A biotechnology research company in Australia has developeda new, patented method to create cell lines that produce totally human monoclonal antibodies and other proteins of therapeuticand commercial interest, such as cytokines and cytokine receptors. Professor Hans Coster reports on a market which could be worth at least $5 billion.
The major advantage of this technology over competing technologies is that the resulting antibodies are naturally human, including the glycosylation (attachment of sugar moieties). This avoids some of the limitations encountered by alternative products, such as humanised mouse antibodies.
The market for human monoclonal antibodies alone is currently estimated to reach $ 5 billion and the demand for other proteins, such as cytokines and cytokine receptors, may be as great or exceed this expectation.
Overcoming the limitations
Historically, monoclonal antibodies, which are specific to one target antigen, have been harvested using mouse cell lines. However, murine antibodies are of limited use as human therapeutics because they can initiate an adverse immune reaction with repeated application. Throughout the world, different technologies have provided a variety of ahumanised' antibodies, but these products are also subject to limitations.
Reduced binding
These problems include reduced binding affinity to their target and the presence of non-human components that have the potential to create an unfavourable immune response in humans.
Moreover, they have abnormal glycosylation, which impairs their ability to provoke the desired immune response and leads to accelerated clearing from the system.
Antibodies can be used in therapy to enhance the normal complement and cell mediated destruction of pathogenic targets (or cancer cells); the humoral immune response, or by attaching a cytotoxic moiety to the antibody.
Cytotoxic moiety
The attachment of a cytotoxic moiety, which in some instances could be a radioactive nucleotide, can have undesirable side effects arising from the presence of such entities in the circulation (as binding of the antibodies to their targets will never be complete).
Clearly totally human antibodies would offer better prospects for therapeutic applications. The biotechnology research company FuCell has developed a method to produce human cell lines which secrete totally human antibodies and other proteins with normal human glycosylation.
The process uses radio frequency electric fields and special electrodes to fuse selected human lymphocyte cells to aimmortal' cancer cells. This creates new hybrid cell lines (hybridomas) that retain the characteristics necessary for cellular division and the mechanisms required for antibody production.
The method employed by FuCell avoids the labour-intensive task of isolating the relevant hybridomas from a heterogeneous mixture of hybrid cells, a process that is required with traditional techniques.
The FuCell technology generates products with fully human amino acid sequence and ensures the correct human glycosylation of the proteins.
A closer look at the technology
The process of creating a human hybridoma secreting antibodies against a specific antigen begins with a proprietary in vitro immunisation of a culture of human lymphocytes.
A considerable advantage of technique is the ability to focus on self-antigens, such as cancer, in addition to pathogenic organisms and viruses. The in vitro immunisation technique is possible with self antigens because in vitro immunised cultures are not subject to the same limitations of antigen modifications, haptens and adjuvants as in vivo immunisation.
The technology for performing selective cell manipulations utilises radio frequency electric fields and the dielectrophoretic (DEP) force response spectra for the cells.
Each cell has a characteristic DEP response spectra which enables the cells to be selectively manipulated. While the electrofusion of cells is by no means new, in the FuCell process this is done on a aone cell on one cell' rather than by the more traditional bulk fusion methods.
aTailored' electric fields
The single pair of cells, consisting of an antibody secreting B lymphocyte and a suitable aimmortal' partner cell line, are manipulated into position using atailored' electric fields, and fusion is electrically induced without global electroporation of the cells (Fig. 1).
The processes for these fusions (Fig. 2) are completely documented for each cell line produced and the entire operation is conducted in a controlled environment to ensure appropriate quality control to health authority standards.
This method has the edge over existing technology, as it enables the production of hybridomas with totally natural, human origin, including human amino acid sequence and glycosylation.
This is an important distinction from humanised mouse products, which contain murine amino acid sequences and lack normal human glycosylation, which adversely impact their effectiveness as therapeutics.
It is anticipated that FuCell's products will afford greater tolerance, a higher binding affinity and have increased residence time in the system.
Integrated antibodies
The fully human antibodies will be able to integrate with the normal immunological response, including complement-mediated and T cell-mediated responses. In addition, the antibodies can also be conjugated, if desired, to cytotoxic or radioactive moieties to effect destruction of their targets in the same way as has been proposed for use with some non-human antibodies.
Research has indicated very substantial potential markets for human monoclonal antibodies as prophylactics, therapeutics and in vivo diagnostics. At present, FuCell is focussing on the production of a human cell line which secretes antibodies against Hepatitis B. This research is supported by the Australian Government Industry Research and Development START Grants Scheme.
FuCell is currently producing fully human hybridomas as standard procedure, with approximately 2 per cent of fusions resulting in a stable human hybrid cell line.
Karyotype testing
This compares very favourably with mouse cell lines, which achieves only a fraction of this success rate. FuCell has performed karyotype testing which confirms that several of their cell lines have retained the requisite portions of the genome of both cell partners.
A library is being compiled containing stable hybrids. These cell lines are producing IgM, IgG or other proteins which are being characterised.
Professor Hans Coster is with FuCell Pty Ltd is based in Sydney, Australia. Fax: +61 2 9385 5981.
