HEK293EBNA1-6E cell line that expresses
a truncated version of the Epstein–Barr nuclear antigen
1 (EBNA1), which increases protein expression [
6, 7
].
However, this cell line is not commercially available
anymore and patents restrict its commercial use.
There are only a few protocols describing the
purification of tetravalent bispecific antibodies and these
methods have only achieved limited expression levels
[
8–10
]. Here, we describe an expression protocol that
uses only commercially available materials and in a
10day process can produce functional tetravalent, bispecific
antibodies at 22 mg protein per liter culture to a low
cost. See Fig. 2 for a schematic overview.
The expression system is based on the Expi293 cells
(LifeTechnologies), that have been adapted to grow in
denser cultures than normal HEK293 cells (the adapted
cells are healthy at 5 million cells (...truncated)