Conjugation Effects on AntibodyâDrug Conjugates - American

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Conjugation effects on antibody-drug conjugates: Evaluation of interaction kinetics in real-time on living cells Sina Bondza, Jonas Stenberg, Marika Nestor, Karl Andersson, and Hanna Björkelund Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500379d • Publication Date (Web): 24 Sep 2014 Downloaded from http://pubs.acs.org on September 29, 2014

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Molecular Pharmaceutics

Conjugation effects on antibody-drug conjugates: Evaluation of interaction kinetics in real-time on living cells Sina Bondza1, Jonas Stenberg1,2, Marika Nestor1,3, Karl Andersson1,2, Hanna Björkelund1,2*

1. Section of Biomedical Radiation Sciences, Department of Radiology, Oncology and Radiation Science, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden 2. Ridgeview Instruments AB, Vänge, Sweden 3. Section of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala University, SE-751 85 Uppsala, Sweden

*

Corresponding author Biomedical Radiation Sciences Uppsala University Dag Hammarskjölds väg 20 SE-751 85 Uppsala, Sweden Phone: +46 708 927 856 Fax: +46 18 471 3432 Email: [email protected] 1 ACS Paragon Plus Environment


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Table of Contents Graphic

Abstract Antibody-drug conjugates (ADC) have shown promising effects in cancer therapy by combining the target-specificity of an antibody with the toxicity of a chemotherapeutic drug. As the number of therapeutic antibodies is significantly larger than those used as ADCs, there is unused potential for more effective therapies. However, the conjugation of an additional molecule to an antibody may affect the interaction with its target, altering association rate, dissociation rate, or both. Any changes of the binding kinetics can have subsequent effect on the efficacy of the ADCs, thus the kinetics are important to monitor during ADC development and production. This paper describes a method for the analysis of conjugation effects on antibody binding to its antigen, using the instrument LigandTracer and a fluorescent monovalent anti-IgG binder denoted FIBA, which did not affect the interaction. All measurements were done in real-time using living cells which naturally expressed the antigens. With this method the binding profiles of different conjugations of the therapeutic anti-EGFR antibody cetuximab and the anti-CD44v6 antibody fragment AbD15171 were evaluated and compared. Even comparatively small modifications of cetuximab altered the interaction with the epidermal growth factor receptor (EGFR). In contrast, no impact on the AbD15171 – CD44v6 interaction was observed upon conjugation. This illustrates the importance to study the binding profile for each ADC combination, as it is difficult to draw any general conclusion about conjugation effects. The modification of interaction kinetics through conjugation opens up new possibilities when optimizing an antibody or an ADC, since the conjugations can be used to create a binding profile more apt for a specific clinical need.

Keywords • • • • • • •

Antibody-drug conjugates Cetuximab EGFR CD44v6 Real-time Cell assay Kinetics 2 ACS Paragon Plus Environment

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Introduction Over the last decades antibody-drug conjugates (ADC) have evolved as new therapeutic options for cancer treatment. These drugs combine the specificity of an antibody with the efficacy of a chemotherapeutic drug. By targeting antigens that are over-expressed on tumor cells the risk of the ADC binding healthy cells is reduced, thus minimizing side-effects.1, 2 Two ADCs are currently in clinical use. Brentuximab vendotin specifically targets CD30 expressed on activated T-cells and B-cells through the antibody brentuximab, and has been approved for treatment of relapsed CD30 positive Hodgkin Lymphomas, as well as systemic anaplastic large cell lymphoma.3 Trastuzumab emtansine is directed towards HER2 (also denoted erbB-2 or neu) and is used in the treatment of HER2-expressing metastatic breast cancer. In both cases, the complete ADC is internalized upon antibody binding. Once inside the cell, vendotin and emtansin bind to tubulin, thereby disrupting the microtubule network.4-6 Furthermore, trastuzumab in itself is approved as a therapeutic antibody against HER2 positive breast cancers and provides additional effects through disruption of downstream signaling, causing reduction of proliferation and angiogenesis.7 A similar approach for targeting tumor cells is radioimmunotherapy, where the antibody is conjugated to a radioactive nuclide. 90Y-labeled ibritumomab tiuxetan and 131I-labeled tositumomab bind the surface protein CD20 on B-cells and are approved for Non-Hodgkin-Lymphomas.8-11 The radionuclide can in these cases activate cell death, similar to a cytotoxic drug. Antibodies with conjugated radionuclides also have an important application in clinical imaging, to detect primary tumors and metastases and for molecular sub characterization.12, 13 To determine the proper dosage of an ADC it is crucial to know its biodistribution and uptake in malignant cells in relation to healthy cells, since this gives an estimation of treatment efficacy and severity of potential side-effects.14 Both aspects could be strongly influenced by the kinetic properties of the ADC. This raises the question if the covalent binding of a comparatively small molecule to an antibody has the potential to change its kinetic properties and thereby its biodistribution and safety profile. The binding of an ADC can be studied through saturation measurements15 or real-time detection technology such as surface plasmon resonance (SPR) technology.16 Saturation measurements have the advantage of being conducted on living cells, but are end-point measurements that lack the time resolution. The information obtained is therefore limited to the binding affinity. Kinetic information about the association and dissociation rate can instead be extracted from SPR data, but these measurements are done in cell free systems with a recombinant target molecule immobilized to a solid support. An alternative method to evaluate the impact of drug conjugation on antibody binding properties is LigandTracer®, which can detect fluorescent or radioactive labels. All measurements are done in realtime with living cells, making it possible to study the kinetic properties while resembling in vivo situations, with the target antigen naturally expressed on the cell surfaces. This generates information rich binding traces with curvatures reflecting the kinetic properties. By fitting the data to an interaction model, the association rate constant ka (M-1s-1), the dissociation rate constant kd (s-1) and the affinity parameter KD (M; the ratio between kd and ka) can be extracted.17 Additionally, measurements with LigandTracer do not contain the blind spots associated with most end-point assays, since the binding is monitored as it occurs, thus reducing the risk of unknown error sources. To evaluate how conjugations can affect antibody binding kinetics, the interaction between the monoclonal human-mouse chimeric antibody cetuximab18 and the epidermal growth factor receptor (EGFR) naturally expressed on tumor cells was selected as a model system. Cetuximab inhibits EGFR 3 ACS Paragon Plus Environment


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and is approved for treatment of EGFR expressing metastatic colorectal cancer19 and advanced squamous cell carcinoma of the head and neck (HNSCC) 20. The development of resistance is common in cancers being treated with cetuximab and new combinations of cetuximab with cytotoxic drugs are currently investigated.21-23 In this study, cetuximab was conjugated to various molecules, such as a radionuclide, a fluorophore and biotin, all of which were used as substitutes for drugs. The binding of cetuximab was measured through the use of a fluorescently labeled monovalent anti-IgG affibody (denoted FIBA, short for Fluorescent IgG Binding Affibody molecule, in this paper). This ensured that the antibody itself remained unmodified and that any observed effects with conjugated cetuximab was due to the conjugation. Since FIBA was small (7 kDa) and only bound to the Fc domain it was considered to have a negligible impact on the binding properties of cetuximab. Additionally, the monovalent nature of the affibody – antibody binding reduced the likelihood of cluster formation which is otherwise a concern when using secondary antibodies. The two conjugations observed to elicit most impact on the cetuximab – EGFR interaction were also tested on the Fab fragment AbD15171, to elucidate whether the observed effects could be observed in a different model system. AbD15171 interacts with CD44v6, a splice variant of the cell adhesive protein CD44 that is considered a promising target for HNSCC.24 CD44v6 is commonly not an internalizing receptor25, 26 and a previous study demonstrated the absence of internalization with A431 cells, the cell line used in this study.27 AbD15171 is one of several fragments found to bind to CD44v6 in a previous screening.28 Since the binding kinetics of the AbD15171 – CD44v6 interaction are known to be significantly faster than the cetuximab – EGFR interaction it was speculated that any change in association or dissociation rate would be easily detectable. This is in contrast to the much slower interactions often observed for therapeutic antibodies,29, 30 for which changes in the binding curvature due to altered kinetics would be less pronounced. This paper describes a new and convenient way of detecting how conjugation of antibodies can affect their binding kinetics in a cell environment, through real-time measurements in LigandTracer and by the use of the monovalent secondary molecule FIBA. The method was used to investigate to what extent an antibody can be altered with conjugation and it was further quantified what these effects correspond to from a time perspective, i.e how the drug-target residence time was affected.

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Experimental Section LigandTracer technology The interactions between cetuximab and EGFR or AbD15171 and CD44v6, both expressed on living A431 cells, were measured in real-time using LigandTracer Green (Ridgeview Instruments AB, Vänge, Sweden). The technology and validation of LigandTracer instruments has been described in detail previously.17, 31, 32 The method requires that targets, in these case receptors expressed on adherent cells, are located on a limited area of a petri dish. The dish is placed on an inclined, rotating support and a buffer containing a labeled ligand (e.g. an ADC) is added to the dish. By continuously comparing the signal of the cell area from the background signal of a cell free area of the dish, a reference subtracted binding curve is created which depicts the amount of bound ligand over time (Fig. 1). All measurements in this paper were done using a blue (488 nm) – green (535 nm) detector, which can detect Alexa 488 and FITC, if not stated otherwise.

Figure 1. Outline of the LigandTracer technology. Cells expressing target receptors are grown on a limited area of a dish, which is placed on an inclined rotating support. Labeled ligand is added, which can bind to the receptors. The signal from the cell area is subtracted with the signal from a background area each rotation, to correct for background fluorescence.

Cell Culture The human squamous carcinoma cell line A431 (CLR 1555, ATCC, Manassas, VA, USA) was cultivated in Ham’s F10 media (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (Sigma Life Science, St. Louis, MO, USA), 2 mM L-glutamine, 100 IU/ml Penicillin and 100 µg/ml Streptomycin (Biochrom AG) and kept in a humidified incubator at 37 °C and 5 % CO2. Cells were seeded at least one day before measurements on local areas in cell culture dishes (NunclonTM, Size 100x20, NUNC A/S, Roskilde, Denmark), by keeping the dishes tilted until the cells had adhered. The medium was replaced with fresh medium and the dishes placed horizontally, to ensure protein coating of all plastic surfaces. All measurements were performed at approximately 100 % confluency.

Labeling of anti-IgG affibody to create FIBA FIBA was produced by labeling the monovalent anti-human lgG affibody® molecule (Cat. No. ab31900, Abcam, Cambridge, UK; binding to human IgG1, IgG2 and IgG4) with a fluorophore. The N-terminal cysteine residue of the affibody was reduced by dissolving 100 µg of the affibody molecule in 300 µl reduction buffer (50 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, pH 7.5) and adding the reducing agent dithiothreitol (DTT) to a concentration of 20 mM. The solution was 5 ACS Paragon Plus Environment


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incubated for 45 min at 37 °C and excess DTT was then removed by purification through a NAP-5 column (Cat. No. 17-0853-02, GE Healthcare, Waukesha, WI, USA). Twenty-fold molar excess of Alexa Fluor 488 C5 Maleimide (Cat. No. A-10254, Life Technologies, Carlsbad, CA, USA) dissolved in DMSO was added drop wise, followed by 2 h incubation at room temperature. Excess fluorophore was removed using another NAP-5 column. Cetuximab (Apoteket, Stockholm, Sweden) was incubated with FIBA over-night at 4 °C prior to each measurement. The concentrations during incubation for cetuximab and FIBA were 130 nM and 520 nM respectively.

Measurement of the FIBA – cetuximab interaction The interaction between cetuximab and FIBA was studied by coating cetuximab to a local area of a polystyrene dish (Cat. No 254925, Nunc, Roskilde, Denmark). The antibody was allowed to adhere overnight and the dish was then washed twice with PBS to remove unbound antibody. The binding of 26.4 nM FIBA in PBS containing 1 % BSA was monitored in real-time in LigandTracer Green. To study the dissociation rate of FIBA from cetuximab, some conjugation measurements (see below) were conducted with FIBA included in the dissociation phase, at the same concentration as during the association phase. These curves were compared to measurements without FIBA in the dissociation phase.

Cetuximab conjugation Cetuximab was incubated with 100 ng of Texas® Red flurophore (Invitrogen, Carlsbad, CA, USA), 100 per µg of cetuximab, in borate buffer pH 9 for 90 min at 37 °C. The labeled protein was purified through a NAP-5 column for removal of unbound fluorophore. Texas Red targets primary amines, i.e. lysines. Tyrosines on cetuximab were labeled with 125I using the chloramine-T protocol.33 In brief, cetuximab was incubated with 125I (0.1-0.3 MBq per µg cetuximab; PerkinElmer, Waltham, MA, USA) and cloramine-T (Sigma, St Louis, MO, USA) for 5 min on ice. The reaction was stopped with sodium metabisulfite (Aldrich, Stockholm, Sweden) and the solution purified through a NAP-5 column to remove excess 125I. For biotinylation, cetuximab was diluted in 100 mM sodium phosphate buffer pH 7.7 to a concentration of 0.2 mg/ml and mixed with ten-fold molar excess of Sulfo-NHS-LC-biotin or NHSbiotin (dissolved in MQ or DMSO respectively; both from ProteoChem, Cheyenne, Wyoming, USA). The mixtures were incubated for 60 min at room temperature and purified through NAP-5 columns. For experiments performed with bound streptavidin (Molecular Probes Inc., Eugene, OR, USA), biotinylated cetuximab was incubated over-night in PBS at 4 °C with four fold molar excess in regard to the amount of biotin that was used for biotinylation.

AbD15171 conjugation AbD15171 (AbD Serotec, Kidlington, UK) was labeled with FITC (Sigma Aldrich, St. Louis, MO, USA) using the same protocol as for Texas Red with cetuximab. Some of the FITC-conjugated 6 ACS Paragon Plus Environment

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AbD15171 was additionally labeled with either Texas Red or 125I, as described above. Conjugated fragments were stored as aliquots in -20 °C until usage.

Analyzing conjugation effects in real-time The interaction between cetuximab and EGFR expressed on A431 cells was measured in real-time using LigandTracer Green. The measurements started with a 30 min baseline measurement, to detect the background fluorescent intensity of the dish prior to addition of cetuximab and to correct for any deviation in the plastic surface of the dish. Cetuximab pre-incubated with FIBA was added step-wise, to a concentration of 2.2 nM and 6.3 nM cetuximab respectively, and measured for 3.5 h for each concentration. The concentrations were selected to obtain a clear curvature during incubation and a visible signal increase upon addition of the higher concentration, which are beneficial for an accurate kinetic evaluation. 34The solution was then replaced with fresh medium and the measurement continued over-night, to detect the dissociation of cetuximab from EGFR. The measurements of the AbD15171 – CD44v6 interaction on A431 cells was performed in a similar fashion, but using 9 and 27 nM AbD15171 for 3 h each. All measurements were performed at room temperature (approximately 22 °C) to reduce possible internalization of EGFR or CD44v6. The interactions were monitored in at least duplicates for each conjugate.

Binding measurements of cetuximab to confirm conjugation Control experiments were conducted to ensure that 125I, Texas Red, Sulfo-NHS-LC-biotin and NHSbiotin had been conjugated to cetuximab. For 125I and Texas Red, the binding of cetuximab to EGFR on A431 cells were monitored in LigandTracer Grey (detecting the gamma radiation of 125I) or in LigandTracer Green with a Yellow (590nm) - Red (632nm) detector (detecting Texas Red). Cetuximab conjugated with Sulfo-NHS-LC-biotin or NHS-biotin was pre-incubated with Texas Red labeled streptavidin (Amersham Biosciences, Little Chalfont, UK) and measured with the same Yellow – Red detector. All measurements were done in the absence of FIBA.

Acid wash measurements to estimate membrane bound and internalized 125I-EGF Internalization of cetuximab was quantified with an acid wash measurement, as described previously.35 In short, A431 cells were incubated with 6.3 nM 125I-cetuximab for 4 h at 37 °C. Cell surface associated 125I-cetuximab was stripped from the membrane by treating the cells with 0.2 M Gly-HCl pH 2.5 containing 0.15 M NaCl and 4 M urea for 6 minutes at 4 °C. 1 M NaOH was added and incubated at 37° C for at least 1 h to fully disrupt the structure of the cells, thus enabling collection of internalized 125I-cetuximab. The activity from the membrane bound and internalized cetuximab proportions were measured using an automatic gamma counter (1480 WIZARD™ 3”, PerkinElmer, Waltham, MA, USA).

Data analysis and statistics The shapes of the real-time binding curves produced in LigandTracer were compared in the evaluation software TraceDrawer 1.6 (Ridgeview Instruments AB, Vänge, Sweden). The curves were normalized 7 ACS Paragon Plus Environment


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to compensate for any differences in signal height caused by e.g. variations in cell number. This allowed a clear visual presentation of differences in binding kinetics, observed as variation in curvature. The signal at the end of the first incubation phase was set to 100 % and the curves scaled accordingly. For examples of curves without normalization, see Supplemental Data S1. Through kinetic fitting using a OneToOne model (corresponding to one monovalent ligand binding to one receptor) for the Cetuximab – EGFR interaction and a OneToTwo model (one monovalent ligand binding to two type of receptors) for the AbD15171 – CD44 v6 interaction, information about the dissociation equilibrium constant KD (corresponding to the affinity), the association rate constant ka and the dissociation rate kd were obtained. Extracted kinetic parameters, i.e. ka, kd and KD, were pair-wise compared using the Student’s t-test.


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Results Analysis of the FIBA – cetuximab interaction The interaction between FIBA and cetuximab was characterized by coating cetuximab to a polystyrene dish and monitoring the binding of FIBA in real-time in LigandTracer. It was found that the FIBA – cetuximab interaction reached equilibrium within two hours using 26.4 nM FIBA (Fig. 2A). After 2.5 hours of FIBA incubation the signal started to decrease, indicating that the rotation of the dish caused cetuximab to detach from the polystyrene surface (Fig. 2A, t > 3 h). This required that the length of the measurements with coated cetuximab had to be kept at minimum, thus it was difficult to include a dissociation measurement of several hours. The rate of FIBA dissociating from cetuximab was instead studied in the conjugation measurements of cetuximab, described in more detail below. In short, A431 cells were incubated with a FIBA-cetuximab solution in LigandTracer, to monitor the binding of cetuximab to EGFR. After 7 h, the solution was replaced with fresh cell culture media, enabling the detection of the dissociation of cetuximab from EGFR. In a subset of measurements the concentration of FIBA was kept constant throughout the run, by adding additional FIBA prior to the dissociation phase (black, Fig. 2B). The binding curves were compared to curves with no FIBA present during the dissociation measurement (grey, Fig. 2B). By keeping the FIBA concentration constant, the black curve displayed only the dissociation of cetuximab from EGFR, in contrast to the grey curve that also included the dissociation of FIBA from cetuximab. These curves were highly similar, suggesting that the interaction between FIBA and cetuximab is stable and that the dissociation of FIBA from cetuximab can be neglected. An equivalent test was done with 125I-cetuximab, with the same conclusions.

Figure 2. Analysis of the FIBA – cetuximab interaction. A) Binding of 26.4 nM FIBA to cetuximab coated to a polystyrene dish. The signal reached equilibrium after 2 h and later started to decrease. B) Dissociation measurement of FIBA bound cetuximab from EGFR on A431 cells (starting at t = 7 h), in the presence (black) and absence (grey) of FIBA in the solution. Curves were normalized to 100 % at t = 3.5 h.



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Binding measurements to confirm conjugation Control experiments were conducted to establish whether the conjugation of cetuximab had been successful. For 125I- and Texas Red-cetuximab this was done by directly monitoring the label, with a gamma detector in LigandTracer Grey (125I) and a Yellow (590nm) - Red (632nm) detector in LigandTracer Green (Texas Red). The biotinylated cetuximab was pre-incubated with Texas Red labeled streptavidin, binding biotin, and monitored in a similar fashion. A clear binding of cetuximab to A431 cells was observed for all conjugations, in the absence of FIBA (see Supplemental Data, S2). This confirmed the success of conjugation in all cases.

Internalization of cetuximab The internalization of EGFR on A431 cells was measured to access its potential impact on kinetc measuerements. After four hours of incubation at 37 °C, 12 % of the cell associated cetuximab was internalized and 88 % still bound to the cell membrane. Thus it was concluded that when kinetic measurements are conducted at room temperature (approx. 22 °C) the internalization of EGFR can be neglected.

Conjugation effects on cetuximab Cetuximab was pre-incubated with FIBA and added to A431 cells for a real-time detection of the cetuximab – EGFR interaction in LigandTracer at room temperature. Each measurement consisted of two consecutive association phases, where the antibody was incubated at different concentrations. By including two concentrations, the kinetic information that could be obtained was increased. All measurements ended with a dissociation phase, where the incubation solution was replaced with medium to follow the residence time. The cells remained adherent to the plastic surface throughout the measurements, as verified by counting cells in the solutions and through visible inspection with a microscope, thus indicating that the cells handled the assay conditions well without any decline in viability. Repeated measurements with unconjugated cetuximab (i.e. unmodified cetuximab with bound FIBA) produced highly similar results, indicating that the system was reproducible (Fig. 3A). The data was fitted to a OneToOne kinetic model, describing one monovalent ligand binding to one target, to obtain information about the kinetic properties such as the association rate constant ka, the dissociation rate constant kd and the equilibrium dissociation constant KD, corresponding to the affinity (Table 1). No unspecific binding of FIBA to A431 cells was observed, as studied in the absence of cetuximab (Fig. 3B, grey). Conjugation of the fluorophore Texas Red to lysines on cetuximab resulted in reduced interaction rates with EGFR, observed as a slower association and dissociation (Fig. 3C, Table 1). The radioactive nuclide 125I had an opposite effect. 125I-cetuximab bound to EGFR with a significantly faster association rate, clearly reaching equilibrium during the 3.5 h incubation of the higher concentration. The impact on the off-rate was even larger and approximately half of the antibody dissociated over the first eight hours (Fig. 3D). All in all, the conjugation of the radioactive nuclide reduced the affinity of the cetuximab – EGFR interaction more than one order of magnitude (Table 1).


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Figure 3. The cetuximab – EGFR model system and conjugation effects. All measurements included a stepwise increase of the cetuximab concentration: 2.2 nM at t = 0 h and 6.3 nM at t = 3.5 h. In A, C and D, cetuximab was pre-incubated with FIBA, which was also included in the medium during the association phase. A dissociation measurement, without cetuximab or FIBA, was started at t = 7 h. Curves were normalized to 100 % at t = 3.5 h. A) Five curves from independent measurements depicting the interaction between unconjugated cetuximab and EGFR B) The binding of 26.4 nM FIBA (grey) or cetuximab pre-incubated with FIBA (black) to EGFR on A431 cells. C) The binding of unconjugated cetuximab (black) and Texas Red labeled cetuximab (grey) to EGFR on A431 cells. The interaction was monitored with a blue (488 nm) – green (535 nm) detector which did not register the Texas Red signal. D) The binding of unconjugated cetuximab (black) and 125 I-cetuximab (grey) to EGFR on A431 cells.

Cetuximab was conjugated to biotin to further investigate how the coupling of molecules can alter binding characteristics. The water soluble Sulfo-NHS-LC-biotin had a small, but visible, effect on the cetuximab – EGFR interaction, reducing the affinity with a factor of four (Fig. 4A, Table 1). When including streptavidin the effect was more pronounced, with an estimated KD value almost ten times higher than with unconjugated cetuximab (Table 1). This lower affinity was also reflected in the binding curve, where the signal increased more for the second concentration of cetuximab than with unconjugated cetuximab, suggesting that EGFR was less saturated after the first concentration (Fig. 4B). The non-water soluble NHS-biotin affected the cetuximab – EGFR interaction, reducing the affinity with almost one order of magnitude (Fig. 4C, Table 1). Streptavidin did not seem to have much additional impact on the interaction (Fig. 4D). Overall, the association rate was slower and the dissociation rate faster in all measurements with biotin, with or without streptavidin.



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Figure 4. The effect of biotin on the cetuximab – EGFR interaction. Cetuximab pre-incubated with FIBA was stepwise increased to 2.2 nM (t = 0 h) and 6.3 nM (t = 3.5 h). A dissociation measurement was started at t = 7 h. The figures shows representative curves of 2-5 measurements per conjugation. Black: Unconjugated cetuximab. Grey: Cetuximab conjugated with Sulfo-NHS-LC-biotin (A, B) or NHS-biotin (C, D). In B and D cetuximab was pre-incubated with streptavidin, which remained in the medium during the association measurement. Curves were normalized to 100 % at t = 3.5 h.


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Table 1. Summary of the kinetic constants of the cetuximab – EGFR interaction for different conjugates of cetuximab.

Unconjugated Texas Redc 125 I Sulfo-NHS-LC-biotin Sulfo-NHS-LC-biotin + SAd NHS-biotin NHS-biotin + SA

ka (M-1s-1) 7.5±0.3*104 5.5±0.3*104 8.6±1.0*104 6.1±1.3*104 3.8±0.1*104 4.8±0.8*104 4.5±1.4*104

kd (s-1) 1.1±0.4*10-6 2.5±2.1*10-7 2.4±0.4*10-5 3.5±1.7*10-6 4.3±0.4*10-6 4.0±1.3*10-6 4.3±0.6*10-6

KD (M) 1.5±0.4*10-11 4.9±3.8*10-12 2.8±0.5*10-10 5.7±1.5*10-11 1.1±0.1*10-10 8.7±3.9*10-11 1.0±0.4*10-10

t1/2 (h)a 175 770 8 55 44 48 44

nb 5 3 3 2 3 3 2

a

t1/2 = ln(2)/kd = the half-life of the interaction, i.e. the time for half of the antibodies to dissociate. n = number of measurements. c The kd value for Texas Red is below the detection limit in this assay. d SA = streptavidin. b

The estimations of ka and kd of all conjugates are presented as mean value ± standard deviation in Table 1, which also include the affinity (KD = kd/ka) and the half-life of the complex, i.e. the theoretical time for half of the antibodies to dissociate from EGFR (t1/2 = ln(2)/kd).36 The dissociation rate for Texas Red is below the detection limit in this assay, resulting in less accurate estimation of kd. All values are significantly different from unconjugated cetuximab (p < 0.05, Student’s t-test), apart from ka of 125I. When presenting the estimated kinetic data in the log(ka)/log(kd) space of an on-off rate plot, it is clear that the different conjugates have little effect on the association rate constant ka, but sometimes a distinct effect on the dissociation rate constant kd (Fig. 5). The dissociation rate constant can vary almost two orders of magnitude as a result of the different conjugations in this particular case.

Figure 5. On-off rate plot depicting the kinetics of the cetuximab – EGFR interaction with different conjugations of cetuximab: A) Unconjugated, B) Texas Red, C) 125I, D) Sulfo-NHS-LC-biotin E) Sulfo-NHS-LC-biotin + streptavidin, F) NHS-biotin, G) NHS-biotin + streptavidin.

Conjugation effects on the AbD15171 – CD44v6 interaction Based on the findings with cetuximab, Texas Red and 125I were selected as the conjugations with highest potential to alter a binding. The interaction between the Fab fragment AbD15171 and CD44v6, known to have a higher association and dissociation rate than the cetuximab – EGFR interaction, was selected to study if the same effects could be observed in a different model system. The fragment was monitored through the detection of FITC, which was directly labeled to AbD15171 since it lacked an Fc domain for FIBA to bind to. The binding of 9 nM (0-3 h) and subsequently 27 nM (3-6 h) AbD15171 to CD44v6 on A431 cells was detected, followed by a dissociation measurement where the unbound antibody fragment was removed and replaced with fresh medium (t = 6 h). In contrast to 13 ACS Paragon Plus Environment


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cetuximab, neither the fluorophore Texas Red nor the radionuclide 125I had a strong impact on AbD15171 (Fig. 6). The antibody fragment bound the CD44v6 with a fast on – fast off manner and most of the protein had dissociated from the target within a few hours. The data did not fit the OneToOne model. Instead, the interaction seemed to have a heterogeneous behavior where AbD15171 could bind to the cells in two different manners. A OneToTwo model fitted the data well and it was observed that the curves were to 75 % the result of a fast-off interaction with an affinity of 60 nM and to 25 % of a slow-off interaction with a KD of 11 nM, with no significant differences between conjugations (p < 0.05).

Figure 6. The effect of 125I and Texas Red on the AbD15171 – CD44v6 interaction. FITC-labeled AbD15171 was stepwise increased to 9 nM (t = 0 h) and 27 nM (t = 3 h). A dissociation measurement was started at t = 6 h. The figure shows representative curves of 3-5 measurements per conjugation. Black: FITC-AbD15171. Light grey: FITC-AbD15171 conjugated with Texas Red. Dark grey: FITC-AbD15171 conjugated with 125I.


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Discussion This paper describes how to monitor changes in binding kinetic properties of antibodies conjugated with small molecules, through the detection of the antibody – antigen interaction in real-time using LigandTracer. The method was applied to the cetuximab – EGFR and AbD15171 – CD44v6 interactions. In order to mimic the in vivo situation more closely, interactions were measured on living cells. This separates the method in this paper with biophysical methods such as SPR which is done with isolated recombinant proteins. On cells the receptor is present in its native form and other factors, such as additional proteins expressed on the cell surface, can have an impact that is relevant and should be taken into consideration. Differences in results from cell assays and SPR data are therefore possible and have been observed previously, with as much as 10-100 times slower interactions on cells.28, 37 When predicting and improving the pharmacokinetic properties of a drug it is important to have knowledge about the on and off rates, since the binding kinetics can provide a more detailed description of the interaction behavior than the affinity alone.38, 39 For example, the time for a biological effect to remain is generally associated with the drug-target residence time, i.e. the dissociation rate.40 Similarly, information about the association rate is relevant for drugs with a high clearance.. All measurements in this paper were done at room temperature to minimize the small, but existing, internalization of cetuximab. Although internalization is crucial for ADC efficacy and sometimes important for radioimmunotherapy, measurements in the absence of internalization enables the study of the association and dissociation rates alone and thus makes it possible to draw conclusions about any changes in the binding characteristics. Information about the binding kinetics is still highly relevant since the ADC must remain bound long enough to trigger internalization. Once the effects on the antibody-target interaction have been established it may however be beneficial to continue the evaluation at 37 °C. A novel FIBA concept was used to follow the binding of cetuximab in a manner that circumvents the need for direct labeling of the antibody. FIBA bound to the Fc domain of the antibody and was expected to have minimal effect on the binding properties. There is however always a risk of affecting the system when including a secondary reagent such as FIBA. To investigate this thoroughly, measurements were conducted to analyze the FIBA – cetuximab interaction. Based on the measurement with FIBA binding to a cetuximab coated dish, it was decided that cetuximab would be pre-incubated with FIBA solution over-night. This ensured that equilibrium was reached prior to the real-time interaction measurements, avoiding any additional binding of FIBA during the cell association of cetuximab. It was observed that the FIBA-cetuximab was highly stable by adding FIBA in the dissociation phase of some measurements, thus any dissociation of FIBA from cetuximab could be neglected. All in all, it was concluded that FIBA has little or no impact on the real-time binding curvature and is thus suitable for the comparison of antibody conjugates. Its binding ability to any antibody with a human IgG Fc region makes it appropriate for a wide range of ADCs. The kinetic constants of the interactions were estimated by fitting the real-time data to a kinetic model. The EGFR expression of A431 is approximately 2 million receptors per cell, according to previous measurements.41 Based on this information, together with cell size and assuming that EGFR are evenly distributed over the cell surface, it was estimated that the receptor density was too low for the cetuximab – EGFR interaction to be bivalent, i.e. cetuximab rarely bound two EGFR at once. Thus, the OneToOne model in TraceDrawer was considered valid and more suitable for the binding of cetuximab than the more complex model for bivalent interactions. 15 ACS Paragon Plus Environment


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When comparing the different conjugations of cetuximab, variations in association as well as in dissociation rates were observed. Some conjugations modified the cetuximab – EGFR interaction to a low extent, such as Sulfo-NHS-LC-biotin, whereas Texas Red caused more pronounced effects. Worth noting is that the estimated kd value of the Texas Red-conjugate corresponds to a half-life of hundreds of hours, thus the dissociation measurement of eight hours was not long enough for an accurate estimation of kd, as indicated by the high standard deviation. Difficulties with accurate estimations of slow off-rates is not unique for LigandTracer, but a problem shared by most instruments detecting protein interactions in real-time. To adapt to the slow dissociation rate of many modern high-affinity binders, LigandTracer detects interactions over several hours which enables the estimation of dissociation rate constants as low as in the order of 1*10-6. This can be compared with most other assays where detection is done over a few minutes, causing a limitation of approximately 1*10-5.42 An accurate estimation of the dissociation rate can provide information about the in vivo behavior for slow interactions where the dissociation rate is the rate limiting step in the body. This is in contrast to the dissociation rates identified with biophysical methods in cell free systems, which are often much faster than the pharmacokinetic elimination from blood and therefore less relevant.43 Apart from the antibody and the cytotoxic drug, the linker is the third component of an ADC. To mimic the use of different linkers, cetuximab was conjugated with the water soluble Sulfo-NHS-LCbiotin and the non-water soluble NHS-biotin, both of which targets primary amines on lysines. NHSbiotin altered the interaction between cetuximab and EGFR to a greater extent than Sulfo-NHS-LCbiotin. This indicates that also changes in the linker have the potential to change the kinetic properties of the ADC. The choice of linker has previously been found important in other aspects as well, such as drug release, compound stability44 and drug resistance mechanisms.45 In an attempt to illustrate the effect of conjugation in a different molecular system, the anti-CD44v6 antibody fragment AbD15171 was conjugated to Texas Red and 125I. The binding of AbD15171 was monitored by labeling the fragment with FITC, since no monovalent anti-Fab molecule was found commercially available and a secondary antibody was considered a risk due to cluster effects. This FITC-label was included and detected also for the Texas Red and 125I conjugates. The two types of interactions that were observed during measurements could be the result of the FITC labeling, which may have caused a damaged subpopulation or increased the amount of aggregates, as speculated previously.46 All AbD15171 conjugates had highly similar binding characteristics to CD44v6, which is surprising in the light that a similar antibody fragment (AbD15179) was observed to be sensitive to conjugation in a previous study.46 This variation in conjugation sensitivity may be explained by the different binding profiles of AbD15171 and AbD15179, where AbD15171 has a faster and more heterogeneous binding to CD44v6 than AbD15179.28 Texas Red was conjugated to lysines just like FITC, thus it is possible that the effects of Texas Red was too similar to the FITC effects to be distinguishable, or the degree of Texas Red incorporation low since some of the lysines were occupied. 125I on the other hand was linked to tyrosines and should not be affected by the FITC labeling. From the cetuximab measurements it was observed that the method described in this paper is sensitive and that changes in kinetic rate constants of less than half an order of magnitude can be detected. The authors therefore believe that the risk of missing any changes in binding properties due to a potential effect of FITC is low, in particular if caused by 125I. The minimal differences between AbD15171 conjugates show that each case is unique and that confirmation of the binding characteristics of conjugated proteins has to be done in each particular case. 16 ACS Paragon Plus Environment

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From the study described in this paper it is clear that the conjugation of molecule to an antibody can affect its binding properties, but that this differs between antibodies and conjugation type. The authors are aware that the conjugations described in this paper are crude models for the sometimes highly controlled conjugations of ADCs, but nevertheless the fact remains that conjugations can modify binding properties, also in controlled cases to specific amino acids.47, 48 Detecting the binding of ADCs to its antigen in real-time is a powerful way of investigating if the kinetic profile has been altered and to pinpoint in what way. This kind of measurement is also relevant in a large scale production line, where the conjugation of a drug or a radionuclide can result in variability between batches. Including a real-time interaction study as a quality control of the kinetics will most probably increase patient safety and the likelihood of correct dosage. A similar quality assurance assay has been described previously, which instead focused on the quantification of biologically active antibody concentrations.49 Important to remember is that changes in binding properties do not necessarily have to be unfavorable. The alteration of the structure of an antibody through conjugation can instead be an advantage. For example, the slower dissociation observed for Texas Red labeled cetuximab may be beneficial for e.g. cancer drugs where a prolonged residence time is often preferable. In other cases a more rapid dissociation rate may be optimal, such as in imaging where the agent should be excreted from the body after the analysis has been done. In other words, the conjugation of an antibody, with drugs or radionuclides, can be done to obtain the most suitable kinetics for a specific clinical setting. This is comparable to the chemical modifications done to optimize biodistribution, by for example reducing renal or hepatic uptake.50, 51 To conclude, ADCs have the potential to improve the effect of a toxic drug while at the same time reducing the risk of hazardous side effects on healthy tissue. However, it should be noted that even comparatively small modifications can change the kinetic properties of an antibody, as shown with cetuximab when binding to EGFR on living cells. This may alter the efficacy and safety of the ADC, but these changes can also lead to favorable improvements of the kinetic properties. Therefore it might be worth testing several alternative conjugations to determine the compound with the best kinetic profile, instead of simply searching for the compound with the smallest difference compared to the unmodified antibody. This paper describes a method for monitoring the interaction in real-time on living cells, which provided detailed information about how the binding pattern of the conjugated antibody had been altered and to what extent.



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References 1.

Scott, A. M.; Wolchok, J. D.; Old, L. J. Antibody therapy of cancer. Nat Rev Cancer. 2012, 12, 278-287.

2.

Sievers, E. L.; Senter, P. D. Antibody-drug conjugates in cancer therapy. Annu Rev Med. 2013, 64, 15-29.

3.

de Claro, R. A.; McGinn, K.; Kwitkowski, V.; Bullock, J.; Khandelwal, A.; Habtemariam, B.; Ouyang, Y.; Saber, H.; Lee, K.; Koti, K.; Rothmann, M.; Shapiro, M.; Borrego, F.; Clouse, K.; Chen, X. H.; Brown, J.; Akinsanya, L.; Kane, R.; Kaminskas, E.; Farrell, A.; Pazdur, R. U.S. Food and Drug Administration approval summary: brentuximab vedotin for the treatment of relapsed Hodgkin lymphoma or relapsed systemic anaplastic large-cell lymphoma. Clin Cancer Res. 2012, 18, 5845-5849.

4.

Krop, I.; Winer, E. P. Trastuzumab emtansine: a novel antibody-drug conjugate for HER2positive breast cancer. Clin Cancer Res. 2014, 20, 15-20.

5.

Deng, C.; Pan, B.; O'Connor, O. A. Brentuximab vedotin. Clin Cancer Res. 2013, 19, 22-27.

6.

Oki, Y.; Younes, A. Brentuximab vedotin in systemic T-cell lymphoma. Expert Opin Biol Ther. 2012, 12, 623-632.

7.

Albanell, J.; Codony, J.; Rovira, A.; Mellado, B.; Gascon, P. Mechanism of action of antiHER2 monoclonal antibodies: scientific update on trastuzumab and 2C4. Adv Exp Med Biol. 2003, 532, 253-268.

8.

Jacobs, S. A. Yttrium ibritumomab tiuxetan in the treatment of non-Hodgkin's lymphoma: current status and future prospects. Biologics. 2007, 1, 215-227.

9.

William, B. M.; Bierman, P. J. I-131 tositumomab. Expert Opin Biol Ther. 2010, 10, 12711278.

10.

Rizvi, S. N.; Visser, O. J.; Vosjan, M. J.; van Lingen, A.; Hoekstra, O. S.; Zijlstra, J. M.; Huijgens, P. C.; van Dongen, G. A.; Lubberink, M. Biodistribution, radiation dosimetry and scouting of 90Y-ibritumomab tiuxetan therapy in patients with relapsed B-cell non-Hodgkin's lymphoma using 89Zr-ibritumomab tiuxetan and PET. Eur J Nucl Med Mol Imaging. 2012, 39, 512-520.

11.

Ivanov, A.; Krysov, S.; Cragg, M. S.; Illidge, T. Radiation therapy with tositumomab (B1) anti-CD20 monoclonal antibody initiates extracellular signal-regulated kinase/mitogenactivated protein kinase-dependent cell death that overcomes resistance to apoptosis. Clin Cancer Res. 2008, 14, 4925-4934.

12.

van Dijk, L. K.; Hoeben, B. A.; Kaanders, J. H.; Franssen, G. M.; Boerman, O. C.; Bussink, J. Imaging of epidermal growth factor receptor expression in head and neck cancer with SPECT/CT and 111In-labeled cetuximab-F(ab')2. J Nucl Med. 2013, 54, 2118-2124. 18 ACS Paragon Plus Environment

Page 18 of 29

Page 19 of 29

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


13.

Goetz, M.; Hoetker, M. S.; Diken, M.; Galle, P. R.; Kiesslich, R. In vivo molecular imaging with cetuximab, an anti-EGFR antibody, for prediction of response in xenograft models of human colorectal cancer. Endoscopy. 2013, 45, 469-477.

14.

Teicher, B. A.; Chari, R. V. Antibody conjugate therapeutics: challenges and potential. Clin Cancer Res. 2011, 17, 6389-6397.

15.

McDonagh, C. F.; Kim, K. M.; Turcott, E.; Brown, L. L.; Westendorf, L.; Feist, T.; Sussman, D.; Stone, I.; Anderson, M.; Miyamoto, J.; Lyon, R.; Alley, S. C.; Gerber, H. P.; Carter, P. J. Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index. Mol Cancer Ther. 2008, 7, 2913-2923.

16.

Golfier, S.; Kopitz, C.; Kahnert, A.; Heisler, I.; Schatz, C. A.; Stelte-Ludwig, B.; MayerBartschmid, A.; Unterschemmann, K.; Bruder, S.; Linden, L.; Harrenga, A.; Hauff, P.; Scholle, F. D.; Muller-Tiemann, B.; Kreft, B.; Ziegelbauer, K. Anetumab Ravtansine - a Novel Mesothelin-Targeting Antibody-Drug Conjugate Cures Tumors with Heterogeneous Target Expression Favored by Bystander Effect. Mol Cancer Ther. 2014.

17.

Björkelund, H.; Gedda, L.; Andersson, K. Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context. PLoS One. 2011, 6, e16536.

18.

Galizia, G.; Lieto, E.; De Vita, F.; Orditura, M.; Castellano, P.; Troiani, T.; Imperatore, V.; Ciardiello, F. Cetuximab, a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody, in the treatment of human colorectal cancer. Oncogene. 2007, 26, 36543660.

19.

Garrett, C. R.; Eng, C. Cetuximab in the treatment of patients with colorectal cancer. Expert Opin Biol Ther. 2011, 11, 937-949.

20.

Cetuximab approved by FDA for treatment of head and neck squamous cell cancer. Cancer Biol Ther. 2006, 5, 340-342.

21.

Smith, R. A.; Yuan, H.; Weissleder, R.; Cantley, L. C.; Josephson, L. A wortmannincetuximab as a double drug. Bioconjug Chem. 2009, 20, 2185-2189.

22.

Roskoski, R., Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol Res. 2014, 79, 34-74.

23.

Gilabert-Oriol, R.; Thakur, M.; von Mallinckrodt, B.; Hug, T.; Wiesner, B.; Eichhorst, J.; Melzig, M. F.; Fuchs, H.; Weng, A. Modified trastuzumab and cetuximab mediate efficient toxin delivery while retaining antibody-dependent cell-mediated cytotoxicity in target cells. Mol Pharm. 2013, 10, 4347-4357.



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

24.

Kundu, S. K.; Nestor, M. Targeted therapy in head and neck cancer. Tumour Biol. 2012, 33, 707-721.

25.

Nestor, M.; Sundstrom, M.; Anniko, M.; Tolmachev, V. Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas. Nucl Med Biol. 2011, 38, 103-112.

26.

Nestor, M.; Persson, M.; van Dongen, G. A.; Jensen, H. J.; Lundqvist, H.; Anniko, M.; Tolmachev, V. In vitro evaluation of the astatinated chimeric monoclonal antibody U36, a potential candidate for treatment of head and neck squamous cell carcinoma. Eur J Nucl Med Mol Imaging. 2005, 32, 1296-1304.

27.

Haylock, A. K.; Spiegelberg, D.; Nilvebrant, J.; Sandstrom, K.; Nestor, M. In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study. EJNMMI Res. 2014, 4, 11.

28.

Nilvebrant, J.; Kuku, G.; Björkelund, H.; Nestor, M. Selection and in vitro characterization of human CD44v6-binding antibody fragments. Biotechnol Appl Biochem. 2012, 59, 367-380.

29.

Andersson, K.; Björkelund, H.; Malmqvist, M. (2010) Antibody-antigen interactions: What is the required time to equilibrium? Available from Nature Precedings. http://precedings.nature.com/documents/5218/version/1. Accessed 2 April 2014.

30.

Barta, P.; Malmberg, J.; Melicharova, L.; Strandgard, J.; Orlova, A.; Tolmachev, V.; Laznicek, M.; Andersson, K. Protein interactions with HER-family receptors can have different characteristics depending on the hosting cell line. Int J Oncol. 2012, 40, 1677-1682.

31.

Bjorke, H.; Andersson, K. Automated, high-resolution cellular retention and uptake studies in vitro. Appl Radiat Isot. 2006, 64, 901-905.

32.

Bjorke, H.; Andersson, K. Measuring the affinity of a radioligand with its receptor using a rotating cell dish with in situ reference area. Appl Radiat Isot. 2006, 64, 32-37.

33.

Hunter, W. M.; Greenwood, F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962, 194, 495-496.

34.

Onell, A.; Andersson, K. Kinetic determinations of molecular interactions using Biacore-minimum data requirements for efficient experimental design. J Mol Recognit. 2005, 18, 307317.

35.

Björkelund, H.; Gedda, L.; Malmqvist, M.; Andersson, K. Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach. Mol Clin Onc. 2013, 1, 343-352.


Page 20 of 29

Page 21 of 29

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


36.

Hulme, E. C.; Trevethick, M. A. Ligand binding assays at equilibrium: validation and interpretation. Br J Pharmacol. 2010, 161, 1219-1237.

37.

Barta, P.; Andersson, K.; Trejtnar, F.; Buijs, J. Exploring time-resolved characterization of the heterogeneity and dynamics of ligand-receptor interactions on living cells. J Anal Oncol. 2014, 3, 94-104.

38.

Markgren, P. O.; Schaal, W.; Hamalainen, M.; Karlen, A.; Hallberg, A.; Samuelsson, B.; Danielson, U. H. Relationships between structure and interaction kinetics for HIV-1 protease inhibitors. J Med Chem. 2002, 45, 5430-5439.

39.

Andersson, K.; Hamalainen, M. Replacing affinity with binding kinetics in QSAR studies resolves otherwise confounded effects. J Chemometrics. 2006, 20, 370-375.

40.

Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov. 2006, 5, 730-739.

41.

Bjorkelund, H.; Gedda, L.; Barta, P.; Malmqvist, M.; Andersson, K. Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with (1)(2)(5)I-EGF. PLoS One. 2011, 6, e24739.

42.

Drake, A. W.; Myszka, D. G.; Klakamp, S. L. Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods. Anal Biochem. 2004, 328, 35-43.

43.

Dahl, G.; Akerud, T. Pharmacokinetics and the drug-target residence time concept. Drug Discov Today. 2013, 18, 697-707.

44.

Alley, S. C.; Okeley, N. M.; Senter, P. D. Antibody-drug conjugates: targeted drug delivery for cancer. Curr Opin Chem Biol. 2010, 14, 529-537.

45.

Kovtun, Y. V.; Audette, C. A.; Mayo, M. F.; Jones, G. E.; Doherty, H.; Maloney, E. K.; Erickson, H. K.; Sun, X.; Wilhelm, S.; Ab, O.; Lai, K. C.; Widdison, W. C.; Kellogg, B.; Johnson, H.; Pinkas, J.; Lutz, R. J.; Singh, R.; Goldmacher, V. S.; Chari, R. V. Antibodymaytansinoid conjugates designed to bypass multidrug resistance. Cancer Res. 2010, 70, 2528-2537.

46.

Stenberg, J.; Spiegelberg, D.; Karlsson, H.; Nestor, M. Choice of labeling and cell line influences interactions between the Fab fragment AbD15179 and its target antigen CD44v6. Nucl Med Biol. 2014, 41, 140-147.

47.

Altai, M.; Strand, J.; Rosik, D.; Selvaraju, R. K.; Eriksson Karlstrom, A.; Orlova, A.; Tolmachev, V. Influence of nuclides and chelators on imaging using affibody molecules: comparative evaluation of recombinant affibody molecules site-specifically labeled with (6)(8)Ga and (1)(1)(1)In via maleimido derivatives of DOTA and NODAGA. Bioconjug Chem. 2013, 24, 1102-1109.



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

48.

Altai, M.; Perols, A.; Karlstrom, A. E.; Sandstrom, M.; Boschetti, F.; Orlova, A.; Tolmachev, V. Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA. Nucl Med Biol. 2012, 39, 518-529.

49.

Wang, E.; Bjorkelund, H.; Mihaylova, D.; Hagemann, U. B.; Karlsson, J.; Malmqvist, M.; Buijs, J.; Abrahmsen, L.; Andersson, K. Automated functional characterization of radiolabeled antibodies: a time-resolved approach. Nucl Med Commun. 2014.

50.

Rosik, D.; Thibblin, A.; Antoni, G.; Honarvar, H.; Strand, J.; Selvaraju, R. K.; Altai, M.; Orlova, A.; Eriksson Karlstrom, A.; Tolmachev, V. Incorporation of a Triglutamyl Spacer Improves the Biodistribution of Synthetic Affibody Molecules Radiofluorinated at the NTerminus via Oxime Formation with F-4-Fluorobenzaldehyde. Bioconjug Chem. 2013.

51.

Strand, J.; Honarvar, H.; Perols, A.; Orlova, A.; Selvaraju, R. K.; Karlstrom, A. E.; Tolmachev, V. Influence of macrocyclic chelators on the targeting properties of (68)Galabeled synthetic affibody molecules: comparison with (111)In-labeled counterparts. PLoS One. 2013, 8, e70028.


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49x27mm (300 x 300 DPI)

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Figure 1. Outline of the LigandTracer technology. Cells expressing target receptors are grown on a limited area of a dish, which is placed on an inclined rotating support. Labeled ligand is added, which can bind to the receptors. The signal from the cell area is subtracted with the signal from a background area each rotation, to correct for background fluorescence. 50x31mm (600 x 600 DPI)


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Figure 2. Analysis of the FIBA – cetuximab interaction. A) Binding of 26.4 nM FIBA to cetuximab coated to a polystyrene dish. The signal reached equilibrium after 2 h and later started to decrease. B) Dissociation measurement of FIBA bound cetuximab from EGFR on A431 cells (starting at t = 7 h), in the presence (black) and absence (grey) of FIBA in the solution. Curves were normalized to 100 % at t = 3.5 h. 113x157mm (600 x 600 DPI)



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Figure 3. The cetuximab – EGFR model system and conjugation effects. All measurements included a stepwise increase of the cetuximab concentration: 2.2 nM at t = 0 h and 6.3 nM at t = 3.5 h. In A, C and D, cetuximab was pre-incubated with FIBA, which was also included in the medium during the association phase. A dissociation measurement, without cetuximab or FIBA, was started at t = 7 h. Curves were normalized to 100 % at t = 3.5 h. A) Five curves from independent measurements depicting the interaction between unconjugated cetuximab and EGFR B) The binding of 26.4 nM FIBA (grey) or cetuximab preincubated with FIBA (black) to EGFR on A431 cells. C) The binding of unconjugated cetuximab (black) and Texas Red labeled cetuximab (grey) to EGFR on A431 cells. The interaction was monitored with a blue (488 nm) – green (535 nm) detector which did not register the Texas Red signal. D) The binding of unconjugated cetuximab (black) and 125I-cetuximab (grey) to EGFR on A431 cells. 114x75mm (600 x 600 DPI)


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Figure 4. The effect of biotin on the cetuximab – EGFR interaction. Cetuximab pre-incubated with FIBA was stepwise increased to 2.2 nM (t = 0 h) and 6.3 nM (t = 3.5 h). A dissociation measurement was started at t = 7 h. The figures shows representative curves of 2-5 measurements per conjugation. Black: Unconjugated cetuximab. Grey: Cetuximab conjugated with Sulfo-NHS-LC-biotin (A, B) or NHS-biotin (C, D). In B and D cetuximab was pre-incubated with streptavidin, which remained in the medium during the association measurement. Curves were normalized to 100 % at t = 3.5 h. 114x75mm (600 x 600 DPI)



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Figure 5. On-off rate plot depicting the kinetics of the cetuximab – EGFR interaction with different conjugations of cetuximab: A) Unconjugated, B) Texas Red, C) 125I, D) Sulfo-NHS-LC-biotin E) Sulfo-NHSLC-biotin + streptavidin, F) NHS-biotin, G) NHS-biotin + streptavidin. 49x30mm (600 x 600 DPI)


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Figure 6. The effect of 125I and Texas Red on the AbD15171 – CD44v6 interaction. FITC-labeled AbD15171 was stepwise increased to 9 nM (t = 0 h) and 27 nM (t = 3 h). A dissociation measurement was started at t = 6 h. The figure shows representative curves of 3-5 measurements per conjugation. Black: FITCAbD15171. Light grey: FITC-AbD15171 conjugated with Texas Red. Dark grey: FITC-AbD15171 conjugated with 125I. 54x36mm (600 x 600 DPI)


Conjugation Effects on AntibodyâDrug Conjugates - American

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