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Improving the Efficacy and Quality of Immunoconjugates Using Novel Enzymatic Methods for the Site-specific and Stoichiometric Modification of Proteins

English title Improving the Efficacy and Quality of Immunoconjugates Using Novel Enzymatic Methods for the Site-specific and Stoichiometric Modification of Proteins
Applicant Schibli Roger
Number 132611
Funding scheme Project funding (Div. I-III)
Research institution Paul Scherrer Institut
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Inorganic Chemistry
Start/End 01.04.2011 - 31.03.2014
Approved amount 220'554.00
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All Disciplines (5)

Discipline
Inorganic Chemistry
Biochemistry
Pharmacology, Pharmacy
Organic Chemistry
Molecular Biology

Keywords (10)

Radiolabeling; Antibody; Tumor Targeting; Enzyme; Transglutaminase; Radioimmunotherapy; Post-expression modification; monoclonal antibodies; protein modification; cancer

Lay Summary (English)

Lead
Lay summary
Monoclonal antibodies (mAbs) have proven to be useful across a spectrum of diseases. Oncology has been a major area of focus for mAb-based reagents. Although unmodified mAbs may show some therapeutic potency, the effects are often not curative. Thus, there is need to improve the antitumor activity of antibodies. The therapeutic efficacy of antibodies can be substantially enhanced by conjugation of cytotoxic compounds, such as chemotherapeutics and particle-emitting radionuclides. Conventional chemical strategies for protein modification are difficult to control and give rise to heterogeneous populations of immunoconjugates with variable stoichiometries. Each differently conjugated antibody molecule of the population has to be considered as an individual species with a characteristic pharmacokinetic, efficacy and safety profile. Therefore, we investigate enzymatic strategies for site-specific and stoichiometric modification of mAbs. The enzyme transglutaminases (TG) have the unique characteristic of forming very stable isopeptidic bonds between the side chains of glutamine and lysine in peptides and proteins. The specificity of TGs limits the number of lysine and glutamine residues of a protein which are accessible for modification since only those with the correct environment are recognized by the enzymes. During the course of the SNF projects No. 112437 and 120158 we have proven that bacterial TG (BTG) enables the modification/functionalization of mAbs with various lysine mimicking substrates with a well-defined stoichiometry. We identified the sole sites for TG-mediated conjugation and could demonstrate that the enzymatic conjugated mAbs showed excellent in vivo behaviour, with high tumour uptake and improved target-to-non-target ratios compared to the chemically modified pendants. Our goal is to increase the effectiveness of radioimmunotherapy (RIT) with immunoconjugates produced via TG-mediated functionalization. We will verify the concept in relevant mouse models in vivo. As radionuclides we propose to use 67Cu, 90Y, 161Tb and 177Lu. Reaction conditions will be identified and optimized in order to allow enzymatic modification of proteins not only with lysine-mimicking but also with glutamin-mimcking substrates (via lysine side chains of the mAb). Sites of modification will be identified via MS techniques. Dual modification of mAbs via lysine and glutamine with different chemical entities will be performed. Novel metal chelating systems suitable as substrates for TG will be prepared. Lysine-mimicking substrates of known chemotherapeutics will be included in our research. This proposal is intended to solve and avoid the most important and critical
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Name Institute

Publications

Publication
Microbial transglutaminase and c-myc-tag: a strong couple for the functionalization of antibody-like protein scaffolds from discovery platforms.
Dennler Patrick, Bailey Laura K, Spycher Philipp R, Schibli Roger, Fischer Eliane (2015), Microbial transglutaminase and c-myc-tag: a strong couple for the functionalization of antibody-like protein scaffolds from discovery platforms., in Chembiochem : a European journal of chemical biology, 16(5), 861-7.
Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models.
Lhospice F, Brégeon D, Belmant C, Dennler P, Chiotellis A, Fischer E, Gauthier L, Boëdec A, Rispaud H, Savard-Chambard S, Represa A, Schneider N, Paturel C, Sapet M, Delcambre C, Ingoure S, Viaud N, Bonnafous C, Schibli R, Romagné F (2015), Site-Specific Conjugation of Monomethyl Auristatin E to Anti-CD30 Antibodies Improves Their Pharmacokinetics and Therapeutic Index in Rodent Models., in Molecular pharmaceutics, 12(6), 1863-71.
A Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates
Dennler Patrick, Chiotellis Aris, Fischer Eliane, Brégeon Delfine, Belmant Claude, Gauthier Luc, Lhospice Florence, Romagne Francois (2014), A Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates, in Schibli, Roger, 25(3), 569-578.
DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl.
Grünberg Jürgen, Jeger Simone, Sarko Dikran, Dennler Patrick, Zimmermann Kurt, Mier Walter, Schibli Roger (2013), DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl., in PloS one, 8(4), 60350-60350.
Enzymatic antibody modification by bacterial transglutaminase
Denner Patrick, Fischer Eliane, Schibli Roger, Enzymatic antibody modification by bacterial transglutaminase, in Ducry Laurent (ed.), Springer, Humana Press, Berlin, Heidelberg.

Collaboration

Group / person Country
Types of collaboration
Ajinomoto Japan (Asia)
- Industry/business/other use-inspired collaboration
DKFZ Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Innate Pharma France (Europe)
- Industry/business/other use-inspired collaboration
ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Gordon Research Seminar: Antibody Biology & Engineering Talk given at a conference Towards Homogenous Antibody-Drug Conjugates - Transglutaminase as a Tool for Site-Specific Antibody Modification 15.03.2014 Lucca (Barga), Italy Schibli Roger; Dennler Patrick; Fischer Eliane; Grünberg Jürgen;
21. Jahrestagung der Arbeitsgemeinschaft Radiochemie/Radiopharmazie (AGRR) Talk given at a conference A Versatile Method to Produce 111In–Labelled Non-Covalent Antibody Conjugates 15.09.2013 Vienna, Austria Grünberg Jürgen; Fischer Eliane; Schibli Roger; Dennler Patrick;
PhD Day, Institute of Pharmaceutical Sciences (IPW), ETH Zurich Talk given at a conference Transglutaminase as a Tool for Site-Specific Protein Modification 05.09.2013 Zürich, Switzerland Grünberg Jürgen; Dennler Patrick; Fischer Eliane; Schibli Roger;
38th FEBS Congress Talk given at a conference Novel Methods for the Site-specific and Stoichiometric Modification of Therapeutic Protein 06.06.2013 St. Petersburg, Russia Schibli Roger;
Biomolecular Research Joint Meeting (JBMR), Department BIO, Paul Scherrer Institute Talk given at a conference Homogeneous Antibody Drug Conjugates - Site-specific Modification by Transglutaminase 16.01.2013 Villigen-PSI, Switzerland Grünberg Jürgen; Schibli Roger; Dennler Patrick; Fischer Eliane;
Pharmacology & Toxicology Poster Day 2012 Poster Towards Homogeneous Antibody Conjugates: Site-specific Modification by Transglutaminase 10.10.2012 ETH Zurich, Switzerland Schibli Roger; Grünberg Jürgen; Fischer Eliane; Dennler Patrick;
5th Swiss Pharma Science Day 2012 Poster Homogeneous Antibody Conjugates: Site-specific Modification by Transglutaminase 23.08.2012 Bern, Switzerland Dennler Patrick; Grünberg Jürgen; Schibli Roger; Fischer Eliane;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
World Medtech Forum Poster 26.09.2012 Luzern, Switzerland Schibli Roger;


Awards

Title Year
ETH Zürich, Pharmacology & Toxicology Poster Day / 1st Poster Prize 2012

Patents

Title Date Number Inventor Owner
Evaluating antibodies, comprises providing first and second antibody sample each comprising antibodies having acceptor glutamine residue in a constant region, and reacting each of first and second antibody sample with a lysine-based linker 13.07.2012 WO2014009426-A2 ; WO2014009426-A3
New antibody or its fragment comprising a functionalized acceptor glutamine residue useful for making pharmaceutical composition for treating autoimmune disease, bacterial or microbial infection, and cancer 21.12.2012 WO2013092983-A2
Enzymatic Conjugation of Antibodies 23.12.2011 WO 2013092998 A1

Associated projects

Number Title Start Funding scheme
120158 Novel enzymatic methods for the site-specific modification of therapeutic proteins 01.05.2008 Project funding (Div. I-III)
128443 Targeted polymers for site-specific drug delivery 01.03.2010 SCOPES

Abstract

In addition to small molecular drugs (e.g. kinase inhibitors such as Glivec), monoclonal antibodies (mAbs) are essential tools in cancer therapy because of their extraordinary selectivity and target affinity. However, despite excellent tumor-targeting properties, the therapeutic potential of such antibodies is often insufficient for a curative effect. This shortcoming of mAbs can be solved by functionalization of such proteins with toxic agents such as anticancer drugs or radionuclides. The promise of (radio)immunoconjugates is to enhance anticancer efficacy of the antibody while minimizing off-target toxicity of the cell killing agent. Thus, monoclonal antibodies (mAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs (immunoconjugates), are becoming a important component of anticancer treatments. Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin™, radiolabeled with 90Y) and tositumomab (Bexxar™, radiolabeled with 131I) have been approved by the FDA for treatment of low-grade B-cell non-Hodgkin’s lymphoma. Recent studies have proven that the median survival time of patients treated with Zevalin™ is upto 3-fold longer than that of patients treated with the non-radioactive pendant MabThera® (Rituximab). However, the antibody modification strategy is still a major challenge for the design and production of immunoconjugates. The products of conventional chemical conjugation strategies are invariably a heterogeneous mixture of antibody species with different numbers of ligands attached at various sites. The lack of site-specificity which results from random conjugation can adversely affect pharmacodynamics, for example, modification in the binding region of the antibody can disrupt antigen binding. Similarly, high ligand loadings can have deleterious effects on the pharmacokinetics of the immunoconjugates. Furthermore, heterogeneity within the preparations challenges batch-to-batch consistency and limits reproducibility. Transglutaminases (TG) have the unique characteristic of forming very stable isopeptidic bonds between the side chains of glutamine and lysine in peptides and proteins. The specificity of TGs limits the number of lysine and glutamine residues of a protein which are accessible for modification since only those with the correct environment are recognized by the enzymes. During the course of the SNF projects No. 112437 and 120158 we have proven that bacterial TG (BTG) enables the modification/functionalization of mAbs with various lysine mimicking substrates with a well-defined stoichiometry (exactly 2 or 4 substrates per antibody depending on the number of accessible Gln residues). Using bottom-up MS for analysis of digested immunoconjugate fragments, we were able to identify Gln295 and/or Gln297 as the sole sites for TG-mediated conjugation (though only after deglycosylation of the mAbs). In vivo biodistribution studies performed with different radiolabeled (67Ga, 64/67Cu and 89Zr) mAbs (anti-L1cam and anti-CD20 mAbs) in tumour-bearing nude mice demonstrated significantly improved biodistribution data for the conjugates prepared with BTG compared to the chemically modified pendants, as accumulation of radioactivity at the tumour sites was higher and non-specific uptake into the excretion organs was lower.Working Hypothesis and Goals: Our previous work has shown, that compared to chemical methods, mAb modification using BTG (i) is site-specific, simple, robust and versatile; (ii) gives rise to immunoconjugates with a defined stoichiometry; (iii) produces immunoconjugate with better pharmacological profiles. In addition, the enzymatic labeling of protein with TG offers the unique opportunity to employ glutamine residue, which cannot easily be functionalized by conventional strategies, as an additional functional group and thus, extends the number of available coupling sites of mAbs. This proposal will further investigate the versatility of this enzymatic methodology. (1) Specifically we will perform tumor therapy studies with radioimmunoconjugates produced by TG-mediated functionalization. We will verify the concept in a mouse model in vivo with our own, in-house produced (anti-L1cam mAbs chCE7) and commercial tumor-targeting antibodies (e.g. Rituximab). As radionuclides we propose to use 67Cu, 90Y, 161Tb and 177Lu. (2) We will modify the primary amino acid sequence of chCE7 and introduce additional glutamines at surface and solvent exposed sites of the protein to avoid deglycosylation of the mAbs before enzymatic modification. (3) Novel (radio)metal chelating systems suitable as substrates for TG will be prepared. (5) Selected mAbs (but also other promising tumor-targeting protein formats) will be systematically modified with known, non-radioactive chemotherapeutic agents with well-defined stoichiometries using TG. We will perform therapy studies (in collaboration with external partners) to identify the optimal drug-to-antibody ratio and compare the potential of our TG-method with conjugates prepared using chemical methods.Expected value of the proposal: Based on our previous results and the results obtained during the new funding period we are keen to prove that this technology has implications in pre-clinical as well as clinical protein-based drug development because of the assumed versatility. Since the problems associated with targeting using immunoconjugates or their production are not limited to radiopharmaceutical development, other groups will also benefit. Thus, the range of potential users profiting from this technology is very broad. The market for targeted imaging probes is expected to expand exponentially in the near future with the growing number of (tumor) targets currently being identified. The technology is also attractive for 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) impetus for the development of new and effective therapeutics and diagnostics for the management of (cancerous) diseases.
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