Influence of the N-Terminal Composition on Targeting

Subscriber access provided by LUNDS UNIV

Article

Influence of the N-terminal composition on targeting properties of radiometal-labeled anti-HER2 scaffold protein ADAPT6. Javad Garousi, Sarah Lindbo, Hadis Honarvar, Justin Velletta, Bogdan Mitran, Mohamed Altai, Anna Orlova, Vladimir Tolmachev, and Sophia Hober Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00465 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Influence of the N-terminal composition on targeting properties of radiometal-labeled antiHER2 scaffold protein ADAPT6. Javad Garousi‡#, Sarah Lindbo†#, Hadis Honarvar‡, Justin Velletta‡, Bogdan Mitran§, Mohamed Altai‡, Anna Orlova§, Vladimir Tolmachev‡*, Sophia Hober† #

These authors contributed equally

‡

Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala, Sweden

†

Department of Protein Technology, KTH - Royal Institute of Technology, SE-10691, Stockholm, Sweden

§

Division of Molecular Imaging, Department of Medicinal Chemistry, Uppsala University, SE-75181, Uppsala, Sweden

*Corresponding author: Prof. Vladimir Tolmachev Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala, Sweden Phone: +46 18 471 34 14 Fax: +46 18 471 34 32 e-mail: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TABLE OF CONTENTS/ABSTRACT GRAPHIC


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT

Radionuclide imaging-based stratification of patients to targeted therapies makes cancer treatment more personalized and therefore more efficient. ADAPTs (ABD Derived Affinity ProTeins) constitute a novel group of imaging probes based on the scaffold of an albuminbinding domain (ABD). To evaluate how different compositions of the N-terminal sequence of ADAPTs influence their biodistribution, a series of human epidermal growth factor receptor type 2 (HER2)-binding ADAPT6 derivatives with different N-terminal sequences were created: GCH6DANS (2), GC(HE)3DANS (3), GCDEAVDANS (4) and GCVDANS(5) and compared with the parental variant: GCSS(HE)3DEAVDANS (1). All variants were sitespecifically conjugated with a maleimido-derivative of a DOTA chelator and labeled with 111

In. Binding to HER2-expressing cells in vitro, in vivo biodistribution as well as targeting

properties of the new variants were compared with properties of the 111In-labeled parental ADAPT variant 1 (111In-DOTA-1). The composition of the N-terminal sequence had an apparent influence on biodistribution of ADAPT6 in mice. The use of a hexahistidine tag in 111

In-DOTA-2 was associated with elevated hepatic uptake compared to the (HE)3-containing

counterpart, 111In-DOTA-3. All new variants without hexahistidine tag demonstrated lower uptake in blood, lung, spleen and muscle compared to the parental variant. The best new variants, 111In-DOTA-3 and 111In-DOTA-5, provided tumor uptakes of 14.6±2.4 and 12.5±1.3 %ID/g at 4 h after injection, respectively. The tumor uptake of 111In-DOTA-3 was significantly higher than the uptake of the parental 111In-DOTA-1 (9.1±2.0%ID/g). Tumor-toblood ratios of 395±75 and 419±91 at 4 h after injection were obtained for 111In-DOTA-5 and 111

In-DOTA-3, respectively. In conclusion, the N-terminal sequence composition affects the

biodistribution and targeting properties of ADAPT-based imaging probes, and its optimization may improve imaging contrast.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION

Targeting of cancer-associated molecular abnormalities is a powerful tool for treatment of disseminated cancer. One way to achieve this is by inhibition of the signaling of overexpressed receptor tyrosine kinases, either by antibodies or by tyrosine kinase inhibitors. A major issue for targeting is the heterogeneity of target expression. Only a part of the tumors may express any given target, and the target expression might change during the course of the disease or in response to therapy.1 Non-invasive visualization of target expression using target-specific companion diagnostic imaging agents might enable stratification of patients for a particular targeted therapy.2 Labeling of therapeutic antibodies with gamma- or positron-emitting radionuclides is a feasible approach for visualization of relevant targets. However, therapeutic antibodies are typically optimized for long residence in circulation. Slow clearance of radiolabeled antibodies from blood and healthy tissues creates a high background, which reduces the sensitivity of the imaging.2 High specificity and sensitivity is an apparent advantage of an imaging companion diagnostic and the use of small proteolytic and engineered antibody fragments has been shown to improve imaging contrast and thereby give the possibility to shorten the optimal time between injection and imaging.3 Theoretical modeling suggested that the tumor accumulation of an imaging agent with high affinity increases with decreased size.4 Indeed, the use of the small immunoglobulin-based imaging agents VHH provided very good contrast images, both in preclinical models and in patients.5,6 However, imaging of small metastases requires extremely high contrast,7 and therefore even smaller targeting molecules than VHH might be needed. Development of such small probes using immunoglobulins is not currently possible. An alternative way for the development of very small binding proteins with high specificity and affinity to desirable targets is to use non-antibody scaffold proteins.8


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Such proteins include a robust amino acid framework providing a constant and stable 3D structure and variable, surface exposed amino acids utilized for selection of binders by molecular display techniques. The scaffold protein approach has enabled the selection of a number of small high-affinity binders. 8 Preclinical experiments have demonstrated that scaffold proteins such as affibody molecules,9 fibronectin domains,10 DARPins,11 knottins,12 and anticalins13 can be successfully used in radionuclide imaging of therapeutic targets. Furthermore, sensitive and specific imaging of human epidermal growth factor type 2 (HER2) using affibody molecules has been demonstrated in clinical studies.14,15 This suggests that the use of non-immunoglobulin scaffold proteins is a promising approach for the development of highly sensitive and specific imaging companion diagnostics probes.

Recently, we have demonstrated the feasibility of radionuclide molecular imaging using a novel type of an engineered scaffold protein, ADAPT (ABD-Derived Affinity ProTein).16,17 ADAPTs are based on the 46-amino acids albumin-binding domain (ABD) of streptococcal protein G.18 This three-helix scaffold provides high stability as well as capacity of highfidelity refolding after thermal denaturation.18 Randomization of 11 surface-exposed amino acids has enabled the development of combinatorial libraries for selection of binders with different specificities. ADAPT clones capable of high affinity binding to cancer-associated proteins such as HER2,19 human epidermal growth factor receptor type 3 (HER3),20 and tumor necrosis factor-α (TNF-α) have been developed.21 In vitro and in vivo studies indicated that one anti-HER2 ADAPT (ADAPT6) labeled with 111In and 68Ga, was able to specifically visualize HER2-expressing xenografts in mice using gamma camera/SPECT and PET.16 Experiences with affibody molecules suggest that the size of the scaffold is essential, but not the only factor that determines the biodistribution of a scaffold protein. Other factors that 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

might affect the biodistribution and pharmacokinetics are for example the composition of the binding site,22,23 presence and composition of a purification tag,24-27 chemical properties of the chelator and its position in the scaffold,28,29 chemical properties of the radionuclide as well as the geometry of the chelator-radionuclide complex.29,30 Systematic studies concerning such influences can be used to identify imaging probes with higher tumor-to-tissue ratios, which consequently enables rational design to generate scaffold proteins with the highest possible sensitivity of imaging. However, due to the relatively recent development of scaffold-based proteins for imaging purposes, the information about the influence of different factors is limited. For example, it has been shown that the use of a hexahistidine purification tag has a negative effect on biodistribution of affibody molecules24,31 and nanobodies.32 In contrast, the use of the more negatively charged histidine-glutamate-histidine-glutamate-histidineglutamate (HEHEHE or (HE)3) tag improved the biodistribution of affibody molecules,25,26 it particularly reduced the hepatic uptake. Reduction of the hepatic uptake when using the (HE)3-tag has also been found for DARPins as well as for two radiolabeled peptides.11,33 However, when ADAPTs, equipped with a hexahistidine- or a (HE)3-tag, were compared in vivo, the advantage of the (HE)3-tagged variant, labeled with 111In, was marginal and the positive effect was only observable shortly after injection.17 One hypothesis explaining the lack of expected effect by the glutamate-containing tag in ADAPT is the composition of the N-terminus of the protein scaffold. When comparing the N-terminal sequences that follow the histidine-containing tags of the affibody molecules and the ADAPTs, the sequence of ADAPT is more negatively charged (DEAVDANS with a net charge of -3.2 (pI 3.44)) than the corresponding sequences of the affibody molecules (AENKFNKE or LQVDNKFN, both with a net charge of -0.2 (pI 6.25) at physiological pH.26 Earlier studies have demonstrated that an increase of negative charge and hydrophilicity of the N-terminus of affibody molecules reduced the hepatic uptake while an increase of positive charge increased the hepatic 6 ACS Paragon Plus Environment

Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


uptake.27,34-36 Thus, the negatively charged N-terminus of the ADAPT might neutralize the effect of the positively charged hexahistidine tag (net charge of 0.5 (pI 7.97)). The goal of this study was to test the hypotheses that: - ADAPT6 variants with shorter N-terminal sequence clear from blood and other tissues more rapidly, - the effect of histidine-containing tags on ADAPTs biodistribution depends on the flanking amino acids; - the amino acid composition of the N-terminal sequence may influence off-target interactions of ADAPTs and modify their biodistribution. For this purpose, a series of ADAPT6 molecules with different N-terminal sequences was created (Figure 1). The rationale was to produce shorter variants, with and without polyhistidine tags, in order to investigate both the impact of the size as well as the charge. All variants underwent site-specific conjugation with a maleimido derivative of DOTA through a cysteine incorporated in the N-terminus and labeled with 111In. The parental variant 111InDOTA-GCSS(HE)3DEAVDANS-ADAPT6 (1) was used for comparison. The influence of the N-terminal sequences on the affinity of labeled ADAPT6 variants to HER2 expressing cancer cell lines and cellular processing of bound conjugates was investigated in vitro, while the biodistribution and targeting properties were evaluated in murine models.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. A. Sequences of ADAPT6 variants evaluated in this study. N-terminal sequences are highlighted by bold font. B. Conjugation of monoamide-maleimido derivative of DOTA to a cysteine.


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION Production, Purification and Conjugation of ADAPT Molecules. A series of five different ADAPT6 variants was produced in Escherichia coli (Figure 1). After incubation of the lysates in 90°C for 10 minutes and a succeeding centrifugation, the proteins were further purified by immobilized metal affinity chromatography (IMAC), with a Ni2+-resin or anion exchange chromatography. All five variants reached a sufficient level of purity, above 90% (data not shown) as determined by analytical reversed-phase high-performance liquidchromatography (RP-HPLC). Attempts to create variants with N-terminal sequences shorter than seven amino acids resulted in negligible levels of soluble proteins, indicating instability of the proteins. We speculate that a certain amount of amino acids are necessary to form the first turn in helix 1.37 This feature has also been observed in other studies concerning this scaffold.38 The chelator DOTA was conjugated to the thiol group of the unique cysteine and the conjugated molecules were successfully purified by RP-HPLC. The purity of all DOTAconjugated proteins was by analytical RP-HPLC confirmed to be more than 95% (data not shown). Analysis by SDS-PAGE also confirmed purity and mass spectrometry analysis confirmed the molecular weights (Table 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 10 of 40

Table 1. Characterization of the ADAPT6 variants. N-terminal sequence

DOTA-GCSS(HE)3DEAVDANS (1) DOTA-GCH6DANS (2) DOTA-GC(HE)3DANS (3) DOTA-GCDEAVDANS (4) DOTA-GCVDANS (5) a

Number of amino acids in Nterminus 18 12 12 10 7

Net charge of Nterminus at pH 7.4 -5.9 -0.6 -3.9 -3.3 -1.3

Theoretical Mw (Da)

Experimental Mw (Da)

Melting temperature (°C)

KD (nM)a

7559.8 6995.3 6971.2 6586.8 6271.6

7560.6 6995.6 6972.1 6587.6 6272.4

61 58 61 57 59

2.7 1.6 2.2 2.1 1.2

determined by SPR.

10

ACS Paragon Plus Environment

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Thermal Stability and Secondary Structure Analysis. The secondary structures of the DOTA-conjugated ADAPT6 variants were measured using circular dichroism (CD) and the new variants showed similar α-helical content as the parental ADAPT6 variant (1) as well as for previously described ADAPT6 variants.16,17 The melting temperatures were high and in the same range for all constructs (Table 1), and all constructs completely refolded after thermal denaturation (Supplemental Figure S1). Biosensor Analysis. Surface plasmon resonance (SPR) was used to determine the equilibrium affinity constants for the interaction between HER2 and the different ADAPT6 variants. The dissociation equilibrium (KD) constants ranged from 1.2-2.7 nM (Table 1), showing minor differences between the parental variant 1 (DOTA-GCSS(HE)3DEAVDANS-ADAPT6) and the new, truncated variants. Thus, the binding ability to HER2 is negligibly affected by the change of length and composition of the N-terminus. Labeling and Stability. The ADAPT6 variants were labeled with 111In in 0.2 M ammonium acetate, pH 5.5, by incubation at 95°C for 35 min. The radiochemical yield was around 99% for all conjugates. A maximum specific activity of 1.2 MBq/µg (7.5-9.1 GBq/µmol) was obtained. Removing traces of free 111In using disposable NAP-5 columns enabled radiochemical purities of 99-100%. All conjugates were stable in vitro, both in PBS and under challenge of 500-molar excess of EDTA (see Table 2).



Table 2. In vitro stability of 111In-labeled ADAPT6 variants. Samples were incubated in PBS or with 500-fold molar excess of EDTA. Protein-associated radioactivity (%) 1h 2h 3h PBS EDTA PBS EDTA PBS EDTA solution solution solution 99.1±0.4 99.9±0.1 100 100 100 100 99±0.1 98.4±0.1 98.6±0.7 99.4±0.1 97.5±0.9 98.6±0.6 100 100 100 100 98.3±0.2 98.7±0.6 100 100 100 100 98.8±1.8 98.4±0.3 100 100 100 100 99.1±0.1 98.4±0.2

111

In-DOTA-1 In-DOTA-2 111 In-DOTA-3 111 In-DOTA-4 111 In-DOTA-5 111

Binding and Processing by HER2-expressing Cells in vitro. The binding of all radiolabeled ADAPT6 variants to HER2-expressing SKOV3 cells was significantly (p

Influence of the N-Terminal Composition on Targeting

Recommend Documents