When you think of microbes what comes to mind? Moldy bread, Penicillin and antibiotics? Vaccines? Fermented food, like yogurt and kombucha? And the latest Probiotics health craze? How about immune therapy? Maybe not so much, but you should that the use of microbes is wide and ever growing. Now researchers are finding ways to use microbes to produce therapeutic products. You should know this because it may help you produce your favorite product.
In this World of Microbes series I have covered some of many facets of microbial use. Including antibiotics and vaccines, and the brave new world of gut flora. I have discussed how vaccines work by priming the immune system to combat an infection, and how gut microbes educate the immune system and help control inflammation. But this is by no means the end of microbe usefulness.
Today I will talk to you about immunotherapy and microbes.
The main type of Immunotherapy uses antibodies. Antibodies are complex proteins that allow the body to recognize non-self molecules (antigens) and to take steps to remove them. They can recognize and bind an almost an infinite number of antigens, and are extensively used as a diagnostic and laboratory tools.
In essence, antibodies acts as a key. As you can see in Figure 1 there are two ends to the antibody: 1) the variable region that is tailor made to bind a specific antigen and 2) the constant region that binds a receptor. Effector cells (a type of immune cell) have receptors that recognize the constant, or Fc region, of the antibody putting them in close proximity to the variable, or Fv region, attached to an antigen. This sets off a cascade of events from the activated effector cells inducing search and destroy of the antigen. Thus ridding the body of any non-self invaders.
Antibody therapy makes use of some or all of the antibody parts. The parts used depend on the therapeutic effect required and the ability to make the whole antibody or antibody fragment to a level of purity and homogeneity that is acceptable as a medicine. An inexhaustive summary of the different types of antibody fragments is given in Expression of Recombinant Antibodies. But, whatever parts you choose to use, you need to first make them.
Microbes for Immunotherapy
Antibodies are large and complex mammalian proteins. Therefore it’s tricky to produce them in prokaryotes. Another big issue with using prokaryotes to produce antibodies is the safety of microbial products for human use. The Food and Drug Administration (FDA) even publishes a special set of rules termed GRAS – Generally Recognized as Safe – which ensure the human safety of microbial products.
Currently the most common method of manufacturing antibodies is through Chinese Hamster Ovary (CHO) cell tissue culture. However, this is an expensive and time consuming manufacturing method, so efforts have been made to express them in microbes. According to Robinson et al. there are currently 151 immunotherapeutics based on antibodies, only two of which are Fab fragments (Figure 1) produced from prokaryotes, the rest being produced using mammalian cell expression systems. Though not yet greatly successful, let’s take a look at the microbes that DO work to make antibodies and antibody fragments.
Figure 1: Antibody structure
Prokaryotic Antibody Production
Prokaryotes have been mainly used to produce fragments of antibodies, the most popular being Fab and scFv fragments (Figure 1). In gram-negative bacteria, such as our ever popular friend E. coli, there are two compartments for protein expression – the cytoplasm and the periplasmic space which is the region between the two cell membranes (Figure 2). The first E. coli success saw secretion of Fv and Fab fragments into the periplasm where an oxidizing environment allowed the correct formation of disulphide bonds (Figure 1). Fragments can also be produced in the cytoplasm by engineering in helper proteins such as chaperones that enable correct protein folding and by varying the expression properties of the plasmids that code for the antibody fragment . Yield can also be dramatically increased by using reactor fermentation systems (where nutrients are continuously added and waste products removed) rather than the traditional closed system of a shaker flask.
Figure 2: Gram-postive and Gram-negative bacterial cell wall (Franciscosp2)
Full length antibodies can also be produced in bacteria [1-3] and this is even being done commercially in E. coli by the company, AbSci using proprietary technology. Published processes rely on the secretion of the antibodies through the inner membrane into the periplasm where folding occurs. This is a problem because the volume of the periplasm is small and limits yield and because compared with the cytoplasm, the periplasm lacks chaperones . Efforts to produce the antibodies in the cytoplasm have been unsuccessful until recently  when full length IgG antibodies were expressed in E. coli at levels similar to those in a CHO system. The need for the addition of carbohydrates to the Fc region, a feature necessary for antibody interaction with Fc receptors on effector cells was avoided (Figure 1) by mutating the glycosylated amino acid and demonstrating effector cell activation. However there were still a number of issues that needed to be addressed such as more efficient folding.
Other species of gram-negative bacteria have been tested but without the same success as E. coli. Gram-positive bacteria have also been successfully used. One advantage of gram-positive bacteria is that they do not produce endotoxin – a highly immunogenic lipopolysaccharide (LPS – see Figure 2) produced by gram-negative bacteria that can cause septic shock; this must be removed if the antibody product is to be considered as a therapeutic and achieve GRAS status. An scFv fragment against the dental caries bacteria Streptococcus mutans produced in the gram positive Lactobacilli showed efficacy in a rat model of dental caries .
Yeast Antibody Production
Lower eukaryotes have also been used to produce antibodies – again they are cheaper and less complex to operate than higher eukaryotic systems such as CHO cells. They have the advantage over prokaryotes of resembling the mammalian protein expression system more closely allowing expression and folding of complex proteins more easily. Additionally they don’t produce endotoxins like gram-negative bacteria do.
Of the yeasts, Pichia pastoris and Saccaromyces cerevisiae dominate the field in antibody production. S. cerevisiae has the advantage of being very well characterized with an abundance of molecular biology knowledge. For antibody fragment production, correct folding and low yields can be a problem. S. cerevisiae also secretes its own proteins along with any that the researcher is trying to over-produce which must be removed [5, 6].
Pichia pastoris on the other hand, does not secrete high levels of its own protein. It is also able to grow to high cell densities and is unique in its ability to grow using methanol as a sole carbon source. This unique characteristic is useful because the presence and absence of methanol tightly controls a promoter that can be used to efficiently and tightly turn on and off recombinant protein expression. However, methanol above certain levels can be toxic and methanol is difficult to use because of its volatility; alternative promoters are under investigation [5, 7].
Both Saccharomyces and Pichia have been used to produce antibody fragments: two from Pichia are in clinical trials  and one from S. cerevisae is commercially produced . One further advantage to Pichia is the ability to humanize the carbohydrate addition (glycosylation) system. Yeast and human glycosylation differ significantly but Pichia pastoris can be engineered to be more human in glycosylation patterns and this is receiving considerable attention .
In addition to yeasts, filamentous fungi such as Trichoderma and Aspergillus, which are currently widely used to produce proteins in the food and biotechnology industries, have also been used to produce antibody fragments and full length antibodies at high yields. However correct folding of the antibody (full length and fragments) remains an issue .
So, while still somewhat shaky in the full length department, both prokaryotic and eukaryotic microbes are successfully being investigated and used as cheaper sources of antibodies, a boon not only for laboratory diagnostics but also for human health and wellbeing.
1. Frenzel, A., M. Hust, and T. Schirrmann, Expression of Recombinant Antibodies. Frontiers in Immunology, 2013. 4: p. 217.
2. Robinson, M.-P., N. Ke, J. Lobstein, C. Peterson, A. Szkodny, T.J. Mansell, C. Tuckey, P.D. Riggs, P.A. Colussi, C.J. Noren, C.H. Taron, M.P. DeLisa, and M. Berkmen, Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria. Nat Commun, 2015. 6.
3. Sidhu, S.S., Full-length antibodies on display. Nat Biotech, 2007. 25(5): p. 537-538.
4. Kruger, C., Y. Hu, Q. Pan, H. Marcotte, A. Hultberg, D. Delwar, P.J. van Dalen, P.H. Pouwels, R.J. Leer, C.G. Kelly, C. van Dollenweerd, J.K. Ma, and L. Hammarstrom, In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotech, 2002. 20(7): p. 702-706.
5. Darby, R.A.J., S.P. Cartwright, M.V. Dilworth, and R.M. Bill, Which Yeast Species Shall I Choose? Saccharomyces cerevisiae Versus Pichia pastoris (Review), in Recombinant Protein Production in Yeast: Methods and Protocols, M.R. Bill, Editor. 2012, Humana Press: Totowa, NJ. p. 11-23.
6. Spadiut, O., S. Capone, F. Krainer, A. Glieder, and C. Herwig, Microbials for the production of monoclonal antibodies and antibody fragments. Trends in Biotechnology, 2014. 32(1): p. 54-60.
7. Delic, M., D. Mattanovich, and B. Gasser, Repressible promoters – A novel tool to generate conditional mutants in Pichia pastoris. Microbial Cell Factories, 2013. 12(1): p. 1-6.
8. Gasser, B. and D. Mattanovich, Antibody production with yeasts and filamentous fungi: on the road to large scale? Biotechnol Lett, 2007. 29(2): p. 201-12.