Site-specific labeling of affinity molecules for in vitro and in vivo studies

83 Pages·2014·5.93 MB·English

by  Anna Perols

Site-specific labeling of affinity molecules
for in vitro and in vivo studies

ANNA PEROLS

Royal Institute of Technology
School of Biotechnology
Stockholm 2014
© Anna Perols
Stockholm 2014

KTH Royal Institute of Technology
School of Biotechnology
AlbaNova University Center
SE-106 91 Stockholm

Printed by Universitetsservice US-AB
Drottning Kristinas väg 53B
SE-100 44 Stockholm
Sweden

TRITA-BIO Report 2014:14
ISSN 1654-2312
ISBN 978-91-7595-252-9
Anna Perols (2014): Site-specific labeling of affinity molecules for in vitro and in vivo
studies. School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden.
Abstract
The thesis is focused on site-specific labeling of affinity molecules for different applications
where two types of binding proteins, Affibody molecules and antibodies, have been used. For the
purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-
functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study,
two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide
chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395).
In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the
111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-
MMA-ZHER2:2395 counterpart, resulting in improved tumor-to-organ ratios. In a second study the
in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in
tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-
ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in
higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant.
In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1,
was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide
bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for
111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-
NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-
ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone
ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the
DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of
helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation
resulted in the highest contrast.
Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-
binding regions might influence the affinity. A method for site-specific labeling of antibodies
using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody
by photoconjugation was optimized. By investigation of positions most suitable for incorporation
of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses
important for both diagnostic and therapeutic applications. In addition, optimized variants were
used in combination with an incorporated click-reactive handle for selective labeling of the
antibody with a detection molecule.
Key words: Affibody molecules, molecular imaging, site-specific labeling, solid phase peptide
synthesis, IgG-binding domains, photoconjugation.
Sammanfattning
Avhandlingen är baserad på arbeten som varit inriktade på inmärkning av två olika typer av
affinitetsmolekyler, dels affibodymolekyler och dels antikroppar. Inmärkningen har skett på ett
kontrollerat sätt som för affibodymolekyler lett till förbättrad tumördiagnostik, och för
antikroppar bibehållen affinitet efter inmärkning.
I första delen av avhandlingen har egenskaper hos Affibodymolekyler som binder den tumör-
associerade biomarkören HER2 med hög affinitet studerats i syfte att förbättra visualiseringen av
HER2-uttryckande tumörer i djurmodeller. För att kunna urskilja en HER2-uttryckande tumör
från övriga organ krävs att den målsökande spårmolekylen ansamlas i tumörvävnaden, och
samtidigt har lågt upptag i omgivande vävnad, dvs. ger en bra kontrast. I dessa studier har olika
typer av kelatkomplex, DOTA, NOTA och NODAGA, testats för att binda den radioaktiva
isotopen 111In till affibodymolekylen. Utvärdering i djurmodeller med tumörer implanterade
visade att både val av kelatkomplex samt var kelatkomplexet positionerades i affibodymolekylen
resulterade i skillnader i kontrasten. Då metastaser vanligen förekommer i skelett för
prostatacancer och i lever för bröstcancer, är det viktigt att dessa organ har ett lågt, icke-specifikt
upptag av affibodymolekyler för att kunna urskilja möjliga metastasbildningar. Studierna
konkluderade att NOTA och NODAGA är lovande kelat-strukturer för visualisering av
prostatacancertumörer vid konjugering C-terminalt med maleimide/tiol-kemi. Uppföljande
studier visade vidare att konjugering via en amidbindning N-terminalt gav förbättrad kontrast
för DOTA för visualisering av metastaser i lever och skelett i jämförelse mot NOTA och
NODAGA, samt att en C-terminal amid-konjugering av DOTA är den mest lovande.
I andra delen av avhandlingen har en metod för inmärkning av antikroppar använts, där den
antikroppsbindande domänen Z har syntetiserats med en fotoreaktiv grupp för att kovalent
kunna korslänkas till en antikropp med UV-ljus, utan att påverka dess bindning. I studien
förbättrades konjugeringseffektiviteten genom att optimera placeringen av den fotoreaktiva
molekylen i domän Z. En optimerad variant användes i en efterföljande studie i kombination
med en funktionell grupp för klick-kemi, för att selektivt kunna koppla en reportergrupp till
antikroppen. Vanligen resulterar antikroppsinmärkning i en heterogen produkt vilket kan
påverka bindningen, men ovanstående studier presenterar ett lovade alternativ för selektiv
inmärkning av antikroppar med bibehållen affinitet.
Sammanfattningsvis visar studierna i denna avhandling att en kontrollerad inmärkning av
affinitetsproteiner är viktigt, dels för användning av Affibodymolekyler i tumördiagnostiskt syfte,
och dels i en metod för selektiv inmärkning av antikroppar utan förlust av dess specificitet.
List of publications
This thesis is based on the following articles or manuscript, which are referred to in the text by
their Roman numerals (I-VI). The articles can be found in the appendix.

I Tolmachev V, Altai M, Sandström M, Perols A, Karlström AE, Boschetti F, and Orlova A.
(2011) Evaluation of a maleimido derivative of NOTA for site-specific labeling of Affibody
molecules. Bioconjug Chem. 22:894-902

II Altai M, Perols A, Karlström AE, Sandström M, Boschetti F, Orlova A, and Tolmachev V.
(2012) Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with
111In using a maleimido derivative of NODAGA. Nucl Med Biol. 39:518-529

III Malmberg J*, Perols A*, Varasteh Z, Altai M, Braun A, Sandström M, Garske U,
Tolmachev V, Orlova A, and Karlström AE. (2012) Comparative evaluation of synthetic
anti-HER2 Affibody molecules site-specifically labelled with 111In using N-terminal DOTA,
NOTA and NODAGA chelators in mice bearing prostate cancer xenografts. Eur J Nucl Med
Mol Imaging. 39:481-492.

IV Perols A*, Honarvar H*, Strand J, Selvaraju RK, Orlova A, Karlström AE, and Tolmachev
V. (2012) Influence of DOTA chelator position on biodistribution and targeting properties
of 111In-labeled synthetic anti-HER2 Affibody molecules. Bioconjug Chem. 23:1661-1670

V Perols A, and Karlström AE. (2014) Site-specific photoconjugation of antibodies using
chemically synthesized IgG-binding domains. Bioconjug Chem. 25:481-488.

VI Perols A, Famme MA, and Karlström AE. Site-specific antibody labeling by covalent
photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions.
Manuscript

* These authors contributed equally to this work.
All papers are reproduced with permission from the copyright holders.
Contributions to the papers
Paper I
Designed, performed and evaluated the protein characterization analyses prior to the
radiolabeling and in vitro and in vivo evaluation. Contributed to the writing and editing of the
paper.
Paper II
Designed, performed and evaluated the protein characterization analyses prior to the
radiolabeling and in vitro and in vivo evaluation. Contributed to the writing and editing of the
paper
Paper III
Synthesized the variants, designed, performed and evaluated the experiments for protein
characterization prior to the radiolabeling and in vitro and in vivo evaluation. Contributed to the
writing and editing of the paper
Paper IV
Synthesized and purified the variants used in the study. Performed all parts of protein
characterization experiments prior to the radiolabeling and in vitro and in vivo evaluation.
Wrote the paper together with co-authors.
Paper V
Synthesized the IgG-binding variants used in the study. Designed, performed the conjugation
optimization studies and analyzed the results. Created the images for the paper and wrote the
paper together with co-author.
Paper VI
Synthesized the IgG-binding variants used in the study. Design, performed and analyzed the
results for the conjugations optimization studies. Created the images for the paper and wrote the
paper together with co-authors.
Related publications

Strand J, Honarvar H, Perols A, Orlova A, Selvaraju RK, Karlström AE, and Tolmachev V.
(2013) Influence of macrocyclic chelators on the targeting properties of 68Ga-labeled synthetic
Affibody molecules: comparison with 111In-labeled counterparts. PLoS One 8: e70028

Rönnlund D, Xu L, Perols A, Gad AK, Karlström AE, Auer G, and Widengren J. (2014)
Multicolor fluorescence nanoscopy by photobleaching: concept, verification, and its application
to resolve selective storage of proteins in platelets. ACS Nano 8: 4358-65

Honarvar H, Strand J, Perols A, Orlova A, Selvaraju RK, Karlstrom AE, and Tolmachev V.
Position for site-specific attachment of a DOTA chelator to synthetic Affibody molecules has a
different influence on the targeting properties of 68Ga- compared to 111In-labeled conjugates.
Molecular Imaging, in press.
Contents
Introduction
Chapter 1 - Proteins ..................................................................................................................... 1
Protein chemistry ........................................................................................................................................... 1
Affinity proteins ............................................................................................................................................ 4
Antibody structure and function ........................................................................................................... 4
Antibody fragments ............................................................................................................................... 7
Antibody generation .............................................................................................................................. 8
Antibody therapy ................................................................................................................................... 8
Alternative scaffolds .............................................................................................................................. 9
Affibody molecules .............................................................................................................................. 10
Chapter 2 - Chemical protein synthesis ............................................................................... 14
From solution to solid phase ........................................................................................................................ 14
Solid phase peptide synthesis (SPPS) .......................................................................................................... 15
Fmoc/tBu strategy ................................................................................................................................ 15
Side chain protecting groups........................................................................................................................ 17
Aggregation ................................................................................................................................................. 20
Microwave synthesis ............................................................................................................................ 20
Limitations in solid phase peptide chemistry ............................................................................................. 22
Chapter 3 - Protein labeling.....................................................................................................23
Chemical approaches .................................................................................................................................. 23
Amino groups ...................................................................................................................................... 23
Thiol groups ......................................................................................................................................... 24
Bioorthogonal reactions for labeling proteins ............................................................................................ 24
Cu(I) catalyzed [3+2] alkyne-azide cycloaddition ............................................................................... 25
Strain-promoted [3+2] alkyne-azide cycloaddition ............................................................................ 27
Alternative ligation strategies .............................................................................................................. 28
Introduction of chemoselective handles in recombinantly produced proteins ................................... 28
Radiolabeling for molecular imaging .......................................................................................................... 29
PET and SPECT ................................................................................................................................... 29
Radiometals and chelators for imaging ............................................................................................... 30
Application-specific considerations ..................................................................................................... 32
Present investigation
Aim of the thesis ........................................................................................................................ 35
Chapter 4 - Functionalization of Affibody Molecules for Detection of HER2 using
Molecular Imaging .................................................................................................................... 36
HER2 as a molecular target......................................................................................................................... 36
Papers I, II and III - The influence of macrocyclic chelators and different conjugation chemistries
for radiolabeling of Affibody molecules in molecular imaging. .................................................................. 37
Papers I and II...................................................................................................................................... 37
Paper III ............................................................................................................................................... 40
Conclusions and future outlook ........................................................................................................... 42
Paper IV – The influence on biodistribution from the positioning of the DOTA chelator in
the Affibody molecule .................................................................................................................................. 43
Conclusions and future outlook .......................................................................................................................... 45
Chapter 5 - Site specific labeling of antibodies using IgG-binding domains ............. 47
Paper V – Site-specific photoconjugation of antibodies using chemically synthesized
IgG-binding domains .................................................................................................................................. 48
Conclusions and future outlook ............................................................................................................51
Paper VI – Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for
alkyne-azide cycloaddition reactions .......................................................................................................... 52
Conclusions and future outlook ........................................................................................................... 55
Concluding remarks and future outlook..............................................................................5 7
Acknowledgements ................................................................................................................... 60
References ................................................................................................................................... 62
List of acronyms
%ID/g Percent injected dosage per gram
ADC Antibody-drug conjugates
ADCC Antibody-dependent cell-mediated cytotoxicity
DAR Drug-antibody-ratio
DNA Deoxyribonucleic acid
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
DTPA Diethylenetriaminepentaacetic acid
E. coli Escherichia coli
EDTA Ethylenediaminetetraacetic acid
EGFR Human epidermal growth factor receptor
Fab Fragment, antigen binding
Fc Fragment crystalizable
FcRn Neonatal Fc receptor
FDA The Food and Drug Administration
FISH Fluorescence in situ hybridization
HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronoium hexa fluorophosphate
HC Heavy chain
HER2 Human epidermal growth factor receptor 2
HF Hydrofluoric acid
HOBt 1-hydroxybenzotriazole
kDa kilo Dalton
LC Light chain
MMA Maleimidoethylmonoamide
NODAGA 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane
NOTA 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid
p.i. post injection
PEG Polyethylene glycol
PET Positron emission tomography
SPECT Single-photon emission computed tomography
SPPS Solid phase peptide synthesis
TETA 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid
TFA Trifluoroacetic acid