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Stabilizing a Tubulysin Antibody-Drug Conjugate to Enable Activity Against Multidrug-Resistant Tumors Leanna R. Staben, Shang-Fan Yu, Jinhua Chen, Gang Yan, Zijin Xu, Geoffrey Del Rosario, Jeffrey T. Lau, Luna Liu, Jun Guo, Bing Zheng, Josefa dela Cruz-Chuh, Byoung-Chul Lee, Rachana Ohri, Wenwen Cai, Hongxiang Zhou, Katherine Ruth Kozak, Keyang Xu, Gail D. Lewis Phillips, Jiawei Lu, John S. Wai, Andrew Gorham Polson, and Thomas H Pillow ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00243 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017
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ACS Medicinal Chemistry Letters
Stabilizing a Tubulysin Antibody-Drug Conjugate to Enable Activity Against Multidrug-Resistant Tumors Leanna R. Staben,† Shang-Fan Yu,† Jinhua Chen,‡ Gang Yan,‡ Zijin Xu,‡ Geoffrey Del Rosario,† Jeffrey T. Lau,† Luna Liu,† Jun Guo,† Bing Zheng,† Josefa dela Cruz-Chuh,† Byoung Chul Lee, †,§ Rachana Ohri,† Wenwen Cai,‡ Hongxiang Zhou,‡ Katherine R. Kozak,† Keyang Xu,† Gail D. Lewis Phillips,† Jiawei Lu,‡ John Wai,‡ Andrew G. Polson,† and Thomas H. Pillow*,† † ‡
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China
Keywords: antibody-drug conjugate (ADC), tubulysin, linker, Pgp, multidrug-resistance ABSTRACT: The tubulysins are promising anti-cancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates or ADCs. Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro. However, we observed metabolism of a critical acetate ester of the drug in vivo, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.
Over the last 40 years, antibody-drug conjugates (ADCs) have been employed to rescue highly potent cytotoxic small molecules that exhibit limited therapeutic activity due to doselimiting toxicities when administered as free drugs1. Tubulin inhibitors in particular have comprised the vast majority of drug types utilized in ADCs in clinical trials2. Almost all of these tubulin inhibitors have been auristatins3 or maytansinoids4 as found in the approved ADCs ADCETRIS® and KADCYLA® respectively. Despite these select clinical successes, the advancement of the next generation of ADCs will rely on improvements in antibody, linker, and drug. While a significant amount of resources has been applied to developing stable, site-specific conjugation strategies5,6, there has been far less effort expanding the diversity of drugs utilized by ADCs. The tubulysins, similar to auristatins in their linear peptidic structure, are a class of highly potent, cytotoxic natural products7,8. They inhibit tubulin polymerization and disintegrate microtubules in dividing cells9. The tubulysins bind to the vinca domain, but unlike monomethyl auristatin E (MMAE), are able to bind to the β subunit alone, perhaps explaining their higher affinity10. One of the most compelling reasons to investigate tubulysins is that they are not effluxed by Pgp11 unlike MMAE and some maytansinoids12. This lack of efflux gives tubulysin the potential to be effective against multidrug-resistant (MDR) cancers. Unfortunately, the high potency was coupled with systemic toxicity and prevented a tubulysin from advancing as a standalone agent. To improve the tolerability early investigations looked into the use of tubulysins for small molecule-drug conjugates (SMDCs) as
well as conjugates with nanoparticles and dendrimers13. Recently, there have been several reports of tubulysins incorporated into ADCs14-19. We were interested in exploring the impact of chemical diversity, increased tubulin binding, and lack of efflux on the efficacy and safety of a tubulysin ADC compared to an MMAE ADC; the latter being well characterized both preclinically and clinically. Unfortunately, the initial linker strategies developed for tubulysin conjugates were unamenable to ADCs13 due to the expected low in vivo stability of unhindered disulfides or hydrazones. We desired a linker with high stability in vivo and the ability to tracelessly release an unmodified tubulysin. The MC-VC-PABC linker, utilized in many clinical ADCs, is highly stable when attached to specific sites on an antibody20 and releases primary or secondary amines when cleaved by lysosomal proteases such as cathepsins21. While tubulysins are not directly amenable to this linker connection as they possess a tertiary amine, removal of the N-methyl group from the pipecolic acid Nterminus of the peptide drug could generate a secondary amine for linker attachment. We employed this strategy with an easily accessible, potent analog tubulysin M10,22 (2) to generate NH-tubulysin M (1) and its carbamate-linked ADC using a cysteine-engineered antibody at site LC-K149C (Figure 1A). Unfortunately, consistent with other recent reports15,18 removal of the methyl group to enable linker attachment resulted in a dramatic loss in potency as 1 was 18-177 fold less potent than 2 (Table 1). As might be expected, this loss of free drug potency abolished ADC activity, as the anti-CD22 carbamate linked-ADC 4 was inactive in vitro against lymphoma cell lines.
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Figure 1. Tubulysin ADCs containing A) carbamate or B) quaternary ammonium salt linker connections are cleaved and eliminate to release tubulysin drugs containing either a 2° amine (1) or a 3° amine (2) respectively.
To prevent this loss of activity, we needed another method for linking tubulysin. We recently described a novel quaternary ammonium salt peptide linker that is amenable to the stable connection and traceless release of tertiary amines23,24. Utilizing this concise, protecting-group free synthesis we generated tubulysin M ADCs in just 3 steps from commercially available starting materials (Figure 1B). The resulting anti-CD22 tubulysin ADC 5 was potent in vitro, in a range similar to MMAE ADC 8 in the CD22-expressing sensitive cell lines BJAB and WSU-DLCL2 (Table 1). Significantly, tubulysin ADC 5 retained activity in a Pgpoverexpressing cell line (BJAB.Luc/Pgp)25 while MMAE ADC 8 was completely inactive. The ADC activity was antigen-specific as evidenced by the lack of activity of 5 against CD22-negative Jurkat cells as well as non-targeting control anti-NaPi ADC 6 being inactive. Furthermore, the requirement for linker cleavage was supported by the inactivity of tubulysin ADC 7, which is epimeric at the citrulline amino acid required for proteolytic linker cleavage. Table 1. In Vitro Cytotoxicity of Tubulysin and MMAE Free Drugs and ADCs Free Drug or ADC #
BJAB IC50 (nM)
BJAB. Luc/Pg p IC50 (nM)
WSU IC50 (nM)
Jurkat IC50 (nM)
1
NH-tubulysin M
2.1
23
2.0
5.0
2
tubulysin M
0.12
0.13
0.11
0.10
3
MMAE
0.42
>30
0.19
0.13
anti-CD22MC-VC-PABCNH-tubulysin M
>190
-
>190
>190
4
anti-CD22MC-VC(S)-PABQtubulysin M
6.8
25
0.27
236
5
anti-NaPiMC-VC(S)-PABQtubulysin M
>253
-
185
>253
6 7
anti-CD22-
>253
-
>253
>253
MC-VC(R)-PABQtubulysin M anti-CD22MC-VC-PABCMMAE
3.3
>380
0.95
>253
8 9
tubulysin Pr
0.14
0.34
0.19
0.26
anti-CD22MC-VC-PABQtubulysin Pr
5.3
19
0.28
-
10
anti-NaPiMC-VC-PABQtubulysin Pr
>253
-
200
>253
11
Due to its potent in vitro activity against sensitive and resistant cell lines, we selected quaternary ammonium saltlinked tubulysin ADC 5 to evaluate in a human lymphoma xenograft in mice. Unexpectedly, 5 showed very modest tumor growth inhibition (TGI) after a single IV dose of 1 mg/kg (57% TGI), whereas MMAE ADC 8 afforded complete tumor regression through day 28 at a matched dose (Figure 2A). This in vitro-in vivo disconnect led us to investigate the in vivo stability of the two ADCs. Both ADCs were comprised of maleimides conjugated to the same site (LC-K149C) of a cysteine-engineered antibody with a drug-to-antibody ratio (DAR) of 2. This antibody site was selected because it forms a highly stable connection to maleimide-containing linker-drugs (Figure S1) 26. Utilizing affinity-capture LC-MS to determine in vivo stability27, we were able to observe a small loss in mass (-43 Da) of the tubulysin ADC 5 over time (Figure S2). This loss is consistent with cleavage of the acetate (Figure 2B) which is known to cause a significant reduction in potency for tubulysins28 and has been recently described with tubulysin ADCs14. Due to the expected loss of ADC activity upon acetate cleavage, we assigned this drug modification as a loss in “drug” in DAR calculations. With this in mind, we observed a significant loss of DAR over time for tubulysin ADC 5 (Figure 2C) with no active “drug” left after 4 days. In contrast, MMAE ADC 8 had no loss in DAR over 4 days. The cleavage of the acetate and corresponding loss in drug activity helps explain the limited efficacy of tubulysin ADC 5.
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Mean(Vol, Vehicle 01 - Vehicle (Histidine Buffer #8), 100 uL, IV once)
A
Mean(Vol, Anti-CD22-MC-VC-PABC-MMAE
B
(8), 1 mg/kg (5), 1 mg/kg
Mean(Vol, Anti-CD22-MC-VC-PABQ-tubulysin Mean(Vol,
1600 1400 1200 1000
400 200 0 0
5
10
15
Day
20
25
30
2.0 1.5
AAnti-CD22-MC-VC-PABC-MMAE (8) AAnti-CD22-MC-VC-PABQ-tubulysin (5)
1.0 0.5 0.0 0
1
2
3
4
Day
Figure 2. In vivo efficacy and stability of a 1st generation tubulysin ADC. (A) Efficacy of quaternary ammonium salt-linked tubulysin M ADC 5 (K149C) compared to MMAE ADC 8 (K149C) in a BJAB.Luc human lymphoma xenograft model in mice. ADCs were given as a single IV dose of 1 mg/kg. (B) Acetate cleavage was observed in vivo in mice. (C) The in vivo stability (expressed as drug-to-antibody ratio or DAR) of tubulysin M ADC 5 over time in mice compared to MMAE ADC 8.
We explored two approaches to improve the in vivo stability of tubulysin ADCs, specifically aiming to minimize or prevent the putatively enzyme-mediated deacetylation. One approach is to utilize cysteine engineering to identify an antibody site to protect the conjugated small molecule from metabolism during circulation14. Toward this effort, we identified an antibody site that decreased tubulysin acetate cleavage and subsequently improved efficacy (manuscript in preparation)29. Yet we preferred a more general solution not requiring antibody engineering. Thus the second approach was to replace the acetate with a stable isostere while retaining potency. Tubulysin analogs with a propyl ether and/or an N-propyl amide afforded low nanomolar potency30. We therefore aimed replace the acetate and modify the amide of tubulysin M (2) to generate tubulysin Pr (9) (Figure 3).
With the new tubulysin Pr analog 9, we were able to remove the metabolic liability, while retaining the low double digit pM potency found with tubulysin M (2) (Table 1). This retained potency was also observed with the quaternary ammonium linked anti-CD22 tubulysin Pr ADC 10, which killed lymphoma cells in a target-dependent manner. An in vivo evaluation of the conjugate was necessary to fully understand how changing the tubulysin structure would impact both ADC stability and efficacy. We evaluated the anti-tumor effects of the anti-CD22 tubulysin Pr ADC 10 in a human lymphoma xenograft model in mice (Figure 4). A
B
Mean(Vol, Vehicle 01 - Vehicle (Histidine Buffer #8), 100 uL, IV once) Mean(Vol, Anti-CD22-MC-VC-PABC-MMAE (8), 0.5 mg/kg Mean(Vol, Anti-CD22-MC-VC-PABQ-tubulysin Pr (10), 0.5 mg/kg Mean(Vol, Anti-CD22-MC-VC-PABQ-tubulysin Pr (10), 1 mg/kg M Anti-NaPi-MC-VC-PABQ-tubulysin Pr (11), 0.5 mg/kg
1600
1200 1000 800 600 400
1000 800 600 400
0 0
5
10
15
Day
Scheme 1. Improving tubulysin amide coupling with sterically hindered substrates.
1200
200
0
Preparation of 9 required an 11-step synthesis (Fig. S4), and step 7 required an amide coupling involving a now increasingly sterically hindered amine (12) with a hindered acid chloride 13 (Scheme 1). The resulting amide was only obtained in a 5% yield, and necessitated a new synthetic route. We found that swapping the Fmoc-amino acid chloride 13 for azido acid chloride 15 resulted in a 14 fold increase in yield of the desired amide and upon quantitative reduction of the azide, provided amine 14 to intersect the original route.
1600 1400
1400
200
Figure 3. Stabilizing tubulysin M (2) by replacing the acetate with a propyl ether. This modification combined with installation of an N-propyl amide in place of the N-Me amide led to design target tubulysin Pr (9).
Mean(Vol, Vehicle 01 - Vehicle (Histidine Buffer #8), 100 uL, IV once) Mean(Vol, Anti-CD22-MC-VC-PABC-MMAE (8), 1 mg/kg Mean(Vol, Anti-CD22-MC-VC-PABC-MMAE (8), 8 mg/kg Mean(Vol, Anti-CD22-MC-VC-PABQ-tubulysin Pr (10), 1 mg/kg Mean(Vol, Anti-CD22-MC-VC-PABQ-tubulysin Pr (10), 2 mg/kg Mean(Vol, Anti-NaPi-MC-VC-PABQ-tubulysin Pr (11), 1 mg/kg
C
Mean Tumor Volume, mm3
600
Mean Tumor Volume, mm3
C
800
Average DAR
Mean Tumor Volume, mm3
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
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25
30
0
5
10
15
20
25
Day
Figure 4. In vivo efficacy of a 2nd generation tubulysin ADC. (A) Structure of tubulysin Pr ADCs. (B) Efficacy of quaternary ammonium salt linked-tubulysin Pr ADC 10 (K149C) compared to MMAE ADC 8 (K149C) in a BJAB.Luc or (C) BJAB.Luc/Pgp human lymphoma model in mice. ADCs were given as a single IV dose.
The stabilized tubulysin Pr ADC 10 resulted in tumor stasis for 21 days when administered at a single dose of 1 mg/kg (Fig. 4B). The response was dose-dependent with modest tumor growth inhibition (77% TGI, day 11) observed at 0.5 mg/kg, and target-specific, as anti-NaPi tubulysin Pr ADC 11 gave no activity (identical to the vehicle control). While the tubulysin Pr ADC 10 was not quite as efficacious as MMAE ADC 8 at a matched 0.5 mg/kg dose (80% TGI, day 11), it had a much-improved efficacy compared to the unstable tubulysin ADC 5 (Figure 2A). Stability was also dramatically improved with no loss of DAR observed for 7 days in vivo (Figure S3).
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One of the most compelling reasons to utilize tubulysins as ADC payloads is their potential to overcome multi-drug resistance. As previously reported we derived lymphoma xenograft models resistant to MMAE ADCs25. These models were characterized to have an upregulation of Pgp while maintaining equivalent levels of target (CD22). We used this model here as it represents acquired resistance to the previous generation of ADCs targeting tubulin. We tested tubulysin and MMAE ADCs head-to-head in the BJAB.Luc-Pgp lymphoma model in mice (Figure 4C). MMAE ADC 8 was dosed up to 8 mg/kg and gave minimal tumor growth inhibition (41% TGI, day 13), which reflected more than 16-fold reduction compared to activity observed in the parental BJAB model. Gratifyingly, the stabilized tubulysin Pr ADC 10 gave clear dose-dependent tumor growth inhibition, resulting in 74% TGI (day 13) at 2 mg/kg, which reflected approximately only 4fold reduction compared to activity in the parental BJAB model. This improved activity of the tubulysin ADC relative to the MMAE ADC in an MDR model is likely a result of the released tubulysin payload not being a substrate for efflux by Pgp. Finally, the tubulysin ADCs were tolerated at all doses with mouse body weights increasing over the duration of the study (data not shown). New classes of highly potent cytotoxic drugs with distinct mechanisms and activity are a source of excitement to synthetic and medicinal chemists. While their incorporation into conjugates for targeted therapy can improve their tolerability and therefore therapeutic potential, new challenges arise. Among these are ways to stably and reversibly connect the drug, and particularly with ADCs, the increased risk for metabolic instability accompanying prolonged half-life. We have employed a peptide linker combined with a quaternary ammonium salt linker connection to provide a stable and bioreversible connection of tubulysins to antibodies. Furthermore, we identified and addressed the metabolism of an acetate ester critical to activity through its replacement with a propyl ether. The resulting conjugate was highly stable and efficacious against both sensitive and multidrug-resistant tumor models. The latter activity substantially differentiates this tubulysin ADC from the clinically validated MMAE ADC and generates excitement for its further investigation.
ASSOCIATED CONTENT Supporting information The supporting information is available free of charge on the ACS Publications website. In vivo stability data, methods for conjugate preparation, in vitro cytotoxicity, in vivo efficacy and stability, experimental procedures for the synthesis of new compounds and the corresponding characterization data (PDF)
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] Present Address §
23andMe, Mountain View, California 94041, United States
Notes The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Yanzhou Liu and Kewei Xu for analytical support and Terry Crawford and Phil Bergeron for assistance during manuscript preparation.
ABBREVIATIONS ADC, antibody-drug conjugate; DAR, drug-to-antibody ratio; MMAE, monomethyl auristatin E; Pgp, p-glycoprotein
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A.; Marquette, K.; Hudson, S.; Doppalapudi, V. R.; Stock, J.; Tchistiakova, L.; Bessire, A. J.; Clark, T.; Lucas, J.; Hosselet, C.; O'Donnell, C. J.; Subramanyam, C. Optimization of Tubulysin Antibody-Drug Conjugates: a Case Study in Addressing ADC Metabolism. ACS Med. Chem. Lett. 2016, 7 (11), 977–982. (15) Leverett, C. A.; Sukuru, S. C. K.; Vetelino, B. C.; Musto, S.; Parris, K.; Pandit, J.; Loganzo, F.; Varghese, A. H.; Bai, G.; Liu, B.; Liu, D.; Hudson, S.; Doppalapudi, V. R.; Stock, J.; O'Donnell, C. J.; Subramanyam, C. Design, Synthesis, and Cytotoxic Evaluation of Novel Tubulysin Analogues as ADC Payloads. ACS Med. Chem. Lett. 2016, 7 (11), 999–1004. (16) Li, J. Y.; Perry, S. R.; Muniz-Medina, V.; Wang, X.; Wetzel, L. K.; Rebelatto, M. C.; Hinrichs, M. J. M.; Bezabeh, B. Z.; Fleming, R. L.; Dimasi, N.; Feng, H.; Toader, D.; Yuan, A. Q.; Xu, L.; Lin, J.; Gao, C.; Wu, H.; Dixit, R.; Osbourn, J. K.; Coats, S. R. A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy. Cancer Cell 2016, 29 (1), 117–129. (17) Thompson, P.; Fleming, R.; Bezabeh, B.; Huang, F.; Mao, S.; Chen, C.; Harper, J.; Zhong, H.; Gao, X.; Yu, X.-Q.; Hinrichs, M. J.; Reed, M.; Kamal, A.; Strout, P.; Cho, S.; Woods, R.; Hollingsworth, R. E.; Dixit, R.; Wu, H.; Gao, C.; Dimasi, N. Rational Design, Biophysical and Biological Characterization of Site-Specific Antibody-Tubulysin Conjugates with Improved Stability, Efficacy and Pharmacokinetics. J. Controlled Release 2016, 236, 100–116. (18) Burke, P. J.; Hamilton, J. Z.; Pires, T. A.; Setter, J. R.; Hunter, J. H.; Cochran, J. H.; Waight, A. B.; Gordon, K. A.; Toki, B. E.; Emmerton, K. K.; Zeng, W.; Stone, I. J.; Senter, P. D.; Lyon, R. P.; Jeffrey, S. C. Development of Novel Quaternary Ammonium Linkers for Antibody-Drug Conjugates. Mol. Cancer Ther. 2016, 15 (5), 938–945. (19) Cohen, R.; Vugts, D. J.; Visser, G. W. M.; Stigter-van Walsum, M.; Bolijn, M.; Spiga, M.; Lazzari, P.; Shankar, S.; Sani, M.; Zanda, M.; van Dongen, G. A. M. S. Development of Novel ADCs: Conjugation of Tubulysin Analogues to Trastuzumab Monitored by Dual Radiolabeling. Cancer Res. 2014, 74 (20), 5700–5710. (20) Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saad, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Nguyen, T.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawli, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R. Conjugation Site Modulates the in Vivo Stability and Therapeutic Activity of Antibody-Drug Conjugates. Nat. Biotechnol. 2012, 30 (2), 184–189. (21) Dubowchik, G. M.; Firestone, R. A.; Padilla, L.; Willner, D.; Hofstead, S. J.; Mosure, K.; Knipe, J. O.; Lasch, S. J.; Trail, P. A. Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin From Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific in Vitro Anticancer Activity. Bioconjugate Chem. 2002, 13 (4), 855–869. (22) Wang, Z.; McPherson, P. A.; Raccor, B. S.; Balachandran, R.; Zhu, G.; Day, B. W.; Vogt, A.; Wipf, P. Structure-Activity and High-Content Imaging Analyses of Novel Tubulysins. Chem. Biol. Drug Des. 2007, 70 (2), 75–86. (23) Lehar, S. M.; Pillow, T. H.; Xu, M.; Staben, L.; Kajihara, K. K.; Vandlen, R.; DePalatis, L.; Raab, H.; Hazenbos, W. L.; Morisaki, J. H.; Kim, J.; Park, S.; Darwish, M.; Lee, B.-C.; Hernandez, H.; Loyet, K. M.; Lupardus, P.; Fong, R.; Yan, D.; Chalouni, C.; Luis, E.; Khalfin, Y.; Plise, E.; Cheong, J.; Lyssikatos, J. P.; Strandh, M.; Koefoed, K.; Andersen, P. S.; Flygare, J. A.; Wah Tan, M.; Brown, E. J.; Mariathasan, S. Novel Antibody-Antibiotic Conjugate Eliminates Intracellular S. Aureus. Nature 2015, 527 (7578), 323–328. (24) Staben, L. R.; Koenig, S. G.; Lehar, S. M.; Vandlen, R.; Zhang, D.; Chuh, J.; Yu, S.-F.; Ng, C.; Guo, J.; Liu, Y.;
Fourie-O'Donohue, A.; Go, M.; Linghu, X.; Segraves, N. L.; Wang, T.; Chen, J.; Wei, B.; Phillips, G. D. L.; Xu, K.; Kozak, K. R.; Mariathasan, S.; Flygare, J. A.; Pillow, T. H. Targeted Drug Delivery Through the Traceless Release of Tertiary and Heteroaryl Amines From Antibody-Drug Conjugates. Nat. Chem. 2016, 8 (12), 1112–1119. (25) Yu, S.-F.; Zheng, B.; Go, M.; Lau, J.; Spencer, S.; Raab, H.; Soriano, R.; Jhunjhunwala, S.; Cohen, R.; Caruso, M.; Polakis, P.; Flygare, J.; Polson, A. G. A Novel Anti-CD22 Anthracycline-Based Antibody-Drug Conjugate (ADC) That Overcomes Resistance to Auristatin-Based ADCs. Clin. Cancer Res. 2015, 21 (14), 3298–3306. (26) Pillow, T. H.; Sadowsky, J. D.; Zhang, D.; Yu, S.-F.; Del Rosario, G.; Xu, K.; He, J.; Bhakta, S.; Ohri, R.; Kozak, K. R.; Ha, E.; Junutula, J. R.; Flygare, J. A. Decoupling stability and release in disulfide bonds with antibody-small molecule conjugates. Chem. Sci. 2017, 8, 366-370. (27) Xu, K.; Liu, L.; Saad, O. M.; Baudys, J.; Williams, L.; Leipold, D.; Shen, B.; Raab, H.; Junutula, J. R.; Kim, A.; Kaur, S. Characterization of Intact Antibody-Drug Conjugates From Plasma/Serum in Vivo by Affinity Capture Capillary Liquid Chromatography-Mass Spectrometry. Anal. Biochem. 2011, 412 (1), 56–66. (28) Patterson, A. W.; Peltier, H. M.; Sasse, F.; Ellman, J. A. Design, Synthesis, and Biological Properties of Highly Potent Tubulysin D Analogues. Chem. Eur. J. 2007, 13 (34), 9534– 9541. (29) Genentech. Cysteine Engineered Antibodies and Conjugates. WO 2016/040856 A2 2016, 1–225. (30) Richter, W. WO 2011/057805 A1 2011, 1–34.
For Table of Contents Use Only Stabilizing a Tubulysin Antibody-Drug Conjugate to Enable Activity Against Multidrug-Resistant Tumors
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Leanna R. Staben,† Shang-Fan Yu,† Jinhua Chen,‡ Gang Yan,‡ Zijin Xu,‡ Geoffrey Del Rosario,† Jeffrey T. Lau,† Luna Liu,† Jun Guo,† Bing Zheng,† Josefa dela Cruz-Chuh,† Byoung Chul Lee, †,§ Rachana Ohri,† Wenwen Cai,‡ Hongxiang Zhou,‡ Katherine R. Kozak,† Keyang Xu,† Gail D. Lewis Phillips,† Jiawei Lu,‡ John Wai,‡ Andrew G. Polson,† and Thomas H. Pillow*,†
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