Development of Fluorophore-Labeled Thailanstatin Antibody-Drug

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Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies Chethana Kulkarni, James E. Finley, Andrew J. Bessire, Xiaotian Zhong, Sylvia Musto, and Edmund Idris Graziani Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00718 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Bioconjugate Chemistry

Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies (Submitted for consideration as a full-length Article in Bioconjugate Chemistry) Chethana Kulkarni,†,┴ James E. Finley,‡ Andrew J. Bessire,§ Xiaotian Zhong,║ Sylvia Musto,¶ and Edmund I. Graziani†,* †

Oncology Medicinal Chemistry, ‡Drug Safety Research & Development, and §Pharmacokinetics, Dynamics, & Metabolism, Pfizer Worldwide R&D, Groton, Connecticut 06340, United States

║

Global Biotherapeutics Technologies, Pfizer Worldwide R&D, Cambridge, Massachusetts 02139, United States

¶

Oncology Research Unit, Pfizer Worldwide R&D, Pearl River, New York 10965, United States

*Corresponding Author: [email protected]

ABSTRACT As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolism—processes that are critical to ADC efficacy and toxicity—imaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a “clickable” ADC bearing an azidefunctionalized linker-payload (LP) poised for “click” reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems.

INTRODUCTION Antibody-drug conjugates (ADCs) constitute a promising and rapidly growing area of targeted therapeutics for oncology. By combining the specificity and desirable pharmacokinetic profile of antibodies with the potency of cytotoxic small molecules, ADCs provide an approach to cancer treatment that may be more effective than current standards of care. Presently, there are two FDA-

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approved ADCs available to patients, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), with an additional 50 ADCs in different phases of clinical development.1,2 The vast majority of these conjugates employ microtubule inhibitors, which show very good cytotoxicity in rapidly proliferating cells, but are less effective against cells that divide slowly. For the current study, we have focused on the thailanstatin class of compounds, which act by inhibiting the spliceosome3,4 and show excellent potency in both proliferating and non-proliferating cancer cells. In addition to possessing a differentiated mechanism-of-action, thailanstatins are effective against some multidrug-resistant cells.5 The relative efficacy—and toxicity—of a given ADC depend largely on where and when the payload is released; these factors, in turn, depend on the uptake, trafficking, and catabolism of the ADC. Numerous questions remain about these critical processes and the ultimate fate of payload drugs in tissues, cells, and plasma. To help address such questions, imaging studies have been performed with antibodies and ADCs labeled with fluorophores6–9 or PET reagents10–12. While these studies have been very informative in elucidating trafficking pathways, concerns persist regarding the impact of the label itself on the structure and properties of the antibody or ADC under investigation. Therefore, for the current work, we have used bioorthogonal “click” chemistry, which allows for in situ labeling with exquisite selectivity in biological systems13 (Figure 1A). Here, we describe the development and evaluation of a clickable thailanstatin ADC (1) bearing a minimally modified, click-chemistry-enabled linker-payload; this ADC may be reacted with a derivatized fluorophore to yield the “pre-clicked” ADC (2) (Figure 1B). Beyond the current study, the methods described here could be applied to other molecules in order to study ADC trafficking and facilitate the design of more effective ADCs.


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Figure 1. (A) An ADC that is click-enabled (“clickable”) may be reacted with a derivatized probe before or after cellular uptake; the term “pre-clicked” refers to click reaction of an ADC with a probe prior to treatment in cells. (B) For this study, the clickable ADC (1) was prepared by labeling a mAb with maleimide-AF488, followed by conjugation to the Azido-Lysine-Thailanstatin clickable LP; the pre-clicked ADC (2) was prepared by reacting ADC 1 with BCN-PEG-TAMRA.

RESULTS AND DISCUSSION



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When designing a linker-payload (LP) capable of participating in bioorthogonal reactions, we chose to incorporate an azide handle, which is significantly smaller than most fluorophores. Azides react with terminal14,15 or strained16 alkynes to yield a highly stable triazole product, or with phosphines via the Staudinger ligation, even in aqueous conditions17. The strain-promoted azide-alkyne cycloaddition reaction has proven particularly useful for many live-cell labeling applications18–20, an important feature for our intended studies of ADC trafficking. Furthermore, the azide group is small and stable, and, like strained alkynes, it is not naturally present in biological systems21. For our clickable LP, we chose to incorporate the azide into the linker region, rather than the payload, to minimize perturbation of the drug itself while allowing for facile substitution of other drugs in place of thailanstatin in future studies. We also intended for the azide group to remain with the payload species throughout ADC catabolism, so that a fluorophore covalently attached to the azide would serve as a marker for payload localization. Briefly, to prepare the clickable LP, the N-hydroxy succinimidyl (NHS)-ester of thailanstatin was reacted with azido-lysine in the presence of Hunig’s base. The resulting free acid was activated as the pentafluorophenyl (PFP)-ester, yielding the desired PFP-Azido-Lysine-Thailanstatin LP (3) (Scheme 1, SI Figure S1A). Scheme 1. Synthesis of PFP-Azido-Lysine-Thailanstatin Linker-Payload.

As described in the detailed synthetic procedures (SI Materials and Methods), the NHS-activated AzidoLysine-Thailanstatin LP was also prepared and characterized (SI Figure S1B). However, it suffered from rapid hydrolysis to the free acid, rendering it ineffective for antibody conjugation. To prepare the clickable ADC 1, a trastuzumab mutant antibody was labeled and conjugated to the LP 3. We chose to work with trastuzumab for our studies because the structural and biological properties of ADCs generated on this antibody are well-characterized22. The mutant antibody we employed possesses a mutation in the heavy chain allowing for site-specific cysteine conjugation, and a second mutation in the light chain (κD185A) allowing for site-selective lysine conjugation23. After a standard reduction/re-oxidation procedure24, the antibody was first labeled with maleimide-Alexa Fluor 488 (AF488) to enable tracking of the antibody independent from the LP. After purification, this fluorophore-labeled mAb (“Tras-AF488”) was conjugated to the PFP-Azido-Lysine-Thailanstatin LP 3 and purified once more to yield the desired clickable ADC 1 (SI Materials and Methods). Additionally, a portion of the clickable ADC 1 was reacted with BCN-PEG-TAMRA25 to yield the dual-labeled, preclicked ADC 2, which served as an important control in imaging studies. In order to determine fluorophore-antibody ratio and drug-antibody ratio (DAR) values for the ADCs under study, the mAb was characterized by LC-MS prior to any conjugation reactions, again after labeling with maleimide-AF488, after conjugation to the LP 3 to yield the clickable ADC 1, and finally


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after reaction with BCN-PEG-TAMRA for preparation of the pre-clicked ADC 2. (Representative mass spectra are presented in SI Figure S2A, SI Figure S3A, SI Figure S4A, and SI Figure S5A, respectively.) Comparison of the spectral masses to the expected masses (SI Table S1) revealed fluorophore-antibody ratio and DAR values of 2. Importantly, the mass spectra for pre-clicked ADC 2 show that the clickable LP reacted with BCN-PEG-TAMRA in quantitative yield, indicating that the azide moiety is accessible in the intact ADC. Characterization by size-exclusion chromatography (SEC) confirmed that no aggregation occurred at any stage (SI Figures S2B, S3B, S4B, and S5B). With the clickable ADC 1 and pre-clicked ADC 2 in hand, we sought to assess the in vitro activity of the ADCs in target-expressing cells. In particular, we were interested in learning more about the potential impact of the azide linker, the TAMRA label, and the AF488 label on ADC efficacy. Measurement of IC50 values in the Her2-expressing N87 cell line showed very good potency for clickable ADC 1, comparable to the potency exhibited by similar thailanstatin conjugates5, thus suggesting that the azide moiety does not impair ADC activity. In contrast, the pre-clicked ADC 2 is approximately 200-fold less active than clickable ADC 1 with equivalent DAR values (Table 1), indicating that the TAMRA label does interfere with ADC function. Table 1. In Vitro Potency of ADCs IC50 in N87 cells (nM) ADC Description DAR

IC50 in HT29 cells (nM)

Clickable ADC 1

2.0

1.61

> 1000

Pre-Clicked ADC 2

2.0

> 300

> 1000

Clickable ADC, no mAb label

1.7

5.80

> 1000

Pre-clicked ADC, no mAb label

2.1

> 300

> 1000

Importantly, the maleimide-AF488 label has no significant impact on ADC activity, with the measured IC50 of the clickable ADC lacking the AF488 tag observed within threefold of the parent compound, 1. In negative control HT29 cells, which do not express Her2, all ADCs yielded IC50 values greater than 1000 nM, demonstrating, as expected, that activity observed in N87 cells specifically depends on targetmediated uptake of the ADC (SI Materials and Methods). As noted above, one of our design goals for the clickable LP 3 was for the azide group, and any fluorophore attached to it, to remain with the payload, thus serving as a marker for payload localization in cells. This design goal is consistent with previously reported observations for ADCs bearing a thailanstatin payload conjugated directly to lysine with no linker; specifically, the released species for such ADCs is a thailanstatin-lysine covalent adduct, resulting from full ADC catabolism.5 In order to identify the released species after catabolism for the ADCs in the current work, a lysosomal degradation assay was performed.26 Briefly, clickable ADC 1 and pre-clicked ADC 2 were each incubated in human liver S9 fraction, and samples were removed and quenched after 24 hr and 48 hr for LC-MS analysis. The results revealed that after 24 hr, the released species for the LP in both ADCs does include the azide handle, whether unreacted (ADC 1) or reacted with BCN-PEG-TAMRA (ADC 2) (Figure 2, SI Figures S6A, S6B, S6C, S6D, and S6E). While it was not possible to directly measure the cytotoxicity of these released species, owing to their low cell permeability, we hypothesize that the additional steric bulk of the TAMRA fluorophore in the released species from the pre-clicked ADC 2 likely interferes with binding to the SF3b subunit of the spliceosome.



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Figure 2. Lysosomal degradation assays revealed that the released species from both the clickable ADC 1 and the pre-clicked ADC 2 is the Lys-capped intact linker-payload.

Negative control reactions in which the S9 fraction was omitted show no signal for either ADC, confirming that degradation is dependent on the presence of the S9 lysosomal enzymes (SI Figure S6F). Additionally, the Cys-capped AF488 released species yielded by degradation of the mAb was identified for both ADCs (SI Figure S6G); negative control reactions again confirmed that the released species is observed only when the S9 fraction is present (SI Figure S6H). With the clickable ADC 1 and the pre-clicked ADC 2 fully characterized, confocal imaging studies were initiated with Her2-expressing SKOV3 cells. Time-course imaging experiments showed that both ADCs bind to the cell surface within 30 min; partial uptake was observed within 4 hr, and complete uptake was visible by the 24-hr mark (Figure 3).


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Figure 3. SKOV3 cells were treated with (A) clickable ADC 1 or (B) pre-clicked ADC 2 and imaged on a confocal microscope. Live-cell imaging after 30 min, 4 hr, or 24 hr of ADC treatment revealed binding within 30 min and robust uptake within 24 hr for both ADCs. A high degree of overlay of the pre-clicked ADC 2 mAb and payload signals is visible at all 3 time-points. (mAb is labeled with AF488; payload is labeled with TAMRA for pre-clicked ADC 2; scale bar in panel (A) applies to all images.)

These studies also suggest that the low activity associated with the pre-clicked ADC 2 is not caused by poor ADC uptake; indeed, these results are consistent with our hypothesis that the reduced activity of the pre-clicked ADC in N87 cells is a result of the intrinsic reduced binding of the released species to its intracellular target. Notably, the merged images for ADC 2 show complete overlay of the antibody and



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payload signals at all time-points. Although it is not possible to conclude from these data that the antibody and payload components remain covalently attached, it is clear that they remain co-localized. Next, we sought to address a key question in our system: whether the clickable ADC 1 can participate in the strain-promoted click reaction in live cells. After 24 hr of treatment with ADC 1 or the negative control, Tras-AF488, SKOV3 cells were exposed to BCN-PEG-TAMRA, washed, and imaged (Figure 4A, 4B).

Figure 4. SKOV3 cells were treated for 24 hr with (A) clickable ADC 1 or (B) Tras-AF488. Live-cell imaging after click reaction with 10 µM BCN-PEG-TAMRA showed a strong TAMRA fluorescence signal for cells treated with clickable ADC 1 and a low background signal for negative control cells treated with Tras-AF488. (C) TAMRA fluorescence signal was quantified across 3 independent fields of view: for each field, the green and red channels were separated, and for each colored image, the average signal intensity was determined in MetaMorph. For each field, the average red signal was divided by the average green signal in order to normalize for the amount of ADC internalized by the cells in that field. (mAb is labeled with AF488; payload is reacted with BCN-PEG-TAMRA; scale bar in panel (A) applies to all images.)


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As evidenced by the corresponding confocal images, clickable ADC 1 reacts in situ and selectively with the cyclooctyne dye, BCN-PEG-TAMRA. Quantitative analysis across independent fields of view showed a 12-fold difference in average TAMRA signal between ADC 1 and the negative control TrasAF488 after treatment with BCN-PEG-TAMRA (Figure 4C). After establishing that live-cell labeling of ADC 1 could be achieved, we undertook time-course imaging studies to examine similarities and differences in cellular uptake between Tras-AF488, ADC 1, and ADC 2. We were also interested in the overlay of these three labeled molecules with a lysosomal signal, because the ultimate fate of most molecules that undergo endocytosis is degradation in the lysosome.27 Fluorophore-labeled dextran molecules have long been used as markers for endocytosis and, depending on the incubation time, for the lysosome in particular. To validate Dextran-Alexa Fluor 647 (Dextran-AF647) as a lysosomal marker in our system, we compared it with LAMP1 antibody staining in immunocytochemistry (ICC) studies and found a high degree of overlay between the signals (SI Figure S7). For time-course experiments with Tras-AF488, ADC 1, and ADC 2, we focused on mAb/lysosome co-localization. Quantitative analysis of microscope images collected across independent experiments showed that both ADCs traffic very similarly to one another, and both exhibit greater and more rapid net internalization than Tras-AF488 (Figure 5). These results suggest that ADCs 1 and 2 preferentially undergo targeting to the lysosome versus recycling to the cell surface, relative to the parent antibody. Given that the pre-clicked ADC 2 is internalized and reaches the lysosome at a similar rate to the clickable ADC 1, the reduced activity of 2 relative to 1 could also result from the inability of the released species of 2 to escape the lysosome and enter the cytoplasm. We therefore set out to determine whether the ultimate fate of the released species from each ADC could be determined.

Figure 5. SKOV3 cells were treated with Tras-AF488, clickable ADC 1, or pre-clicked ADC 2 and imaged on a confocal microscope at the following time-points: 30 min, 4 hr, 8 hr, 16 hr, and 24 hr. For each time-point after 30 min, cells treated with clickable ADC 1 were also reacted with BCN-PEG-TAMRA. All cells were treated with Dextran-AF647 for lysosomal visualization and with Hoechst stain for nuclear visualization. (A) All images were analyzed in MetaMorph to determine mAb/lysosome co-localization values, which were normalized by



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number of lysosomal puncta per field of view to account for slight differences in uptake of Dextran-AF647. n=6 independent fields of view collected across 2 independent experiments. (B) Representative images are presented for cells treated with clickable ADC 1 and Dextran-AF647 (mAb is labeled with AF488 and depicted in green; lysosomes are visualized with Dextran-AF647 and depicted in magenta; scale bar in 30-min image applies to all images; note that 405-nm signal corresponding to Hoechst staining is not shown in these images so as to improve clarity of mAb and lysosome signals). See SI Materials and Methods for complete description of treatment of cells, image acquisition, and data analysis.

We thus performed extended time-course studies, in which SKOV3 cells were treated for 96 hr with Tras-AF488, ADC 1, or ADC 2, then imaged (SI Figure S8). We observed significant persistence of the AF488 signal for all three molecules, and a high degree of overlay between the antibody (AF488) and payload (TAMRA) components of ADC 2. The persistence of the TAMRA signal in the lysosome even after 96 hr suggests that the presence of the fluorophore tag within the released species of ADC 2 could inhibit uptake by amino acid (lysine) transporters in the lysosomal membrane. Based on the total set of imaging results, we hypothesize that the large difference in activity between ADC 1 and ADC 2 is likely due to differences in lysosomal escape and/or target engagement of the released species for each ADC. Future studies employing more specialized experimental strategies, such as a cell-free spliceosomal assay or sophisticated fluorescence microscopy/electron microscopy instrumentation, could shed light on the validity of these hypotheses. In summary, we designed and developed a novel thailanstatin-based ADC bearing a clickreactive linker-payload to enable in situ fluorophore labeling of the ADC. A unique double-mutant trastuzumab antibody allowed for independent labeling of the mAb with an AF488 label at a Cys site, and conjugation to the azide-containing, PFP-activated thailanstatin LP at a Lys site. In in vitro activity studies, the clickable ADC 1 showed very good potency in Her2-expressing cells, but addition of a BCN-PEG-TAMRA label to ADC 1, yielding ADC 2, caused a 200-fold loss of activity. This large difference in activity underscores the importance of examining the effect that a fluorophore label may have on a mAb or ADC, particularly in the LP portion. Live-cell imaging studies showed robust uptake of both ADCs, and quantitative image analysis showed greater net internalization of the ADCs versus Tras-AF488, as evidenced by lysosomal overlay. Importantly, the ability of the clickable ADC 1 to react with a cyclooctyne probe even within the complex cell environment suggests that a similar, clickchemistry-based approach could be followed to facilitate imaging studies of other ADCs as well. As such, the methods developed in these proof-of-principle studies could be useful for gaining insight into ADC uptake and trafficking behaviors in different contexts, with the ultimate goal of designing more effective ADCs.

MATERIALS AND METHODS Please see the Supporting Information file for a complete description of all experimental procedures.

SUPPORTING INFORMATION DESCRIPTION


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Supporting Information. Detailed Materials and Methods; characterization data for linkerpayload, mAbs, and ADCs; spectral data for lysosomal degradation assay; additional confocal imaging results. (“Brief statement in non-sentence format listing the contents of the material supplied as Supporting Information.”)

AUTHOR INFORMATION Corresponding Author: Edmund Graziani *Email: [email protected] Mail: 445 Eastern Point Rd Pfizer Mail Stop 8220-3524 Groton, CT 06340 Phone: (860) 715-6768 Present Addresses ┴ New England Biolabs, 240 County Road, Ipswich, MA 01938, United States Notes The authors declare the following competing financial interest(s): All authors are or were employees of Pfizer when the research was conducted.

ABBREVIATIONS • • • • • • • • •

ADC: antibody-drug conjugate BCN: bicyclononyne ICC: immunocytochemistry LP: linker-payload mAb: monoclonal antibody NHS: N-hydroxy succinimidyl PEG: polyethylene glycol PFP: pentafluorophenyl TAMRA: tetramethyl-6-carboxyrhodamine

ACKNOWLEDGEMENTS We thank Chris am Ende for providing BCN-TAMRA, Jesse Teske for synthetic guidance, and Sujiet Puthenveetil, Nathan Tumey, Jeff Casavant, Colleen Doshna, and Frank Loganzo for helpful advice. C. K. was supported by the Pfizer Worldwide Research & Development Post-Doctoral Fellowship Program.



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Development of Fluorophore-Labeled Thailanstatin Antibody-Drug

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