PhD student profiles

     
     
Felix Schumacher

Felix Schumacher
Felix studied for his PhD on the ISMB's Wellcome Trust 4-year Interdisciplinary PhD Programme, beginning in Autumn 2008.

Project title
Chemical modification of proteins

Principal investigator: Dr James Baker, Department of Chemistry, UCL
Co-investigators: Professor Stephen Caddick, Department of Chemistry, UCL

   
Background
My name is Felix Schumacher and I am a member of the first intake of students who have done their PhD in the 4-year interdisciplinary Wellcome Trust programme at UCL. I grew up and went to school in Munich in southern Germany. After a year of (at that time mandatory) army service I did my undergraduate studies in biochemistry at the Technische Universität Munich in the group of Prof Johannes Buchner with a focus on protein folding. Following that, I obtained a Master’s Degree (also in biochemistry) at the University Hospital “Rechts der Isar” under the supervison of PD Dr Gisela Keller working on drug resistance and epigenetic markers of cancer cells. With this somewhat diverse background the idea of a PhD program based on interdisciplinary appealed to me from the very beginning. 
 
Rotations
As for most of my fellow PhD students I started the program with a rotation in the field where I had acquired expertise during my undergraduate studies – structural biology. In the lab of Prof Steve Perkins I expressed and purified various fragments of factor H, a regulator of the complement cascade, and tested the influence of zinc on the oligomerisation of this protein by ultracentrifugation and X-ray scatter experiments. Linked to this project my next rotation was under the supervision of Prof David Jones. Here we used bioinformatics tools such as protein docking programs and algorithms for the prediction of metal binding sites to evaluate the potential of zinc induced dimerisation of factor H. Finally I joined the group of Dr James Baker where I learned the basics of organic chemistry and synthesis and also carried out the first successful bioconjugation of the small peptide hormone somatostatin with disubstituted maleimides. Intrigued by the potential of the project, the friendly atmosphere in the lab and the chance to learn a new set of scientific skills I decided to stay with Dr Baker for the next three years of the program.
 

PhD Project
The research I carried out for my PhD project was centred on the development and evaluation of a new method for bioconjugation. This is a process where a chemical entity is attached, most often covalently, to a biomolecule of interest to change one or more of its natural properties or to add new functionality. For example biocompatible polymers can be coupled to proteins to increase their half life in the human blood or radioactive compounds attached to antibodies to trace their distribution in a patient. Although today very common in the development of new biopharmaceuticals and novel research and diagnostic techniques the chemical toolbox of efficient bioconjugation reactions is still surprisingly limited. Doing chemistry at the interface to biology requires any good method to fulfil a number of requirements of which especially high selectivity and site-specificity are critical to obtain defined and active products.

Our approach to carry out bioconjugation on polypeptides is to target their solvent accessible disulfide bonds. These covalent linkages between the side chains of two cysteine amino acids are fairly common in proteins of therapeutic interest and afford two highly reactive thiol groups upon reductive cleavage. As the native function of many disulfide bonds is structural maintenance and stabilisation of the protein fold an ideal method for their modification has to re-form the connection between the two cysteines. To this end, we synthesised bis-reactive compounds – doubly substituted maleimides – which would react with both free thiols of a reduced disulfide bond and thereby re-connect them by insertion of an artificial two-carbon bridge (see Scheme 1). Before this process a functional molecule would have been installed on the maleimide, which thus would efficiently become attached to the protein of interest.

figure 1
Scheme 1: Concept of functional bridging of protein disulfide bonds
 
After a few experiments on the chemistry to understand the behaviour and reactivity of our next generation maleimides we firstly realised the concept of “functionalization by bridging” on the small peptide hormone somatostatin. We successfully conjugated a range of functional molecules including fluorophores, polymers and spin labels to its single disulfide bond and demonstrated, in collaboration with a group at UCL Medicine, stability and full biological activity of the synthesised somatostatin derivatives. This model system was also used to optimise the conjugation process and test variations of the substituted maleimide.

We then applied the developed methods to modify and functionalise the disulfide bonds of insulin as well as various antibody fragments (see Scheme 2) and full antibodies.
figure 1
Scheme 2: Efficient modification of an antibody fragment (ds-scFv) via maleimide-based disulfide bridging
 
In most cases our new conjugation method was found to be fast, high yielding, selective and site-specific. Moreover we were able to show in collaboration with groups from the Cancer Institute, UCL ISMB, the London Centre of Nanotechnology and MRCT that the synthesised bioconjugates were active, stable and displayed the desired functionalities in a wide range of biochemical and biophysical assays. Here I was able to learn many new techniques and the ease with which it was possible to work with people or equipment from other research groups is in my opinion one of the great strengths of the interdisciplinary program, facilitated by the friendly climate at UCL and the structure of the participating institutions.
In conclusion I was able to develop and prove the efficiency of the envisaged bioconjugation technology during my PhD. Therefore I am very pleased to see various aspects of my work continued by other brilliant PhD students and postdoctoral researches.
 
thiologistics
As the successful development of our new bioconjugation platform enabled us to prepare a number of biologicals with apparent implications for therapeutic and diagnostic use, the commercial aspect of my PhD project soon was realised. With support from UCL Business Dr Baker and our long term collaborators on this project, Prof Stephen Caddick and Dr Mark Smith (UCL Chemistry), had already filed a number of patents when I started to work with maleimides. These, together with two more patent applications based on my research, created the basis for the foundation of ‘Thiologics’ (http://www.thiologics.com), a spin-off company from UCL aiming at the commercialisation of the next generation maleimide platform with a focus on the synthesis of superior antibody-drug conjugates. This opportunity to experience the translation of academic research towards business goals has shown me that I would like to pursue a career in industrial sectors related to my PhD research.
 

Publications and patent applications

*Cox MB, Riggs DL, Hessling M, Schumacher F, Buchner J, Smith DF. 2007. FK506-binding protein 52 phosphorylation: a potential mechanism for regulating steroid hormone receptor activity. Mol Endocrinol 21(12):2956-2967.

Smith ME, Schumacher FF, Ryan CP, Tedaldi LM, Papaioannou D, Waksman G, Caddick S, Baker JR. 2010. Protein modification, bioconjugation, and disulfide bridging using bromomaleimides. J Am Chem Soc 132(6):1960-1965.

Schumacher FF, Nobles M, Ryan CP, Smith ME, Tinker A, Caddick S, Baker JR. 2011. In situ maleimide bridging of disulfides and a new approach to protein PEGylation. Bioconjug Chem 22(2):132-136.

Chudasama V, Smith ME, Schumacher FF, Papaioannou D, Waksman G, Baker JR, Caddick S. 2011. Bromopyridazinedione-mediated protein and peptide bioconjugation. Chem Commun (Camb) 47(31):8781-8783.

*Nan R, Farabella I, Schumacher FF, Miller A, Gor J, Martin AC, Jones DT, Lengyel I, Perkins SJ. 2011. Zinc binding to the tyr402 and his402 allotypes of complement factor h: possible implications for age-related macular degeneration. J Mol Biol 408(4):714-735.

*Mutze K, Langer R, Schumacher F, Becker K, Ott K, Novotny A, Hapfelmeier A, Hofler H, Keller G. 2011. DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer. Eur J Cancer.

Ryan CP, Smith ME, Schumacher FF, Grohmann D, Papaioannou D, Waksman G, Werner F, Baker JR, Caddick S. 2011. Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem Commun (Camb) 47(19):5452-5454.

Jones MW, Strickland RA, Schumacher FF, Caddick S, Baker JR, Gibson MI, Haddleton DM. 2012. Highly efficient disulfide bridging polymers for bioconjugates from radical-compatible dithiophenol maleimides. Chem Commun (Camb) 48(34):4064-4066.

Baker JR, Schumacher FF, Smith ME, Caddick S; 2012. Chemical modification of antibodies by selective bridging of accessible disulfide bonds.

Jones MW, Strickland RA, Schumacher FF, Caddick S, Baker JR, Gibson MI, Haddleton DM. 2012. Polymeric dibromomaleimides as extremely efficient disulfide bridging bioconjugation and pegylation agents. J Am Chem Soc 134(3):1847-1852.

Felix F. Schumacher, Vishal A. Sanchania, Berend Tolner, Zoë V. F. Wright, Chris P. Ryan, Mark E. B. Smith, John M. Ward, Stephen Caddick, Christopher W. M. Kay, Gabriel Aeppli, Kerry A. Chester & James R. Baker. 2013. Homogeneous antibody fragment conjugation by disulfide bridging introduces ‘spinostics’. Scientific Reports 3, Article number: 1525. doi:10.1038/srep01525

*these publications are not from my PhD project

 

 

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