Project

Back to overview

Novel enzymatic methods for the site-specific modification of therapeutic proteins

English title Novel enzymatic methods for the site-specific modification of therapeutic proteins
Applicant Schibli Roger
Number 120158
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pharmazeutische Wissenschaften ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Inorganic Chemistry
Start/End 01.05.2008 - 30.04.2010
Approved amount 142'900.00
Show all

All Disciplines (4)

Discipline
Inorganic Chemistry
Organic Chemistry
Pharmacology, Pharmacy
Biochemistry

Keywords (7)

Radiolabeling; Antibody; Tumor Targeting; Enzyme; Transglutaminase; Radioimmunotherapy; Post-expression modification

Lay Summary (English)

Lead
Lay summary
Hintergrund: Cancer is responsible for more than 20 % of deaths in developed countries. Monoclonal antibodies (MAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs, are becoming a significant component of anticancer treatments. However, chemical post-translation modification of protein can lead to inactivation or severe effects on the pharmacokinetics of the immunoconjugate. For the next generation of immunoconjugates, advances in protein modification are therefore required in order to allow better control over the location and stoichiometry of the conjugation process. Transglutaminases (TGase) have the unique characteristics to form very stable isopeptidic bonds between the side chains of glutamine and lysine in peptides and proteins. Due to the specificity of TGases only a limited number of lysine and glutamine residues of a protein with the correct environment are accessible for modification by the enzymes. Ziel: Our previous work using the high affinity anti-L1 antibody chCE7 has shown, that compared to chemical methods, MAb modification via TGase can be: (i) more domain-specific, (ii) more reliable, giving rise to immunoconjugates with defined stoichiometry, (iii) simple and robust (iv) versatile. In addition, the enzymatic labeling of protein with TGase offers the unique opportunity to employ glutamine residue as an additional functional group and hence, enables extending the currently available number of coupling sites of MAbs. This proposal is intended to further investigate the versatility of this enzymatic methodology. We will perform in vivo biodistribution and imaging experiments (PET/SPECT). Results will be compared to previously, chemically modified chCE7 to prove equal or superior characteristics of the enzymatically modified conjugated with respect to their in vivo targeting capacity or pharmacokinetics. This represents the first important milestone. We will further analyze the domain/site-specificity of the functionalization in case of the MAb chCE7agl. We will perform protein MS analyses as well as point-mutation of the protein in order to characterize the minimal (structural and chemical) requirements for an efficient, reproducible enzymatic modification. We will verify the concept in vitro and in vivo with other tumor targeting antibodies. In addition we will prepare immunoconjugates potentially useful for in vivo targeted MRI imaging in order to prove the versatility of the enzymatic approach. Bedeutung: The market for targeted imaging probes is expected to expand in the near future exponentially with the growing number of (tumor) targets identified currently. Implication of this novel technology can be foreseen in pre-clinical as well as clinical protein-based drug development because of the assumed versatility. Thus, the number of potential users profiting from this technology is very broad. We are convinced that a method, which allows the production of novel immunoconjugate with a well defined stoichiometry, will be of highest importance. Furthermore, the technology has potential for scale-up production of immunoconjugates.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Name Institute

Associated projects

Number Title Start Funding scheme
112437 Novel enzymatic methods for the site-specific modification of therapeutic proteins 01.05.2006 Project funding (Div. I-III)
132611 Improving the Efficacy and Quality of Immunoconjugates Using Novel Enzymatic Methods for the Site-specific and Stoichiometric Modification of Proteins 01.04.2011 Project funding (Div. I-III)
116185 Combination of radioimmunotherapy with growth inhibition for antibody-based therapy of L1 expressing metastases 01.05.2007 Project funding (Div. I-III)

Abstract

Background: Cancer is responsible for more than 20 % of deaths in developed countries (www.cancer.org). Monoclonal antibodies (MAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs (immunoconjugates), are becoming a significant component of anticancer treatments. Radiolabeled monoclonal antibodies have proven to play a crucial role in detection and treatment of aggressive metastatic diseases. Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin™, radiolabeled with 111In or 90Y) and tositumomab (Bexxar™, radiolabeled with 131I) have been approved by the FDA for treatment of low-grade B-cell non-Hodgkin’s lymphoma. Chemical post-translation modification of protein generally results in random conjugation of the MAb, which can lead to inactivation or severe effects on the pharmacokinetics of the immunoconjugate. For the next generation of immunoconjugates, advances in protein modification are therefore required in order to allow better control over the location and stoichiometry of the conjugation process, resulting in more uniform and defined products for the improved delivery of radioisotopes to tumors. Transglutaminases (TGase) have the unique characteristics to form very stable isopeptidic bonds between the side chains of glutamine and lysine in peptides and proteins. Due to the specificity of TGases only a limited number of lysine and glutamine residues of a protein with the correct environment are accessible for modification by the enzymes. This is an appealing feature of TGases for the development of a domain/site-specific modification of MAb. In the course of the SNF project No. 112437 we could recently show for the first time, that TGases enable the modification/functionalization of the anti-L1cam antibody chCE7agl with fluorescent probes and also with different metal chelating system useful in radioimmunodiagnosis and therapy in a single step and under mild conditions. The modification resulted in conjugates with well defined stoichiometry (exactly 4 ligands per antibody) and very domain-specific (almost exclusively functionalization via the heavy chain of the MAbs). The immunoconjugate retained full biological activity in vitro. With chemical methods, such specificity has never been achieved with intact antibodies to the best of our knowledge. This could represent a milestone in post-translation modification of MAbs. Working Hypothesis and Goals: Our previous work using the high affinity anti-L1 antibody chCE7 has shown, that compared to chemical methods, MAb modification via TGase can be: (i) more domain-specific, (ii) more reliable, giving rise to immunoconjugates with defined stoichiometry, (iii) simple and robust (iv) versatile. In addition, the enzymatic labeling of protein with TGase offers the unique opportunity to employ glutamine residue as an additional functional group and hence, enables extending the currently available number of coupling sites of MAbs. This proposal is intended to further investigate the versatility of this enzymatic methodology. With the newly generated immunoconjugates of chCE7agl we will perform in vivo biodistribution and imaging experiments (PET/SPECT). Results will be compared to previously, chemically modified chCE7 to prove equal or superior characteristics of the enzymatically modified conjugated with respect to their in vivo targeting capacity or pharmacokinetics. This represents the first important milestone. We will further analyze the domain/site-specificity of the functionalization in case of the MAb chCE7agl. We will perform protein MS analyses as well as point-mutation of the protein in order to characterize the minimal (structural and chemical) requirements for an efficient, reproducible enzymatic modification. We will verify the concept in vitro and ev. in vivo with other (commercial) tumor targeting antibodies. Furthermore, we will investigate and test two new enzymes (guinea pig TGase and human coagulation factor XIII) for their potential use in protein modification. New metal chelating system (tailor-made for e.g. 111In, 99mTc/188Re), which are recognized as substrates of TGase, will be synthesized and tested. In addition we will prepare immunoconjugates potentially useful for in vivo targeted MRI imaging in order to prove the versatility of the enzymatic approach. Ultimately, decoration of MAbs with two independent signatures (PET/SPECT and MIR) should be produced for multimodality imaging. Excepted value of the proposal: Implication of this novel technology can be foreseen in pre-clinical as well as clinical protein-based drug development because of the assumed versatility. Thus, the number of potential users profiting from this technology is very broad. Since the problems of targeting strategies using immunoconjugates or their production respectively are not only limited radiopharmaceutical development other groups will also benefit. The market for targeted imaging probes is expected to expand in the near future exponentially with the growing number of (tumor) targets identified currently. The technology is also attractive for the industry. We are convinced that a method, which allows the production of novel immunoconjugate with a well defined stoichiometry, will be of highest importance. Furthermore, the technology has potential for scale-up production of immunoconjugates. Overall we are convinced that this new method will provide an additional dynamic impulse toward development of new and effective therapeutics and diagnostics for the future management of (cancerous) diseases.
-