Site-Specific Conjugation of Tumour-Targeting Antibodies Using Transglutaminase

141 Pages·2010·6.91 MB·English

ETH Library
Site-specific conjugation of
tumour-targeting
antibodies using transglutaminase
Doctoral Thesis
Author(s):
Jeger, Simone
Publication date:
2009
Permanent link:
https://doi.org/10.3929/ethz-a-005963273
Rights / license:
In Copyright - Non-Commercial Use Permitted
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DISS. ETH 18696

Site-Specific Conjugation of Tumour-Targeting
Antibodies Using Transglutaminase

A dissertation submitted to
ETH ZURICH
for the degree of
DOCTOR OF SCIENCES

Presented by
SIMONE JEGER
Master of Pharmaceutical Sciences, University of Basel
born 15.04.80
citizen of Meltingen SO

Accepted on the recommendation of
Prof. Dr. Roger Schibli, examiner
Prof. Dr. P. August Schubiger, co-examiner

2009

Science may set limits to knowledge,
but should not set limits to imagination.

Bertrand Russell

To my parents

Acknowledgements

First of all, I wish to thank Prof. Roger Schibli for giving me the opportunity to perform
my doctorate in his group. Working under his supervision not only allowed me to gain
deep insight into radiopharmaceutical sciences but also encouraged me to continue
working in research.
My thanks are due to Prof. P. August Schubiger for agreeing to co-referee my thesis.
I am especially grateful to Kurt, Christine und Alain. I want to thank you not only for
your support in the lab and for teaching me from your huge experience but also for the
very nice time we spent together! It was always a great pleasure to work with you!
Harriet, I want to thank you for all your help with writing my thesis and for synthesizing
the magic sugar! Ever since you left PSI, I have missed our music sessions in the lab
and our trials to get the best schaum in the cafeteria! I am grateful to Jürgen and
Susan who helped me with the chCE7agl-antibody preparation, cell culture and animal
experiments. Many thanks to Michael and Claudia for performing PET imaging and to
Alexander for producing the 89Zr. My thanks are also due to Peter Hunziker from the
Functional Genomics Centre Zurich for the MALDI-TOF MS analyses and helpful
discussions. Furthermore, I would like to thank Prof. Walter Mier and Dikran Sarko
from the UniversitätsKlinikum in Heidelberg for providing the (Poly)-DOTA-polylysine
compounds.
I wish to thank my dear colleagues Judith, Cristina, Evelyne, Eva, Carla, Sabrina,
Sandra, Alexandra, Christian, Aileen, Tatjana and Phoebe for many scientific and non-
scientific discussions and the nice time we spent together inside and outside the lab.
Finally, my heartfelt thanks go to my parents Margreth and Toni, Marco, Anja and all
my friends who have supported me a lot during the last years.
This work was supported by the Swiss National Science Foundation (112437).
Contents

Summary ......................................................................................................................3
Zusammenfassung........................................................................................................7
Abbreviations..............................................................................................................11
1 Introduction.............................................................................................................15
1.1 Tumour-targeting therapeutic monoclonal antibodies......................................15
1.1.1 Antibody-drug conjugates....................................................................16
1.1.2 Radioimmunoconjugates.....................................................................17
1.1.3 Chelating agents..................................................................................21
1.1.4 Anti-L1-CAM mAb chCE7....................................................................23
1.1.5 Anti-CD20 mAb rituximab....................................................................24
1.2 Antibody conjugation methods ........................................................................26
1.2.1 Lysine residues....................................................................................26
1.2.2 Reduced disulfide bonds .....................................................................27
1.2.3 Glycoengineering.................................................................................29
1.2.4 Enzymatic conjugation.........................................................................30
1.2.5 Conjugation of ligands attached to polymers .......................................30
1.2.6 Towards site-specificity and low stoichiometry.....................................32
1.2.7 Towards homogeneity.........................................................................33
1.3 Structural features and analysis of antibodies.................................................34
1.3.1 Structure of antibodies.........................................................................34
1.3.2 Antibody glycosylation.........................................................................35
1.3.3 Mass spectrometry in protein analysis.................................................39
1.4 Transglutaminases..........................................................................................41
1.4.1 Transglutaminases in nature................................................................41
1.4.2 Bacterial transglutaminase...................................................................42
1.4.3 Proposed catalytic mechanism of transglutaminases...........................42
1.4.4 Hydrolysis of the glutamine-containing substrate.................................43
1.4.5 Protein modification using transglutaminase........................................43
1.4.6 Transglutaminase-mediated modification of antibodies........................45
1.5 Aim of the thesis..............................................................................................47
2 Results and Discussion..........................................................................................49
2.1 Chemical antibody conjugation........................................................................49
2.2 Synthesis of new substrates for bacterial transglutaminase.............................51
2.3 Conjugation of new substrates to human IgG1-antibodies using bacterial
transglutaminase.............................................................................................56
2.3.1 Reaction conditions for BTG-mediated antibody conjugation...............56
2.3.2 Hydrolysis of glutamine-containing substrates.....................................56
2.3.3 Structural differences between the lysine-containing substrates..........57
2.3.4 (Poly)-DOTA-polylysine.......................................................................58
2.3.5 Top-down LC/MS analysis...................................................................60
2.3.6 Bottom-up LC/MS analysis ..................................................................61
2.3.7 Mechanistic insights.............................................................................64
2.3.8 Structural insights................................................................................65
2.3.9 Dual antibody modification...................................................................66
2.3.10 Antibody biotinylation...........................................................................67
2.3.11 Antibody glycosylation.........................................................................67
2.4 In vitro and in vivo evaluation of 67Ga-deferoxamine-chCE7agl.......................68
2.5 In vitro and in vivo evaluation and SPECT imaging of 67Ga-deferoxamine-
chCE7agl and 67Ga-deferoxamine-chCE7aglQ295N.......................................72
2.6 PET imaging of 89Zr-deferoxamine-chCE7agl..................................................77
2.7 In vitro and in vivo evaluation of 177Lu-DOTA-chCE7agl..................................81
2.8 In vitro and in vivo evaluation of 64/67Cu-CPTA-rituximab.................................86
3 Conclusions and Outlook........................................................................................93
4 Experimental Section..............................................................................................97
4.1 Synthesis of chelating agents..........................................................................97
4.2 Antibodies.....................................................................................................103
4.3 Enzymatic conjugation...................................................................................107
4.4 Chemical conjugation....................................................................................110
4.5 Radiolabelling procedures.............................................................................112
4.6 In vitro studies...............................................................................................114
4.7 In vivo studies................................................................................................116
References................................................................................................................121
Curriculum Vitae........................................................................................................133
Publications and Presentations.................................................................................135
3
Summary

The growing knowledge of cancer-related antigens allows the design of highly specific
monoclonal antibodies. However, despite excellent tumour-targeting properties, the
therapeutic potential of such antibodies is often insufficient for a curative effect.
Nevertheless, the tumour-targeting ability of these proteins can be exploited by
functionalization with toxic therapeutic 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.
The incorporation of metallic radionuclides into antibodies requires first that an efficient
chelating system is conjugated to the protein. 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 as a result of random conjugation can
adversely affect pharmacodynamics, for example, modification in the binding region of
the antibody could disrupt antigen binding. Similarly, unfavourable 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.
To address these shortcomings, we have established a novel enzyme-mediated
strategy for site-specific and stoichiometric antibody modification. Using an enzyme of
the transglutaminase (TG) family we have succeeded in conjugating a range of
functional molecules to tumour-targeting antibodies. TGs catalyze covalent protein
crosslinking by forming isopeptide bonds between lysine donor residues of one protein
and acceptor glutamine residues of another protein. We derivatized the chelators
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 4-(1,4,8,11-
tetraazacyclotetradec-1-yl)-methyl benzoic acid (CPTA) and deferoxamine with lysine
and glutamine-containing residues. While we observed undesired TG-mediated
deamidation of the glutamine-containing substrates, the lysine-mimicking ligands were
recognized by the enzyme and successfully conjugated to glutamine side chains of
various deglycosylated human IgG1-antibodies. Top-down mass spectrometry (MS) of
the novel immunoconjugates revealed total product homogeneity. A small number of
ligands was stoichiometrically and reproducibly conjugated to the antibodies. Using
bottom-up MS for analysis of digested immunoconjugate fragments, we were able to
identify Gln295 (Kabat numbering) in the conserved glycosylation region as the site for
4
TG-mediated conjugation. This glutamine residue is only accessible to the enzyme
upon deglycosylation, since it is shielded by the N-glycans attached to Asn297 in
native antibodies. In the aglycosylated antibody chCE7agl Asn297 has been replaced
by Gln to abort N-glycosylation. The modified antibody provides a second enzymatic
conjugation site at position 297. The number of ligands, which were stoichiometrically
and site-specifically conjugated by TG, was either two or four for the deglycosylated
and aglycosylated antibodies, respectively.
Following enzymatic conjugation of metal chelators to the aglycosylated anti-L1-CAM
antibody chCE7agl and the deglycosylated anti-CD20 antibody rituximab, the
immunoconjugates were stably radiolabelled with different nuclides for in vitro and in
vivo assessment of the pharmacodynamics and pharmacokinetics of the novel
compounds. In vivo biodistribution studies were performed with 67Ga-deferoxamine-
chCE7agl and 64/67Cu-CPTA-rituximab in tumour-bearing nude mice. The
homogeneous conjugates, which were prepared using the enzymatic conjugation
strategy and had a well-defined ligand/antibody ratio, were compared with analogous
conjugates prepared using chemical conjugation methods, for which only an average
ligand/antibody ratio could be determined. The results indicated better therapeutic
indices for the conjugates prepared with TG, as accumulation of radioactivity at the
tumour sites was higher while toxic, nonspecific uptake into the excretion organs was
lower.
To further investigate the influence of stoichiometry on the pharmacokinetic profile of
conjugated antibodies we designed a mutant of chCE7agl (chCE7aglQ295N) which
was conjugated to two deferoxamine chelators using TG. The biodistribution of the
67Ga-radiolabelled immunoconjugate was compared with that of 67Ga-chCE7agl-
deferoxamine bearing four chelators in tumour-bearing nude mice. The decreased
number of deferoxamine chelators per antibody did not result in an improvement in the
in vivo behaviour when compared with the analogue with a greater number of
chelators. These results led to the conclusion that product homogeneity rather than low
stoichiometry would appear to be a more significant determinant for
radioimmunoconjugate potency and tolerability.
The promising results obtained in the organ distribution studies could be directly
translated to SPECT (67Ga-radiolaballed conjugates) and PET (89Zr-radiolaballed
conjugates) imaging. Both chCE7agl-deferoxamine conjugates (prepared either using
the enzymatic or chemical conjugation strategies) showed excellent visualization of
metastatic and solid tumours in xenografted animals.