FRET Reagent Reveals the Intracellular Processing of Peptide-Linked

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A Novel FRET Reagent Reveals the Intracellular Processing of Peptide-linked Antibody-drug Conjugates Byoung-Chul Lee, Cécile Chalouni, Sophia Doll, Sam Nalle, Martine Darwish, Siao Ping Tsai, Katherine R Kozak, Geoffrey Del Rosario, Shang-Fan Yu, Hans Erickson, and Richard Vandlen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00362 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 2, 2018

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Bioconjugate Chemistry

A Novel FRET Reagent Reveals the Intracellular Processing of Peptide-linked Antibody-drug Conjugates Byoung-Chul Lee1,7,#, Cecile Chalouni2,#, Sophia Doll1,2,6, Sam C. Nalle3,7, Martine Darwish1, Siao Ping Tsai4, Katherine R. Kozak4, Geoffrey Del-Rosario5, Shang-Fan Yu5, Hans Erickson1, & Richard Vandlen1* 1

Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA 2 Department of Pathology, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA 3 Department of Cancer Immunology, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA, 4 Department of Biochemical and Cellular Pharmacology, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080 USA 5 Department of Translational Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080 USA 6 Present address: Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82152 Planegg, Germany. 7 Present address: 23andMe, 349 Oyster Point Blvd, South San Francisco, CA 94080 USA # These authors contributed equally to this work. *Correspondence should be addressed to RV ([email protected])

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ABSTRACT: Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.

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INTRODUCTION

Antibody-drug conjugates (ADCs) are emerging as the next generation anti-cancer therapeutic agents. ADCs harness the power of monoclonal antibody specificity to target the delivery of highly potent drugs to the tumor site. Highly cytotoxic payloads are conjugated via linkers to tumor-targeting antibodies with an expectation of improving the efficacy, safety and, therefore, the therapeutic window of the cytotoxic payload. Kadcyla™ and Adcetris™ are two FDA-approved ADCs that have shown remarkable clinical activities at tolerated doses.1-4 Kadcyla™ targets HER2-positive breast cancers with a maytansinoid DM1 conjugate5 and Adcetris™ targets CD30positive Hodgkin lymphoma with a monomethyl auristatin E (MMAE) conjugate.6 FDA approval of these ADCs has drawn significant attention to this therapeutic approach and a large number of ADCs are now being tested in pre-clinical and clinical phases with various target antibodies, different linkers, and various payloads.7-20 Despite the success of ADCs in the clinic, detailed intracellular activation mechanisms of the ADCs are largely unknown. The current understanding of ADC processing is that ADCs get localized and degraded in endosomes and lysosomes of target tumor cells after receptor-mediated endocytosis. The released payloads diffuse into the cytoplasm or nucleus where they interact with their targets.21-23 However, detailed and quantitative assessments of the process of ADC activation are needed to more fully understand the intracellular pathways utilized by the ADCs and to develop more efficacious ADCs. Previous imaging studies of ADC internalization were mostly conducted using fluorescently tagged antibodies.24-26 Since the payloads were not labeled it was not possible to follow when the payload was released and where it went in the cell. By adding FRET reporter groups to the linker on either side of the linker cleavage site of the ADCs, it is possible to follow cellular uptake and linker processing and to track the distributions of both the antibody and released payloads. Upon linker cleavage, donor fluorescence is expected to appear at the cellular location where the released payload is located. We created a series of probes to allow simultaneous tracking of both the antibody and payload. Another FRET probe was created with the addition of the

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cytotoxic drug DM1 to the linker-drug construct. ADCs with this linker-drug were used to verify that the introduction of FRET components did not alter the normal ability of DM1-ADCs to kill tumor cells. We chose to target two growth factor receptors, HER2 and tomoregulin (TenB2 or TMEFF2), with ADCs using two human cancer cell lines, SK-BR-3 and PC3, respectively, to explore the intracellular uptake and processing mechanisms of these ADCs. SK-BR-3 is a HER2-positive breast cancer cell line while PC3 is a prostate cancer cell line. PC3 cell lines have been engineered to express either the prostate cancer-specific growth factor receptor tomoregulin-2 or HER2 at the cell surface.27 We compared the antigen-mediated internalization and processing of TenB2 and HER2 in the same PC3 cell background. In addition, we compared the processing of the same receptor, HER2, in the different cell backgrounds (SK-BR-3 and PC3 cells), to better understand drivers of the antigen-mediated antibody uptake pathway. A brief summary of characteristics for cell lines used in this study is described in Table 1. Using our FRET probes, we were able to quantitatively probe various aspects of intracellular ADC processing including antibody binding to cell surface, linker processing and cytosolic transport of the released payload. Table 1. Characteristics of cell lines used in this study

Cell lines used in this study

Engineered

Cell surface antigen and copy number

Internalization Route

HER2, 2.0 x 106

Recycling

Comments

References

In vitro model for SK-BR-3

TenB2-PC3 HER2-PC3

No

Yes

TenB2, 1.7 x 106

Lysosome

Yes

HER2, 1.0 x 105

Lysosome

32, 34, 35 HER2-positive breast cancer In vitro model for prostate cancer Control for TenB2-PC3

RESULTS

Construction of antibody FRET-peptide conjugates 4


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We synthesized a series of FRET peptides with and without the cytotoxic agent maytansinoid DM1 at the C-terminus of the peptide linkers (Fig. 1, Fig. S1 and Table S1).28 All linkers contained an N-terminal maleimide for selective thiol conjugation to engineered cysteines in the antibodies. The FRET peptide named FRET1 contains DM1 at the C-terminus (Fig. 1). DM1 is a highly potent microtubule inhibitor and has been used widely as a payload in various therapeutic ADCs.18

Figure 1. FRET-labeled antibody conjugates. (a) Construct design. One of the FRET peptides, FRET1, was conjugated to the THIOMABTM antibody. A FRET pair of TAMRA and fluorescein was added to the val-cit linker with C-terminal DM1. In this

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construct, TAMRA quenches the donor fluorescence. Donor fluorescence appears upon linker cleavage. In our imaging experiments, if the TAMRA and fluorescein signals reside in the same pixel, the resultant pixel will show yellow in the overlaid images of the red and green channels. (b) Chemical structure of other FRET peptides. No DM1 was attached to these FRET peptides. These FRET peptides were utilized for the linker cleavage assay. FRET pairs of TAMRA and fluorescein, or TAMRA and Alexa Fluor® 488 were used in these linkers. In addition to the val-cit containing FRET peptides, we synthesized FRET4 and FRET5 peptides with poly-L-alanine and non-natural poly Dalanine linkers as controls, respectively. The FRET5 peptide was used as a negative control for the non-protease cleavable linker. (c and d) The val-cit linker is cleaved by various cysteine cathepsins. The anti-TenB2 FRET2 conjugate was treated with cathepsins for 2 hours at (c) pH 5.0 and (d) pH 7.2. Fluorescence emission from the fluorescein was obtained by excitation at 495 nm. The fluorescence maximum at 525 nm showed the fluorescence recovered by cathepsin-mediated linker cleavage and subsequent de-quenching of the donor fluorescence. The second fluorescence maximum at 580 nm was originated from the excitation of energy-transferred TAMRA. Cysteine cathepsins B, K, L and S, but not the aspartate protease cathepsin D, increased the donor fluorescence at pH 5.0 and 7.2 with a rank order of Cat B ≈ K ≈ S > Cat L.

Other FRET peptides, FRET2 to FRET5, contain only a FRET pair in the linker without the cytotoxic payload. In the FRET1 to FRET3 constructs, a cathepsincleavable dipeptide of valine-citrulline (val-cit) was inserted between a fluorescence donor, fluorescein (or Alexa Fluor® 488) and an acceptor tetramethylrhodamine (TAMRA). The val-cit dipeptide has been used in therapeutic ADCs as a linker to connect cytotoxic drugs to antibodies,18-19 and is sensitive to cleavage by cathepsins in lysosomes.29 In addition, we synthesized FRET4 and FRET5 as controls (Fig 1b). FRET4 contained tetra-alanine residues as a control for non-specific peptide processing and FRET5 contained non-natural tetra-D-alanine residues for the negative control of a non-cleavable peptide linker. The fluorescent acceptor TAMRA efficiently quenches the fluorescence from the donor due to the short distance between the FRET pairs and the overlap of the emission spectrum of fluorescein or Alexa Fluor® 488 with the absorption spectrum of TAMRA. This pair of FRET probes has been utilized for enzyme activity assays.28 The fluorescence signal from the donor is expected to appear upon cleavage of the val-cit linker (Fig 1a).

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We conjugated these FRET peptides to anti-HER2 and anti-TenB2 A118C THIOMABTM antibodies, which have been developed to introduce two engineered cysteines per antibody for selective drug conjugation (Fig. 1a, 1b and Table S1).30,31 Using an anti-TenB2 antibody conjugate with the FRET2 payload, we monitored the fluorescence recovery of the donor and assessed linker cleavage by cathepsins. Cysteine cathepsins B, K, L and S increased the donor fluorescence in pH 5.0 and 7.2 buffers (Fig. 1c and 1d), while no donor fluorescence was detected using a negative control lysosomal enzyme cathepsin D, an aspartate protease which does not cleave the val-cit dipeptide. This demonstrated the sensitivity and selectivity of the linker to cysteine cathepsins. Cathepsin B, K and S were more efficient than cathepsin L at equi-molar concentrations in the linker cleavage assay (Fig. 1c and 1d), suggesting that the sensitivity of the val-cit linker was variable within the family of cysteine cathepsins.

Cytotoxicity of FRET-labeled ADCs in HER2 and TenB2 cancer cell lines To explore processing of FRET-labeled ADCs in cultured cells, we chose two cell lines SK-BR-3, a human breast cancer cell line, and PC3, a prostate cancer cell line, that had been transfected to express high densities of the surface receptors HER2 and TenB2, respectively (see Table 1).32,33 In these cell lines, receptor copy numbers for HER2 and TenB2 in SK-BR-3 and TenB2-transfected PC3 cells were similar with approximately 2.0 x 106 and 1.7 x 106 receptors per cell, respectively.32 However, the copy number of HER2 in HER2-transfected PC3 cell lines was relatively low with approximately 1.0 x 105 receptors per cell.32 In the literature, HER2 and TenB2 have been reported to have different internalization pathways during receptor-mediated endocytosis. TenB2 has a short, 33 amino acid residue intracellular domain while HER2 has a tyrosine kinase domain of 580 amino acids. In SK-BR-3 cells, HER2 has been shown to recycle mostly back to the cell surface during receptor-mediated uptake of the anti-HER2 antibody.34,35 In contrast, the TenB2 antigen proceeded directly to the lysosomal pathway in PC3 cells with minimal recycling.26,27,32 We hypothesized that our novel FRET-labeled ADCs may provide insight on whether these two different internalization pathways impact ADC processing.

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First, we carried out cell potency assays for anti-HER2 and TenB2 FRET-labeled ADCs in our model cancer cell lines SK-BR-3 and PC3 cells in order to verify that the bulky fluorescent dyes in the linker did not alter the normal processing and cellular toxicity activities of DM1-ADCs. The IC50 values of FRET1 conjugates for killing of SK-BR-3 and PC3 cells were 0.19 and 0.73 nM (28.5 and 109.5 ng/ml), respectively (Fig. 2a). The IC50 values of these anti-HER2 FRET1 conjugates were potent and similar to those of other anti-HER2 DM1 conjugates, suggesting that these fluorescent dyes have minimal impact on the cellular activities of ADCs.5,36,38 Since the potency of the FRET1 ADC on the PC3 cells was about 4-fold less than on the SK-BR-3 cells, we tested the free drug potency using S-methyl DM1 on these two cell lines in order to ascertain that the difference in potency was due to inherent drug sensitivity differences. S-methyl DM1 has been used for the DM1 sensitivity assays5,39 and presumably bypasses the lysosomal pathway and enters the cytoplasm, its site of action, directly. The IC50 values of S-methyl DM1 obtained in this study for SKBR-3 and TenB2-PC3 cells were 1.3 and 7.5 nM, respectively (Fig. 2b). The 6-fold difference of free drug potency between SK-BR-3 and TenB2-PC3 cells correlated well to that seen with the FRET1 ADC conjugates, suggesting that the intrinsic DM1 sensitivity of the cells may be responsible for the different ADC potencies seen with the FRET1 conjugates. However, we cannot exclude other possibilities that led to the difference in the ADC potency, such as differences in membrane permeability, antigen density, intracellular processing or endolysosomal permeability of the released payload.

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Figure 2. In vitro activity of FRET-labeled ADCs and free drug. (a) In vitro potency of FRET1 conjugates in SK-BR-3 and TenB2-PC3 cells using anti-HER2 and TenB2 antibody conjugates, respectively. FRET2 conjugates were used as controls. After 3 days of the treatment, Alamar Blue reagent was used to assess cell viability. The IC50 values of FRET-labeled ADCs for SK-BR-3 cells and TenB2-PC3 cells were 0.19 and 0.73 nM (28.5 and 109.5 ng/ml), respectively. (b) S-methyl DM1 potency in SK-BR-3 and TenB2-PC3 cells. The IC50 values of the S-methyl DM1 for SK-BR-3 cells and TenB2-PC3 cells were 1.3 and 7.5 nM, respectively.

Intracellular processing of the linker assessed by flow cytometry In order to quantify the difference in antigen density and intracellular processing of the linker, we utilized our antibody-FRET3 peptide conjugates with the FRET pair TAMRA and Alexa Fluor® 488 without a cytotoxic payload in conjunction with flow cytometric analysis. This design of the antibody conjugates allowed precise monitoring of the linker cleavage without impacting cellular physiology during the experiment. We were able to assess intracellular linker processing quantitatively by measuring the ratio of the fluorescence intensity of the donor from a FRET conjugate to a reference conjugate which had only the fluorescent donor. This ratio is assumed to represent the level of processing of the linker. Previously, radioisotopic labeling of the payload and its extraction with organic solvents from the cell and the culture media have been used to assess the time-dependent intracellular processing of ADCs.37-40 However, our FRET method is easier to use and does not need laborious processes such as organic extraction. Importantly, we can assess the intracellular processing in live cells by flow cytometry or fluorescence microscopy.

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In our flow cytometric assay, the amount of cell-surface bound antibody labeled with Alexa Fluor® 488 was slightly higher in SK-BR-3 cells compared to TenB2-PC3 cells (bottom panels of Fig 3a) as the mean fluorescence intensity of the Alexa Fluor® 488 conjugate on SK-BR-3 (n=2, 12673±779) was higher than that of the Alexa Fluor ® 488 conjugate on TenB2-PC3 cells (n=2, 9390±270). This data is in line with previous calculations of antigen density by cell-based Scatchard analysis as mentioned above.32 However, using the relevant FRET constructs, we found that linker processing is different in SK-BR-3 and TenB2-PC3 cells. While 74% of the linker was cleaved in TenB2-PC3 cells after 20 hours, only 41% of the linker was cleaved in the SK-BR-3 cells (Fig. 3b). These data suggested that TenB2-PC3 cells internalize and cleave a valcit linked ADC relatively faster than SK-BR-3 cells.

Figure 3. Intracellular processing of linkers assessed by FACS and IncuCyte® Zoom. FACS analysis of time-dependent linker cleavage are shown in (a) and (b). In the left panel of (a), SK-BR-3 cells were treated with anti-HER2 FRET3 (top) and Alexa Fluor 488 conjugate as a reference (bottom), respectively. In the right panel of (a), TenB2PC3 cells were treated with anti-TenB2 FRET3 (top) and Alexa Fluor® 488 conjugate as a reference (bottom), respectively. (b) Rate of the linker cleavage in SK-BR-3 cells and TenB2-PC3 cells calculated from the data in (a). The ratio of mean fluorescence intensity of the FACS data (n=2) in (a) between the FRET3 and Alexa Fluor® 488 conjugate was used to calculate the % linker cleavage. (c) Time-dependent intracellular processing of anti-HER2 FRET3, FRET4 and FRET5 conjugates in SK-BR-3 cells was assessed by IncuCyte® Zoom. The cells were treated with the conjugates at 4oC, washed with cell culture media three times and then incubated at 37oC. Fluorescence images were taken at different time points for quantitative analysis. FRET4 contains

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poly L-alanine linker for the control of non-specific peptide processing. FRET5 contains non-natural poly D-alanine linker for the control of non-peptidic cleavage.

In a previous study using Trastuzumab-DM1 (with direct linkage of DM1 to lysine residues on the antibody), complete antibody digestion in a HER2-positive breast cancer cell was necessary to release the payload resulting in approximately 50% release of lysine-DM1 after 20 hours of treatment.37

This rate of payload release was

comparable to the rate of val-cit cleavage observed in our study using SK-BR-3 cells (Fig. 3), suggesting that payload release by the val-cit linker may offer no obvious advantage over release via hydrolysis of the polypeptide backbone in this particular case of anti-HER2 antibody drug conjugates. To further test this hypothesis of non-specific peptide processing, we compared processing of the val-cit linker to poly-D-alanine and poly-L-alanine linkers using antiHER2 conjugates in SK-BR-3 cells. In this study, we conducted quantitative fluorescence imaging with the IncuCyte® Zoom. There was no processing of the nonnatural poly-D-alanine conjugate as expected for the negative control of non-peptide cleavage (Fig. 3c). We found that there was no significant difference of FRET peptide processing between val-cit and poly-L-alanine linker utilizing the FRET3 and FRET4 conjugates in SK-BR-3 cells (Fig. 3c). This data strengthens our hypothesis of general peptide processing in lysosomes. As described above, the intracellular processing of the linker was 1.8 times more efficient in TenB2-PC3 cells than in SK-BR-3 cells. Considering the roughly equivalent amount of cell-surface bound antibody and the difference in observed intracellular processing, we anticipated that TenB2-PC3 cells would be more or equally sensitive to the drug conjugate than SK-BR-3 cells. In fact, the anti-HER2 FRET1 conjugate was 3.8 times more potent on SK-BR-3 cells than the anti-TenB2 FRET1 conjugate in cell proliferation assays, as mentioned earlier (Fig. 2a). Unexpectedly, linker cleavage was not the determining factor for ADC potency in our systems.

Imaging studies for the intracellular payload release

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To gain insight into the details of ADC internalization and the trafficking of the released payload, we carried out live-cell fluorescence imaging using these FRET constructs. We believed that the fluorescent dyes would have a minimal impact on the internalization mechanism of ADCs because the anti-HER2–DM1 FRET1 conjugate was as potent as other DM1 conjugates with a similar range of IC50 values as described above.5,37,38 In less than 5 minutes after cell treatment with the FRET1 conjugates, we observed binding of those ADCs at the cell surface of SK-BR-3 and TenB2-PC3 cells as measured by the red fluorescence from TAMRA (Supporting Movies 1 and 2). In these movies, no measurable green fluorescence above the noise level was detected on or near the cell surface during the course of the live-cell imaging, suggesting that the linker was stable on the cell surface or diluted rapidly into the media. Alexa Fluor® 488-only conjugates, which were used as reference fluorescence for the FACS experiments, continued to show cell surface binding of antibodies in both SK-BR-3 and TenB2-PC3 cells during 15 hours of pulse-chase treatment (Fig. S2a and S2d). In contrast, the cells treated with the FRET3 conjugates had no fluorescence from released Alexa Fluor® 488 at the cell surface in either SK-BR-3 or TenB2-PC3 cells. The fluorescence from the released Alexa Fluor® 488 appeared only in internalized vesicles. However, TAMRA signal was still detected at the cell surface, especially in SK-BR-3 cells (Fig. S2c), suggesting that there are recycling endosomes with antibody still bound to the antigen, but with the linker processed. Release of payload was observed within 20 minutes following incubation, primarily inside endosomal vesicles, in both SK-BR-3 and TenB2-PC3 cells. Simultaneously, an increase of green fluorescence in the cytosol was observed, particularly in SK-BR-3 cells (Supporting Movie 1), showing that the released fluorescent DM1 had traversed to the cytosol. The signal intensity of the green fluorescence in the cytosol increased over time. After 15 hours of treatment, the cytosolic release of fluorescent DM1 was 3-fold more pronounced in SK-BR-3 cells than in TenB2-PC3 cells (Fig. 4). This might suggest that the lysosomal membrane permeability of the payload is intrinsically different between cells, and the permeability is enhanced in SK-BR-3s compared to

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TenB2-PC3s. But we cannot exclude other possibilities such as the difference in drug efflux and cytosolic retention.

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Figure 4. Cytosolic transport of the released payload. FRET1 conjugates were utilized for the live-cell imaging of SK-BR-3 and TenB2-PC3 cells (see the accompanied movie files). Snapshot images are shown here after 15 hours of continuous treatment with the FRET1 conjugates. Arrows are indicated for the representative cytosols that contain fluorescein-labeled DM1. The green fluorescein-labeled DM1 fluorescence only appears upon the linker cleavage, but the fluorescence acceptor red TAMRA yields fluorescence constantly regardless of linker cleavage. For the SK-BR-3 cells treated with anti-HER2 FRET1 conjugate, (a) fluorescence image from green channel and (b) overlaid images of green and red channels are shown. For the TenB2-PC3 cells treated with anti-TenB2 FRET1 conjugate, (c) fluorescence image from green channel and (d) overlaid images of green and red channels are shown as well. (e) Fluorescence intensities (A.U.) of the released payload, the fluorescein-labeled DM1, in the cytosol of SK-BR-3 and TenB2-PC3 cells are compared in this graph. Five snapshots after 15 hours treatment of the FRET-labeled ADC were used to obtain the averaged fluorescence intensity per cell. Note that significant amounts of released payload remained in the intracellular vesicles in TenB2-PC3 cells for an extended period of time as shown in (c), (d) and (e).

There was a remarkable difference in the ADC internalization pathways between SK-BR-3 and TenB2-PC3 cells. In TenB2-PC3 cells, the anti-TenB2 FRET1 conjugates were internalized into small vesicles near the cell membrane. Those became congregated into a perinuclear area during the early stage of uptake of 2 to 3 hours (Supporting Movie 2). After this time, the conjugate-containing vesicles became spread throughout the cytosol and near the plasma membrane (Supporting Movie 2). We observed the same pattern of initial clustering using the anti-TenB2 FRET3 conjugates without the payload (Fig. 5d to 5f). Thus, the clustering of the internalized vesicles at the early stage of antibody uptake was intrinsic to the internal subcellular architecture of the PC3 cells, and probably not due to the disruption of the microtubule network that might have been caused by the DM1 toxin.

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Figure 5. Importance of cell background for antigen-mediated antibody intracellular uptake. Alexa Fluor® 488-labeled anti-HER2 and TenB2 antibodies were utilized for the live-cell imaging of SK-BR-3, HER2-PC3 and TenB2-PC3 cells. Images were taken at (a) 10 min, (b) 1 hr and (c) 15 hrs of treatment of Alexa Fluor® 488-labeled anti-HER2 antibody in SK-BR-3 cells. In the second row, images were taken at (d) 10 min, (e) 1 hr and (f) 15 hrs of treatment of Alexa Fluor® 488-labeled anti-TenB2 antibody in TenB2-PC3 cells. In the third row for HER2-PC3 cells, images were taken at (g) 10 min, (h) 2 hr and (i) 15 hrs of treatment of Alexa Fluor® 488-labeled antiHER2 antibody. The clustered vesicles are indicated by arrows in (e) and (h).

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We attempted to identify the clustered architecture of the internalized vesicles at the 2-3 hr time frame, but there was no co-localization of the anti-TenB2 FRET3 conjugate fluorescence with any of endocytosis markers tested, including EEA1, transferrin receptors, LAMPs and LysoTracker® (data not shown). However, we observed the co-localization of anti-TenB2 FRET3 conjugate with LysoTracker® after the internalized vesicles were spread throughout the cytosol after 15 hrs (Fig. S3b), which is in line with the previous observation that the TenB2 antigen eventually is localized in lysosomes in TenB2-PC3 cells.27,32,36 In contrast, there was no directed clustering of internalized vesicles in SK-BR-3 cells (Fig. 5a to 5c and Supporting Movie 1). We observed co-localization of the anti-HER2 FRET3 conjugates with LysoTracker®, suggesting the conjugate reaches lysosomes (Fig. S3a). The endosomal vesicles in SK-BR-3 cells were spread throughout the cytosol during the course of ADC internalization with a cell surface staining of red TAMRA (Fig. 5a to 5c and Supporting Movie 1), which is consistent with previous studies indicating endosomal recycling of HER2 in SK-BR-3 cells. To address whether this unique internalization pathway in PC3 cells depends on cell background or antigen itself, we carried out live-cell imaging with HER2-transfected PC3 cells. We compared the internalization of TenB2 and HER2 in the same cell background of PC3, and the same antigen HER2 in the different cell backgrounds of SK-BR-3 and PC3. We used the Alex Fluor® 488-only conjugates in these experiments. We found that HER2 was internalized similarly as TenB2 in HER2engineered PC3 cells. Both receptors were initially clustered in the cell perinuclear area with subsequent spreading throughout the cytosol thereafter (Fig. 5g to 5i and Supporting Movie 3). These data suggest that cellular background plays an important role for the antigen-mediated antibody uptake in these cell lines.

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FRET ADCs reagents allow visualization of the payload and antibody components of ADCs in vivo We extended our imaging analyses to mouse xenograft models in order to monitor ADC dynamics in vivo (Fig. 6). TenB2-PC3 cells were injected into CB17 SCID-beige mice and allowed to establish tumors. The mice were then injected intravenously with anti-TenB2 FRET2 or control anti-HER2 FRET2 conjugates. In this mouse xenograft model, we used the FRET2 conjugates without the toxic payload in order to follow the tumor growth and monitor the intracellular processing of the val-cit linker in the xenograft tissue. When the xenograft was analyzed at 4 and 24 hours after i.v. injection of the anti-TENB2 conjugate, we identified yellow vesicles in the overlaid images of red TAMRA and green fluorescein channels, indicating the in vivo cellular uptake of antibody conjugates and linker processing, consistent with the expectation that the antibody conjugate was targeted to the tumor cells and internalized by endocytosis. The conjugate-containing vesicles were co-localized with the lysosomal marker LAMP1 (Fig. S4). There was no sign of antibody internalization and processing in the tumors when the control anti-HER2 FRET2 conjugate was administered in this Ten-B2 PC3 xenograft model. These results demonstrate that the in vitro cell-based model was correlative to the in vivo tumor model for ADC internalization and processing.

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Figure 6. Intracellular uptake and processing of the anti-TenB2 FRET2 conjugate in the mouse TenB2-PC3 xenograft tumors. Fluorescence images of tissue sections are shown from tumors harvested 4 hours (a, b, and c) and 24 hours (d, e, and f) after i.v. injection of the conjugate. Images from green fluorescein (a and d), red TAMRA (d and e) and overlaid (c and f) channels are shown in this figure. Arrows indicate representative vesicles containing both released fluorescein and TAMRA.

DISCUSSION

A multitude of molecular events, e.g., antigen density, antibody affinity, rate of internalization, intracellular pathway of antigen-mediated antibody uptake, levels of processing enzymes, lysosomal permeability of payload, sensitivity of target to drug, etc., are involved in the cellular processing and intracellular activities of ADCs, all of which may influence the cytotoxic potency of toxic payloads. In many cases, the antigen density on the cell surface is important for the potency. However, the molecular event(s) that drive or limit ADC potency might not be the same between different targets. In some cases, it has been reported that the efficacy of some ADCs was not driven by the antigen copy number nor antigen-binding affinity.41-45

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FRET-peptide linked antibody conjugates were developed as tools to investigate the intracellular processing of ADCs in order to better understand cellular factors that influence ADC potency and efficacy. Previous imaging studies of ADC internalization pathways were mostly conducted using fluorescently labeled antibodies.24-26 The ability to track released payloads was not possible with those constructs. By FRET labeling of the linkers and drugs, we could monitor the processing of the linkers and further track the released payloads inside cells. By combining flow cytometry and fluorescence imaging, we were able to study various aspects of the ADC activation process quantitatively. Unexpectedly, linker cleavage was not a determining variable for ADC potency in our systems (Fig. 3). In TenB2-PC3 cells, even though a majority of the linker was cleaved within 1 day of treatment as shown with the anti-TenB2 FRET3 conjugate (Fig. 3), the cytotoxic potency of the anti-TenB2 FRET1 conjugate was less than that of the anti-HER2 FRET1 conjugate when tested on SK-BR-3 cells (Fig. 2a). By studying the activity of the toxic payload, DM1, on both cell types, it was clear that PC3 cells were inherently less sensitive to the action of the drug (Fig. 2b). Thus the rate of linker cleavage in different cells may not directly correlate to the final activity of the payload toxin. We observed a greater cytosolic transport and retention of the released payload in SK-BR-3 cells than in TenB2-PC3 cells (Fig. 4). This enhanced cytosolic retention of the payload in SK-BR-3 cells might be related to the nature of the HER2 receptormediated endocytosis pathway. In SK-BR-3 cells, HER2 antigens were shown to recycle mostly back to the cell surface during receptor-mediated uptake of the antiHER2 antibody.34,35 In contrast, TenB2 antigens do not show as much recycling and they proceed primarily to the lysosomes.27,32,36 It still remains to be resolved whether or not the intracellular uptake pathway matters for the ADC potency. Recently, a specific DM1 transporter SLC46A3 has been identified in lysosomal membranes by Hemblett, et al.46 It is possible that this DM1 transporter enhanced the cytosolic transport of the released DM1 payload specifically in SK-BR-3 cells.

Levels of the

transporter in the engineered PC3 cells is unknown. Interestingly, our study of antigen-mediated antibody internalization suggests that the internalization pathway may depend more on the cellular background than on the

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antigen in our model cell lines (Fig. 5, and supporting Movies 1 to 3). We compared the internalization of TenB2 and HER2 in the same cell background of PC3 cells, and the same antigen HER2 in the different cell backgrounds of SK-BR-3 and PC3 cells. In PC3 cells, HER2- and TenB2-mediated internalization pathways are similar, both showing rapid lysosomal accumulation (Fig. 5 and supporting Movies 2 and 3). However, in SK-BR-3 cells, the HER2-mediated internalization pathway shows substantial differences from the HER2-mediated internalization pathway in PC3 cells (Fig. 5 and supporting Movies 1 and 3). Thus, the cell background plays an important role for the antigen-mediated antibody uptake. Common cell lines like 293 or HeLa cells are often transfected with ADC target antigens. Our study suggests that the antigen-mediated antibody uptake in transfected cells may utilize different pathways compared to cells expressing antigens endogeneously and thus caution needs to be exercised in interpreting internalization data from transfected cells. Recently our FRET technology has been utilized in the clinical development of an antibody-antibiotic conjugate therapeutic for treating intracellular methicillin-resistant staphylococcus aureus. We applied our FRET probe technology to monitor the uptake of an anti-bacterial antibody FRET conjugate bound to bacteria into phagolysosomes of macrophages47 and to observe the kinetics of linker cleavage and subsequent distribution of the released payload. Efficient linker processing was observed in this system,47 providing support for the concept of using antibodies to deliver antibiotics and other drugs that are normally not cell permeable into cells that harbor significant populations of intracellular bacteria. In summary, our FRET ADC technology provides a facile, robust, and non-invasive method for evaluating intracellular ADC processing and has provided new insights into the internalization and trafficking of various cell surface receptors in various cell types. This technology is amenable to utilization in high-throughput assays as well. We believe that our FRET approach can help unravel some of the complexity of the mechanisms of actions of the ADCs and help to develop more efficacious ADCs in the future. An interesting observation in our ADC imaging studies was that a significant amount of released payload remained in the intracellular vesicles, especially in TenB2PC3 cells for an extended period of time (Fig. 4c, 4d, and 4e). This observation

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suggests an area of possible improvement of molecular engineering to enhance ADC potencies by increasing the membrane permeability of released payloads from intracellular vesicles such as endosomes and lysosomes in tumor cells and by enhancing the speed of action or potency of antibody drug conjugates.

EXPERIMENTAL PROCEDURES

Cell Lines and Reagents

The SK-BR-3 cell line was obtained from the in-house facility at Genentech, Inc. The PC3 human prostate cell line was obtained from the National Cancer Institute and two subclones, TenB2-PC3 and HER2-PC3 were developed at Genentech to express a high copy number of TenB2 and HER2 proteins, respectively, by stable transfection.32,33 The cells were cultured with DMEM with 10% FBS, 2 mM glutaMax and 2% Pen Strep for SK-BR-3 cells, and with RPMI-1640 with 10% FBS, 2 mM Lglutamine and 2% Pen Strep for TenB2- and HER2-PC3.

Synthesis of maleimide FRET peptides with C-terminal DM1 payload The maleimide-containing FRET peptides were synthesized by standard Fmoc solid-phase chemistry using a PS3 peptide synthesizer (Protein Technologies, Inc.) as shown in the Fig S1 (Supporting Information). Fmoc-Lys(Boc)-Wang or Rink amide resin (0.1 mmol) were used to generate the C-terminal carboxyl or carboxamide FRET peptides, respectively. Fmoc-Lys(Mtt)-OH was used as the first N-terminal residue to allow removal of the 4-methyltrityl (Mtt) group on the resin for additional side-chain chemistry. The Mtt group was removed by three consecutive 30 min washes of 1% TFA in dichloromethane with 3% triisopropylsilane (TIS) and the fluorescence acceptor, 5(6)-carboxy tetramethylrhodamine (TAMRA) was added and allowed to react for 20 hrs. The N-terminal Fmoc group was removed by addition of 20% piperidine in dimethylformamide (DMF) and an additional standard 2-(1HBenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) amide

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coupling step was conducted with maleimidocaproic acid. The TAMRA-labeled intermediate peptides were cleaved from the resin with 95:2.5:2.5 trifluoroacetic acid (TFA)/TIS/water (v/v/v) for 2 hours at room temperature with gentle shaking. The cleavage solution was filtered and evaporated under a stream of nitrogen to remove the TFA. The crude intermediates were dissolved in a mixture of water and acetonitrile and were subjected to further purification by reversed-phase HPLC with a Jupiter C4 column (5 µm, 10 mm x 250 mm) from Phenomenex. After lyophilization, the purified intermediates were then reacted with 10 eq. of NHS-fluorescein in 50/50 phosphate buffered saline (PBS)/ DMF (v/v) to label the free amine at the C-terminal lysine. For the FRET peptide containing the TAMRA-Alexa Fluor 488 pair, tetrafluorophenylester (TPF)-Alexa Fluor 488 was used. The FRET peptides were then purified and lyophilized as described above. To attach the cytotoxic payload DM1 to the C-terminal carboxyl group, aminoethylmaleimide (AEM) was used to modify the thiol group of DM1 to produce a free amine. DM1 was reacted with 10 eq. of AEM in 50/50 PBS/DMF (v/v) solution. The product of AEM-DM1 was purified by HPLC as described above. The final product of FRET1 was generated by a standard HBTU amide coupling of the AEM-DM1 to the FRET peptide. 1 eq. of AEM-DM1 was used in this reaction with 1 eq. HBTU, 0.1 eq. hydroxybenzotriazole (HOBT) and 2 eq. N,Ndiisopropylethylamime (DIEA). After 17 hours of reaction, the final product was purified by HPLC as described above and lyophilized. All reaction mixtures and final products were analyzed and confirmed by LC-MS.

Construction of FRET-labeled ADCs We used anti-HER2 and anti-TenB227 A118C THIOMABTM antibodies. As described previously,21,22 we attached FRET peptides or Alexa Fluor® 488 C5 maleimide (ThermoFisher Scientific) to the anti-HER2 and anti-TenB2 THIOMABTM,, A118C variants. After purification, the antibody conjugates were analyzed by LC-MS and analytical size-exclusion chromatography to verify coupling and degree of aggregation. Cathepsin cleavage assay

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Human cathepsins B, D, K, L and S were obtained from EMD Millipore Chemicals. Anti-TenB2 FRET2 conjugate (1 uM) was treated with 50 nM of various human cathepsins for 2 hours in pH 5 (0.1M sodium citrate) and pH 7.2 (0.1M sodium phosphate) buffers with 3 mM DTT and 2.5 mM EDTA. Fluorescence emission spectra were measured after excitation of samples at 495 nm using a Tecan Safire plate reader (Tecan Group Ltd). Cell culture and mouse xenograft studies For mouse xenografts with TenB2-PC3 cells, female CB-17 SCID-beige mice (Charles River Lab; San Diego, CA) were inoculated subcutaneously in the flank area. When the xenograft tumors reached an average tumor volume of 200-300 mm3, animals were randomized into groups of 6 mice each and received a single intravenous injection of 5 mg/kg anti-TenB2 FRET2 conjugate or the control of anti-HER2 FRET2 conjugate. Tumors (n=2/group) were subsequently collected at 4 hr, 24 hr and 7 days after i.v. dosage, embedded and kept frozen in optical curating temperature (OCT) media for cryo-sectioning. Thin-section slices were mounted on cover slides for fluorescence microscopy.

In vitro potency assays The in vitro potency of FRET1 conjugates and free drug S-methyl DM1 were assessed by cell viability assays. SK-BR-3 and PC3-TenB2 cells were grown in 96-well plates and treated with anti-HER2 or anti-TenB2 FRET1 conjugates or S-methyl DM1, respectively. After 3 days, cell viability was assayed by fluorescence of Alamar Blue reagent (Bio-Rad Laboratories, Inc.) using a Tecan Safire plate reader (Tecan Group Ltd). FACS Analyses Single cell suspensions were prepared from SK-BR-3 and TenB2-PC3 cell lines. The cell suspensions were pulse incubated with 100 nM of antibody at 4°C for 10 min to allow binding before washing and incubation at 37°C for the indicated time points.

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Flow cytometry was performed with a FACS Canto II (BD) and analyzed with FlowJo version 9.4.11 (Tree Star). Percent linker cleavage was calculated by dividing the median fluorescence intensity (MedFI) of an internalization control antibody by the MedFI of the signal from the cleaved FRET antibody. Shown are means ± SEM of pooled data from 2 independent experiments. Quantitative linker processing by IncuCyte® Zoom The linker processing assay used the IncuCyte® Zoom live-cell imaging system (Essen Bioscience, Ann Arbor  MI). SK-BR-3 cells were plated at 4,000 cells/well seeding density in a 384-well CellCarrier plate (PerkinElmer, Waltham, MA). Plates were incubated overnight after seeding and on the second day, the plates were briefly cooled on ice, and the anti-HER2 FRET3, FRET4 and FRET5 conjugates were diluted and added to the plate using an ECHO acoustic dispenser (Labcyte, Sunnyvale, CA) followed by incubation for 1 hr on ice to allow binding. The cells were washed three times with phosphate buffer saline (PBS) and Hanks F-12 K media with 10% FBS and 1 % penicillin and streptomycin were added back to the plate. The plate was then placed in an IncuCyte® Zoom live cell imaging system. Time-lapse fluorescence and phase-contrast images were obtained at two- or three-hour intervals for 48 hours. The total fluorescence intensity was then calculated by IncuCyte® Zoom software. Live-cell fluorescence imaging and analysis LEICA SP5 confocal microscope was used to obtain live-cell fluorescence images. The microscope was equipped with an environmental chamber connected to a Ludin cube temperature controller and a Ludin brick CO2 controller, which allowed live-cell imaging for longer periods of time. Time-lapse fluorescence images were collected every minute for first one hour and then every 10 minutes for the next 17 hours. Hybrid detectors, a 63x oil immersion, N.A 1.4 lens, and laser excitation lines at 488 nm and 543 nm were used for the confocal microscopy. The 633 nm laser line was used to obtain fluorescence images for the co-localization experiment with Alexa Fluor 647labeled transferrin or LysoTracker Deep Red. The fluorescence intensity of the cytosolic fluorescein-DM1 was measured by the image analysis software Volocity.

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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis details for FRET peptides, additional cellular imaging observations and antibody conjugate masses as noted in the text (PDF) AUTHOR INFORMATION Corresponding Author •

E-mail: [email protected]. Phone: +1 650-225-2648. Fax: +1 650-225-5945

•

Notes The authors were all employees of Genentech at the time of these studies and declare no competing financial interest.

ACKNOWLEDGMENTS We would like to thank the Protein Purification Group at Genentech for providing antiHER2 and anti-TenB2 THIOMABTMs and the Department of Pathology for tissue sectioning.

ABBREVIATIONS FRET ADC TDC TAMRA DM1 MMAE HER2 Ten-B2

fluorescence resonance energy transfer antibody drug conjugate THIOMAB antibody-drug conjugate tetramethylrhodamine maytansinoid monomethyl auristatin E Human Epidermal Growth Factor Receptor 2 Tomoregulin

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TABLE OF CONTENTS GRAPHIC

TAMRA

FRET1

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DM1 Fluorescein Incuba( on of SK-BR-3 cells with Hercep( n-FRET1 linker drug

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FRET Reagent Reveals the Intracellular Processing of Peptide-Linked

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