Article pubs.acs.org/bc
Investigation of Hydrophilic Auristatin Derivatives for Use in Antibody Drug Conjugates Brian A. Mendelsohn,* Stuart D. Barnscher,† Josh T. Snyder, Zili An, Jennifer M. Dodd, and Julien Dugal-Tessier Agensys Inc. an affiliate of Astellas Pharma Inc., 1800 Stewart Street, Santa Monica, California 90404, United States S Supporting Information *
ABSTRACT: Antibody drug conjugates offer a targeted cancer treatment for the delivery of potent cytotoxic drugs. Derivatives of the natural product dolastatin 10 containing pyridines and other basic amines were examined with the objective of determining if a more hydrophilic auristatin derivative would be potent enough for use as part of an ADC. This may be advantageous if a less hydrophobic drug makes a better ADC. A pyridine derivative, monomethyl auristatin PYE, showed the greatest potency when tested in vivo. While only a modest tumor growth inhibition was observed when the HCC1954 human breast cancer xenografts were treated with“non-cleavable” linker ADCs, tumor regression was seen when treated with an enzymatically degradable “cleavable” linker ADC when conjugated to trastuzumab. Based on these studies, monomethyl auristatin PYE shows promise for use as an ADC payload.
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platinum-sensitive ovarian carcinoma10), no significant clinical activity was observed at the maximum dose tolerated. The same result was obtained for the auristatin TZT-1027 (auristatin PE, 2b).11 Since these early studies, the auristatins have had great success as the cytotoxic component of antibody drug conjugates (ADCs). An ADC made up of monomethyl auristatin E (MMAE, 2d)12 conjugated to a CD30 specific antibody was approved for the treatment of relapsed Hodgkin’s lymphoma in 2011.12,13 Multiple auristatin ADCs are currently being evaluated in the clinic for a variety of indications including solid tumors, B-cell malignancies, prostate cancer, and breast cancer.2,14 The auristatins used in these ADCs include MMAE, monomethyl auristatin F (MMAF, 2e),15 and a 2-aminoisobutyric acid derivative (PF-06380101, 2f).16 The auristatin scaffold is relatively hydrophobic as shown by the high calculated log D7.4 values (dolastatin 10, 5.42; MMAE, 2.99). This places some limits on the ADCs that can be generated, as it has been shown that the more hydrophobic the drug linker, the more it tends to aggregate during conjugation and storage.17−19 Auristatin-based ADCs with high drug-toantibody ratios (DAR) not only tend to aggregate more, but also have higher clearance in vivo, probably due to increased hydrophobicity.20 Decreasing the hydrophobicity of the drug and/or linker of the ADC also decreases the clearance rate in vivo.21
INTRODUCTION Dolastatin 10 (1) and its synthetic derivatives the auristatins (Figure 1) have generated a great deal of interest as potent
Figure 1. Dolastatin 10 and selected potent auristatins.
cytotoxic agents since the first report by Pettit et al.1,2 These tubulin destabilizing peptide-like molecules showed promise for cancer treatment and studies were carried out to assess the use of dolastatin 10 (1) as a single-agent chemotherapy drug. Although it was advanced to a number of clinical studies for a variety of cancers (metastatic soft tissue carcinoma,3 hormone-refractory metastatic prostate adenocarcinoma,4 colorectal,5 breast,6 melanoma,7 nonsmall-cell lung cancer,8 pancreaticobiliary,9 and © XXXX American Chemical Society
Received: September 15, 2016 Revised: December 16, 2016
A
DOI: 10.1021/acs.bioconjchem.6b00530 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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either 100 >100 0.06
>100 >100 >100 >100 >100 >100 >100 >100
some tumor growth inhibition (67%) but this was not statistically significant when compared to H3−1.4-32 on day 23 (p = 0.0895). Based on these results it would appear that the carbamatecontaining linkers act more like traditional amide noncleavable linkers and are not able to release the free payload by cleavage at the carbamate.
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CONCLUSIONS The hydrophilicity of auristatin containing ADCs has been previously shown to be an important factor in governing
Figure 7. In vivo efficacy of amide linked (A) and carbamate linked (B) ADCs. (A) Female ICR/SCID mice bearing size-matched HCC1954 human breast carcinoma xenografts of 200 mm3 were given a single dose intravenously at 10 mg/kg on day 0 (n = 10). The tumor volumes for all animals on day 18 are shown with the mean and the standard error. (B) Female ICR/SCID mice bearing size-matched HCC1954 human breast carcinoma xenografts of 200 mm3 were dosed intravenously at 10 mg/kg as a single dose on day 0 (n = 6). Tumor volumes for all animals are shown at day 23 with the mean and the standard error. G
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Figure 8. In vivo efficacy of a cleavable ADC. Human breast carcinoma HCC1954 cells (3 × 106 cells per mouse) were implanted into the mammary fatpad of female CB17/SCID mice. Treatments with Trastuzumab-36 and Kadcyla at 5 mg/kg as a single dose were started when tumors reached 200 mm3 (n = 10). cHmLys.1c3.G2k-36 is the nonbinding control ADC and the Vehicle is 20 mM Histidine/5% Trehalose, pH 5.2. (A) Graph shows mean tumor volume over time with standard error for each cohort. (B) Individual tumor volumes on day 21 in each cohort with mean and standard error.
their properties.21 In particular, it has been shown that increasing the hydrophilicity decreases the tendency to form protein aggregates17−19 and decreases the clearance rate of the ADC from circulation.20,21,35 Compounds with greater hydrophilic character may reduce possible bystander activity due to lower membrane permeability, although based on the in vitro activity observed of these payloads we do not expect this to be significantly altered. Methods to quantitatively measure bystander effect(s) are not well established.39 Some negatively charged auristatin derivatives (e.g., MMAF) are known to have increased aqueous solubility compared to neutral auristatins (e.g., MMAE).15 In this study we synthesized and tested several positively charged amine-based auristatins such as auristatin PYE, which has been reported to have tumor growth inhibition activity.23 Future studies can examine the effects of bystander activity of hydrophilic compounds such as those described herein. Our screening cascade for the series of synthesized compounds began with two in vitro assays: an in vitro cytotoxicity assessment and a tubulin polymerization assay. These were followed by vivo screening of free drugs and the best payload was
then evaluated for use as part of an ADC. The results of the in vitro cytotoxicity screen show that some weakly basic aromatic side-chain groups were tolerated with minimal loss of activity. Pyridines and anilines retained activity but the more strongly basic and aliphatic groups had diminished cytotoxic activity. All of the derivatives were analyzed for inhibition of tubulin polymerization but no correlation was observed between this assay and the in vitro cytotoxicity assay. Several compounds were evaluated in the cytotoxicity assay but only those with an IC50 < 1 nM were shown to be active in vivo. Two compounds, the monomethyl derivative of auristatin PYE (12) and aniline 22 showed the greatest efficacy in vivo, although 22 also showed signs of toxicity at the 4 mg/kg dose (pronounced body weight loss). One limitation in screening small molecules by this method is the possible failure to identify active compounds that do not have passive cell permeability. Such compounds are restricted from passively entering cells so no cytotoxic effect may be elicited. However, they may still be effective as ADCs as internalization of the antibody will transport the compound into the cell. This lack H
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aggregate by SEC (column: Tosoh TSKGelSW3000xl 7.8 mm × 30 cm, 5 μm). All ADCs were reduced with 100 mM DTT at pH 9 for 10 min and analyzed by reverse-phase HPLC (column: PLRP-S 1000 Å, 8 μm, 50 × 2.1 mm) to separate heavy and light chains on the basis of drug load. Drug to antibody ratio (DAR) was calculated using a weighted average of the heavy and light chain species with various drug loads. Hydrophobic Interaction Chromatography. Trastuzumab-36 and unconjugated trastuzumab were separated on the basis of drug load to assess hydrophilicity of the drug moiety using a previously published method39 (see Supporting Information). Tubulin Inhibition. The inhibition of tubulin polymerization was evaluated using an HTS-Tubulin Polymerization Assay Kit (Cytoskeleton Inc.; Catalog No. BK004P) on bovine brain tubulin. Tubulin was seeded at approximately 400 μg per well in 100 μL of general tubulin buffer, and then treated with 10 μM final concentration of compound in duplicate at the initiation of the assay. Tubulin polymerization assays were carried out at 37 °C for 60 min after the addition of test compounds. Tubulin polymerization was determined by absorbance spectroscopy using the optical density value at 340 nm each minute after the addition of test compounds. The extent of tubulin polymerization by the compound-treated tubulin was compared to that of buffer-treated tubulin. In Vitro Cytotoxicity. Cytotoxic activity of compounds and ADCs was measured on PC3 (human prostate carcinoma), HCC-1954 (human mammary ductal carcinoma), and SW780 (human bladder transitional cell carcinoma) cancer cell lines. Cells were seeded in clear tissue-culture treated 96-well plates at approximately 1000−3000 cells per well in 50 μL of growth media (RPMI-1640 + 10% heat-inactivated fetal bovine serum) and incubated overnight in a humidified incubator at 37 °C with 5% CO2 to allow them to attach. Stock solutions of the test compounds in DMSO or ADCs in PBS were diluted with growth media (RPMI-1640 + 10% heat-inactivated fetal bovine serum). The next day, 50 μL of a 2× stock of vehicle control (DMSO) or compounds/ADCs at varying concentrations were added to each well in triplicate. The plates were incubated in the humidified tissue culture incubator with 5% CO2 at 37 °C for 4 to 6 days after addition of compounds to measure cytotoxicity. After 4 to 6 days, 20 μL of PrestoBlue Cell Viability Reagent (Life Technologies, Carlsbad, CA, Catalog No. A13261) was added to each well. Plates were then incubated at 37 °C for 1 to 2 h to allow for PrestoBlue uptake. Fluorescence was recorded at 540ex/590em using a Biotek Synergy H4 plate reader. Data for compounds tested in this assay are graphed as percent survival compared to untreated control wells. In Vivo Efficacy. All experimental protocols were approved by Agensys’ Institutional Animal Care and Use Committee (IACUC). Either SW780 human bladder cancer cells (2 × 106 cells) or HCC1954 human breast cancer cells (3 × 106 cells) were injected subcutaneously into the flank (SW780) or the mammary fatpad (HCC1954) of individual SCID mice (Taconic Farms). Animals were dosed when tumor size reached 200 mm3. Tumors were size matched and animals were randomized into treatment and control groups. Tumor-bearing mice were treated intravenously and tumor volume was assessed twice weekly by caliper measurement. A statistical analysis of the tumor volumes for the last day before animal sacrifice were analyzed by the Kruskal−Wallis test and pairwise comparisons were made using the Tukey’s test procedures (two-sided)41 on the ranked data to protect the experimental error rate.
of direct cytotoxicity has been observed with other charged auristatins such as MMAF which is ineffective as a free drug but highly potent as an ADC payload. Some of the more basic compounds from our library, such as piperazine 20, are predicted to have poor cell permeability; however, any correlations between an ADC payload’s structural characteristics and in vitro cytotoxicity experiments remain an area underexplored in the scientific literature. The payload with the best activity and the least toxicity in vivo was compound 12. It was successfully attached to a variety of linkers and then conjugated to trastuzumab for evaluation as an ADC. ADCs with noncleavable drug-linkers showed no activity in vitro and showed only moderate or no efficacy in vivo. Carbamate linkers, like the noncleavable ADCs, had no in vitro or in vivo activity. The similar behavior of the noncleavable and carbamate-linked ADCs suggests that the carbamate linkage, which we hypothesized would release free drug after intracellular degradation of the carbamate linkage, appears not to release the free drug after internalization under the conditions tested. Further studies assessing the metabolites generated by these linkers are necessary to fully evaluate this hypothesis. For instance, it may be possible to increase the release of free drug by adding a spacer to move the carbamate away from the N-methyl group on the auristatin P1 making it more accessible for enzyme degradation. ADCs with an enzyme-cleavable mc-Val-Cit-Paba linker, such as trastuzumab-36, showed tumor regression when tested in vivo compared to the ADCs using noncleavable linkers. This result demonstrates the ability of payloads containing basic nitrogen groups to be successfully used as part of an ADC when combined with a cleavable linker. A study of the relationship between aggregate and DAR was carried out on a human IgG2 antibody since, in our experience, they often have a greater propensity to aggregate than do human IgG1 antibodies. The degree of protein aggregation did not increase during conjugation of mc-12 (30) or mc-Val-Cit-Paba-12 (36) in an increasing-DAR titration study. This payload may be a good candidate to use with an antibody that has a tendency to aggregate and may help control the properties of the resulting ADC. Overall, the results of this study show that using compound 12 as part of an ADC may enable us to overcome some of the problems seen when using more hydrophobic molecules. This is encouraging and it opens up the possibility to study other auristatins containing heterocycles, including methyl imidazoles, thiazoles, thiophenes, oxazoles, and indoles as potential ADC payloads.
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EXPERIMENTAL METHODS Conjugation Methods. Monoclonal antibodies were incubated with a defined molar ratio of TCEP to antibody ranging from 1.7:1 to 8.5:1 at pH 7.4 for 2−4 h. Maleimide drug linkers were conjugated to reduced antibody by adding approximately a 2:1 molar ratio of drug linker to free thiols followed by incubation at room temperature for 1−2 h at pH 7.4. Bromoacetamide drug linkers were conjugated to free thiols by adding approximately 20 mol equiv per antibody and incubating at room temperature for up to 24 h at pH 8.5. Conjugation reactions were done in the presence of 5−20% DMSO to ensure drug linker solubility. Following conjugation, N-acetyl-L-cysteine was added to quench unreacted drug linker. For efficacy testing, the resulting ADCs were purified using 20 mM histidine, pH 6.0, containing 5% sucrose with disposable PD10 desalting columns. For aggregation studies the unpurified ADCs were analyzed for I
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(5) Saad, E. D., Kraut, E. H., Hoff, P. M., Moore, D. F., Jr., Jones, D., Pazdur, R., and Abbruzzese, J. L. (2002) Phase II study of dolastatin-10 as first-line treatment for advanced colorectal cancer. Am. J. Clin. Oncol. 25, 451−453. (6) Perez, E. A., Hillman, D. W., Fishkin, P. A., Krook, J. E., Tan, W. W., Kuriakose, P. A., Alberts, S. R., and Dakhil, S. R. (2005) Phase II trial of dolastatin-10 in patients with advanced breast cancer. Invest. New Drugs 23, 257−261. (7) Margolin, K., Longmate, J., Synold, T. W., Gandara, D. R., Weber, J., Gonzalez, R., Johansen, M. J., Newman, R., Baratta, T., and Doroshow, J. H. (2001) Dolastatin-10 in metastatic melanoma: a phase II and pharmokinetic trial of the California Cancer Consortium. Invest. New Drugs 19, 335−340. (8) Krug, L. M., Miller, V. A., Kalemkerian, G. P., Kraut, M. J., Ng, K. K., Heelan, R. T., Pizzo, B. A., Perez, W., McClean, N., and Kris, M. G. (2000) Phase II study of dolastatin-10 in patients with advanced nonsmall-cell lung cancer. Ann. Oncol. 11, 227−228. (9) Kindler, H. L., Tothy, P. K., Wolff, R., McCormack, R. A., Abbruzzese, J. L., Mani, S., Wade-Oliver, K. T., and Vokes, E. E. (2005) Phase II trials of dolastatin-10 in advanced pancreaticobiliary cancers. Invest. New Drugs 23, 489−493. (10) Hoffman, M. A., Blessing, J. A., and Lentz, S. S. (2003) A phase II trial of dolastatin-10 in recurrent platinum-sensitive ovarian carcinoma: a Gynecologic Oncology Group study. Gynecol. Oncol. 89, 95−98. (11) Patel, S., Keohan, M. L., Saif, M. W., Rushing, D., Baez, L., Feit, K., DeJager, R., and Anderson, S. (2006) Phase II study of intravenous TZT-1027 in patients with advanced or metastatic soft-tissue sarcomas with prior exposure to anthracycline-based chemotherapy. Cancer 107, 2881−2887. (12) Doronina, S. O., Toki, B. E., Torgov, M. Y., Mendelsohn, B. A., Cerveny, C. G., Chace, D. F., DeBlanc, R. L., Gearing, R. P., Bovee, T. D., Siegall, C. B., et al. (2003) Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21, 778−784. (13) Senter, P. D., and Sievers, E. L. (2012) The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol. 30, 631−637. (14) Chari, R. V., Miller, M. L., and Widdison, W. C. (2014) Antibodydrug conjugates: an emerging concept in cancer therapy. Angew. Chem., Int. Ed. 53, 3796−3827. (15) Doronina, S. O., Mendelsohn, B. A., Bovee, T. D., Cerveny, C. G., Alley, S. C., Meyer, D. L., Oflazoglu, E., Toki, B. E., Sanderson, R. J., Zabinski, R. F., et al. (2006) Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjugate Chem. 17, 114−124. (16) Maderna, A., Doroski, M., Subramanyam, C., Porte, A., Leverett, C. A., Vetelino, B. C., Chen, Z., Risley, H., Parris, K., Pandit, J., et al. (2014) Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications. J. Med. Chem. 57, 10527−10543. (17) Hollander, I., Kunz, A., and Hamann, P. R. (2008) Selection of reaction additives used in the preparation of monomeric antibodycalicheamicin conjugates. Bioconjugate Chem. 19, 358−361. (18) King, H. D., Dubowchik, G. M., Mastalerz, H., Willner, D., Hofstead, S. J., Firestone, R. A., Lasch, S. J., and Trail, P. A. (2002) Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains. J. Med. Chem. 45, 4336−4343. (19) Burke, P. J., Senter, P. D., Meyer, D. W., Miyamoto, J. B., Anderson, M., Toki, B. E., Manikumar, G., Wani, M. C., Kroll, D. J., and Jeffrey, S. C. (2009) Design, synthesis, and biological evaluation of antibody-drug conjugates comprised of potent camptothecin analogues. Bioconjugate Chem. 20, 1242−1250. (20) Hamblett, K. J., Senter, P. D., Chace, D. F., Sun, M. M., Lenox, J., Cerveny, C. G., Kissler, K. M., Bernhardt, S. X., Kopcha, A. K., Zabinski, R. F., et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063−7070. (21) Lyon, R. P., Bovee, T. D., Doronina, S. O., Burke, P. J., Hunter, J. H., Neff-LaFord, H. D., Jonas, M., Anderson, M. E., Setter, J. R., and Senter, P. D. (2015) Reducing hydrophobicity of homogeneous
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00530. Crystallographic data (CIF) Experimental procedures for all compounds and intermediates, curves for tubulin polymerization, graphs of mean tumor size for all days for in vivo data, and NMR spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Brian A. Mendelsohn: 0000-0002-6339-2377 Present Address
† Zymeworks Inc., 540−1385 West 8th Avenue, Vancouver, BC, Canada V6H 3V9
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS All research was funded by Agensys, Inc. (an affiliate of Astellas Pharma). We would like to thank the Agensys Target and Drug Validation group for their assistance with cytotoxicity and tubulin binding assays, the Agensys Pharmacology group for assistance with in vivo experiments, the Agensys ADC Chemistry group for assistance with high resolution mass spectrometry and for helpful review of the manuscript. We would like to acknowledge Dr. Saeed Khan from the J. D. McCullough Laboratory of X-ray Crystallography at UCLA for assistance with crystallography. We would like to thank Professor George R. Pettit and Dr. Jeanette L. Grant for helpful discussions and manuscript review.
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ABBREVIATIONS ADC, antibody drug conjugate; DAR, drug to antibody ratio; DEPC, diethyl cyanophosphonate; DIEA, diisopropylethylamine; mc, maleimidocaproyl; mc-Val-Cit-Paba, maleimidocaproyl-valine-citrulline-paba; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; MMAPYE, monomethyl auristatin PYE; SEC, size exclusion chromatography; TCEP, tris(2-carboxyethyl)phosphine
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