Template:FAU Erlangen/abstract

iGEM Erlangen 2019

B.A.A.C.C.
Bispecific Antibody Against Colorectal Cancer

Abstract

Colorectal cancer is the second-largest cause of cancer-related death. While the primary tumor is relatively easy to remove surgically, metastases present dangerous complications. The major hurdles in treating the secondary tumors are locating them, eliminating only the aberrant cells, and reducing the negative treatment side-effects (Kuipers et al. 2015; Steeg 2016). Thus our iGEM Team at FAU Erlangen will create a bispecific antibody as a targeting marker, enabling T-lymphocytes to target colorectal cancer cells. Our protein is designed as a Bispecific T-cell Engager (BiTE) as novel pathway for T-lymphocytes, a component of the body’s immune system to actively attack cancer cells. The challenge being to test several different approaches for producing bispecific antibodies in E. coli regarding the production efficacy, the ease of the procedure, and the stability and binding affinity of the product. Using the tools provided by synthetic biology, we hereby strive to gain new insights that will prove beneficial in streamlining the production process of bispecific antibodies and reducing production costs.

Since the application of bispecific antibodies is a relatively new approach, current protocols for production are not yet standardized. In most cases mammalian cells are used for the expression of these proteins. However, this expression system is associated with very high costs and proves to be very time-consuming. The use of the bacterial host Escherichia coli poses an alternative for the expression of recombinant protein. It is well published and provides many advantages over the expression in mammalian cells, such as a far cheaper cultivation due to the higher proliferation rate and faster protein production. Despite these benefits, producing proteins of mammalian origin in E. coli has been shown to be difficult since post-translational modifications are often vital to protein folding and function and cannot be replicated in these bacterial systems. Especially for complex proteins like antibodies, misfolding is often detrimental for their solubility and performance (Spadiut et al. 2014; Liu und Huang 2018; Lee und Jeong 2015).
Devising a reliable and well applicable procedure to successfully express a functional bispecific antibody in bacterial cells is crucial in fully accessing their therapeutic potential by facilitating cost- and time-effective research. However, published protocols vary immensely and offer no conclusive approach to producing recombinant, bispecific antibodies in bacteria.

For our experiments we chose to create a bispecific antibody against CD3 on T-lymphocytes and GPA33. GPA33 can be found on 95% of all colorectal cancer cells, thereby allowing them to be specifically targeted (Heath et al. 1997). By also binding CD3, the bispecific antibody can bring cytotoxic T-lymphocytes (CTLs) into direct contact with the cancer cells, which activates the CTLs to release their cytotoxic granules and kill the malignant cell (Osada et al. 2010; Gruber et al. 1994). Our protein is designed as a Bispecific T-cell Engager (BiTE) and consists of two distinct single-chain variable Fragments (scFv) that are connected via a flexible linker (Sedykh et al. 2018).
We are going to use a pET27b plasmid with a T7 promoter and lacZ control, as well as a pACYC184 plasmid with a ptac promoter in the E. coli strains BL21, BL21 Star and Tuner. By using a variety of expression systems, we can determine the most efficient one for our purposes. It is well published that periplasmic expression of recombinant protein offers an oxidizing environment that is beneficial for correct protein folding, as well as a reduced number of proteases that may degrade our protein (Liu und Huang 2018; Thie et al. 2008). For this reason, we plan to use a PelB leader sequence to transport our constructs to the periplasm of E. coli for synthesis.

In summary, we pursue three approaches:

In our first approach, we plan on expressing a complete BiTE molecule that will serve as reference for our other production strategies.

In our second approach, we express the two scFvs separately as fusion proteins with SpyTag or SpyCatcher, respectively. This system relies on a split domain of the FbaB protein from Streptococcus pyogenes to form a covalent, isopeptide bond upon co-culturing. This allows us to avoid the possible problem of producing a large, complex protein that could result in the formation of insoluble aggregates. Furthermore, the SpyTag/SpyCatcher system is fashioned after a modular concept. If this approach proves to be successful, it will be possible to express several effector subunits with varying affinities or functions, i.e. different antigen specificities or enzymatic activity, and connect them. This will allow for a flexible modular system that might be adapted with regards to current requirements in a fast and easy manner.

In our last approach, we intend to produce CD3- and GPA33-specific antibody fragments, so called Fabs, and link them together via disulfide bonds. This chemical conjugation is achieved by using a bis-dibromomaleimide cross-linker, providing a versatile method to produce bispecific antibodies (Hull et al. 2014).

To test the binding activity of our products, we will perform killing assays with blood derived CTLs. We will further test their stability at different temperatures and pH conditions. Altogether, the assays will provide a comparative evaluation of the properties of our different products and permit the assessment of their respective value for clinical or scientific application.

By trainnig a neural network, it will also be possible to predict potential allergenicity of our bispecific antibodies. This will also prove beneficial as a prognostic tool to produce other recombinant proteins and their possible clinical application.
Furthermore, by modelling the different types of linkers with MD-Simulation, we want to predict the stability of the linkers used in the wet lab.

We are a team that is tackling problems of the emerging field of molecular immunology - no team in Erlangen has done this before. Here in Erlangen, we have an optimal professional network, since the newly established Masters-Program has been opened to sustain the ongoing research in this field, from which we can benefit greatly. Furthermore, our Team has an interdisciplinary approach as we have sub-teams working on chemical ligation of Fab-Fragments and a deep-learning approach to predicting immune reactions concerning different allergens. Our interdisciplinary approach is comprehensive as it covers the cure as well as the side effects by predicting these with our neural network software.

This project is the result of a kickstart weekend in January 2018 with almost all iGEM members of the current team. During this weekend we developed a bunch of promising project ideas. The idea of working with Bispecific Antibodies Against Colorectal Cancer suggested by the students of the program Integrated Immunology excited all members of the team, since it gives the possibility to integrate the knowledge of the students of the fields of informatics, chemistry and computational biology.

References

  • Gruber, M.; Schodin, B. A.; Wilson, E. R.; Kranz, D. M. (1994): Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli. In: Journal of immunology (Baltimore, Md. : 1950) 152 (11), S. 5368–5374.
  • Heath, J. K.; White, S. J.; Johnstone, C. N.; Catimel, B.; Simpson, R. J.; Moritz, R. L. et al. (1997): The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily. In: Proceedings of the National Academy of Sciences of the United States of America 94 (2), S. 469–474. DOI: 10.1073/pnas.94.2.469.
  • Hull, Elizabeth A.; Livanos, Maria; Miranda, Enrique; Smith, Mark E. B.; Chester, Kerry A.; Baker, James R. (2014): Homogeneous bispecifics by disulfide bridging. In: Bioconjugate chemistry 25 (8), S. 1395–1401. DOI: 10.1021/bc5002467.
  • Kuipers, Ernst J.; Grady, William M.; Lieberman, David; Seufferlein, Thomas; Sung, Joseph J.; Boelens, Petra G. et al. (2015): Colorectal cancer. In: Nature reviews. Disease primers 1, S. 15065. DOI: 10.1038/nrdp.2015.65.
  • Lee, Yong Jae; Jeong, Ki Jun (2015): Challenges to production of antibodies in bacteria and yeast. In: Journal of bioscience and bioengineering 120 (5), S. 483–490. DOI: 10.1016/j.jbiosc.2015.03.009.
  • Liu, Yongkang; Huang, He (2018): Expression of single-domain antibody in different systems. In: Applied microbiology and biotechnology 102 (2), S. 539–551. DOI: 10.1007/s00253-017-8644-3.
  • Osada, T.; Hsu, D.; Hammond, S.; Hobeika, A.; Devi, G.; Clay, T. M. et al. (2010): Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T-cell killing mediated by CEA/CD3-bispecific T-cell-engaging BiTE antibody. In: British journal of cancer 102 (1), S. 124–133. DOI: 10.1038/sj.bjc.6605364.
  • Sedykh, Sergey E.; Prinz, Victor V.; Buneva, Valentina N.; Nevinsky, Georgy A. (2018): Bispecific antibodies: design, therapy, perspectives. In: Drug design, development and therapy 12, S. 195–208. DOI: 10.2147/DDDT.S151282.
  • Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph (2014): Microbials for the production of monoclonal antibodies and antibody fragments. In: Trends in biotechnology 32 (1), S. 54–60. DOI: 10.1016/j.tibtech.2013.10.002.
  • Steeg, Patricia S. (2016): Targeting metastasis. In: Nature reviews. Cancer 16 (4), S. 201–218. DOI: 10.1038/nrc.2016.25.
  • Thie, Holger; Schirrmann, Thomas; Paschke, Matthias; Dübel, Stefan; Hust, Michael (2008): SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. In: New biotechnology 25 (1), S. 49–54. DOI: 10.1016/j.nbt.2008.01.001.