BIOTECHNOLOGY Human hybridoma technology for the production of monoclonal antibodies A new method of creating cell lines has been developed for the production of "totally human" monoclonal antibodies. Professor Hans Coster and Dr David Monaghan, FuCell Pty Ltd n 1975, Köhler and Milstein succeeded in tissue of immunised subjects. After many years of generating mouse monoclonal antibodies unsuccessful research, the prospect of a quick I(MAbs). Monoclonal antibodies were hailed as solution from the new technology of genetic a powerful new way to treat viral infections or engineering led to the abandonment of the human cancer. However, the hybridoma process, so hybridoma approach. The new strategy was to successful in mouse cells and of such immense genetically engineer the mouse MAbs so that they value, could not be readily replicated using mimicked their human counterparts. However, in human cells. the intervening 15 years or so, molecular biologists Efforts to replicate the mouse hybridoma results have directed their energy - and literally billions of using human cells were frustrated by a lack of dollars of public and private funding - into suitable partner cells which had the requisite genetically engineering mouse MAbs, only to come metabolic deficiencies (for example, HAT sensitivity), up with a handful of humanised MAbs that have as well as ready access to the spleen reached the market. 76 Innovations in Pharmaceutical Technology BIOTECHNOLOGY Now a new, patented method of creating cell lines has been developed that could change the face of antibody production by providing totally human antibodies and other proteins with natural human glycosylation. This platform technology has been developed by the Australian company, FuCell Pty Ltd. Monoclonal antibodies One type of cell in the immune system of mammals, the B lymphocyte cell, functions by producing and secreting a molecule known as an antibody. These antibody molecules act by binding to specific molecular features (called “antigens”) which occur on the surfaces of invading virus particles and bacteria, as well as cancer cells. This initiates the killing of the invading pathogen, or it signals other cells of the mammal’s immune system to bind and kill that invading pathogen or cancer cell. Upon recognition of the antigen on the invading pathogen, the B lymphocyte cell - which has the right antibody - proliferates to make millions of copies of itself, and these cells then secrete large quantities of antibody of the right specificity to effect neutralisation of the invading pathogen. Unfortunately, B lymphocytes cannot be grown on a large scale in artificial culture in bioreactors to allow the manufacture of antibodies for therapeutic use; this is because the cells will only divide a finite number of times before the culture dies. Antibodies can, however, be manufactured in culture from cells which are hybrid cells produced by the fusion of a suitable B lymphocyte cell (secreting the right antibody) with an immortal cancer (for example, myeloma) cell. Such hybrid cells both produce the desired antibody and are immortal; they are known as hybridomas. The antibody molecules that are secreted by such cells are known as monoclonal antibodies. Monoclonal antibodies are antibodies produced by one cell line which are specific to one target (antigen); historically, they have been produced using mouse cell lines. Antibodies derived from mouse hybridomas are of limited use as human therapeutics, since they produce an adverse immune reaction with repeated use. There are currently two approaches used in the development of antibodies for therapeutic use in humans. The first of these involves the “humanisation” of mouse antibodies, and the second is based on the production of human antibodies in a transgenic mouse. The first method is a recombinant DNA approach, as exemplified by the products under development by Protein Design Labs (PDL). With this method, the xenogeneic portions of the mouse MAb are replaced by human immunoglobulin Innovations in Pharmaceutical Technology 77 BIOTECHNOLOGY Figure 1. A series of micrographs showing the fusion process between a human cancer cell and a human B lymphocyte. The two cells are brought together using electric fields which are then ‘pulsed’ causing the two cells to fuse into one hybrid cell. structures in an effort to construct humanised moieties) - this impairs their ability to monoclonal antibody molecules. The latest provoke the desired immune response and advances - although technically impressive - also leads to more rapid clearance from ... molecular remain imperfect with substantial refinements still the system, biologists have required in order for the engineered products to achieve the desired level of efficacy, without • non-human components which have the directed their adverse side effects or limitations in vivo. potential to create an unfavourable immune energy ... into The second method involves the use of response in humans, and genetically “transgenic” mice which contain segments of the human genome coding for antibodies. The •a low yield for these various methods engineering antibodies, as exemplified by the products of of production. mouse MAbs, Medarex and Abgenix, are chimeric antibodies - The FuCell technology only to come up that is, they are constructs that combine DNA segments from non-mating species, usually from At FuCell, we have discovered an entirely new with a handful mouse and human genomes. method of generating hybrid cells (hybridomas), of humanised In addition to these two methods, there are also that overcomes all the problems limiting the “plantibodies” produced by plants transfected with MAbs that have current methods of making such hybridoma cells complementing human immunoglobulin genes to for the production of monoclonal antibodies for reached the produce Mendelian offspring which manufacture human therapeutic purposes. market antibodies in the bulk tissue. Fragments of human The new technology: antibodies have also been produced - such as domain antibodies (DAbs) and single-chain •Pre-selects only those cells (B lymphocytes) variable fragments (scFvs) - where just the binding- which are expressing the desired antibody, site protein is generated. Many companies have developed humanised •Simultaneously brings those cells into antibodies using a variety of different technologies. individual contact with individual cells of These humanised products are subject to the chosen immortal cancer (myeloma) cells, limitations, including: •a reduced binding affinity to their target, •Fuses the pairs of these two selected cells - and only these - to produce the desired • abnormal glycosylation (attachment of sugar immortal hybrid cell (hybridoma), and 78 Innovations in Pharmaceutical Technology BIOTECHNOLOGY Figure 2. An overview of the FuCell fusion apparatus. A single user can operate the apparatus. All the necessary fields are pre-programmed and the micro-manipulators used for cell transfer, as well as the generation of the electric fields, are under computer control. • Allows the use of almost any immortal find the desired hybridoma. All that is required is fusion partner cell; the method does not to "grow up" the hybridomas produced in culture; require post-fusion selection, as the only this greatly reduces the cycle of time involved in the cells remaining are hybridomas expressing process of making hybridomas. antibodies to the target antigen. The FuCell product is described as “totally human” to emphasise its natural human origin; This procedure obviates the need to screen and both the amino acid sequence and the separate (“clone”) large numbers of fused cells to glycosylation is human. This is distinct from many 80 Innovations in Pharmaceutical Technology BIOTECHNOLOGY Figure 3. A close-up of the FuCell fusion apparatus. On the left is the femto-pipette used for transferring single cells, and on the right is a pair of micro-electrodes used for applying the electric fields used during the fusion process. mouse-derived products, which are sometimes (cytoplasm) that is highly conductive, due to the described as “fully human” but which, nonetheless, accumulation of ions (such as K+), and a relatively retain a significant percentage of mouse high dielectric constant; the surrounding The FuCell components in their amino acid sequences and, in membrane has a very low conductivity and a lower all cases, lack normal human glycosylation. The dielectric constant. The degree of distortion of the product is FuCell products, therefore, will potentially be field, both inside and outside of the cell, is a very described as better tolerated, less rapidly cleared from the strong function of the frequency of the applied “totally system and have high affinity binding. As a electric field. As a result, when placed in a therapeutic, they will have the ability to utilise the non-uniform electric field, cells will experience a human” to normal immunological pathways of complement- force whose magnitude and direction will vary - in emphasise its and cell-mediated destruction of the targets. If a complicated manner - with the frequency of the desired, the antibodies can also be conjugated to applied field. This effect can be exploited to natural human cytotoxic or radioactive moieties to effect destruction selectively manipulate living cells using AC electric origin; both the of their targets - as has also been proposed for use fields created via suitable micro-electrodes. The amino acid with some non-human antibodies. movement