An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Published OnlineFirst September 23, 2014; DOI: 10.1158/1535-7163.MCT-14-0487-T

Molecular Cancer Large Molecule Therapeutics Therapeutics

Suzie J. Scales1, Nidhi Gupta1, Glenn Pacheco2, Ron Firestein3, Dorothy M. French3, Hartmut Koeppen3, Linda Rangell3, Vivian Barry-Hamilton1, Elizabeth Luis4, Josefa Chuh5, Yin Zhang6, Gladys S. Ingle1, Aimee Fourie-O'Donohue5, Katherine R. Kozak5, Sarajane Ross2, Mark S. Dennis6, and Susan D. Spencer2

Abstract Mesothelin (MSLN) is an attractive target for antibody–drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule- disrupting drugs monomethyl auristatin E (MMAE) and MMAF, ﬁnding the most effective to be MMAE with a lysosomal protease-cleavable valine–citrulline linker. The humanized (h7D9.v3) version, aMSLN-

MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin(SS1P)intransfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of aMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose- dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of aMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial. Mol Cancer Ther; 13(11); 2630–40. 2014 AACR.

Introduction thelial cancers, including ovarian, pancreatic, and meso- Mesothelin (MSLN) is a glycoprotein limited to the thelioma (see references in Supplementary Table S1). surface of the single mesothelial cell layer lining the peri- The high tumor versus normal expression makes cardia, pleura, and peritoneum (1). It is expressed as an mesothelin an attractive target for cancer therapy. Indeed, approximately 75 kDa fusion protein with the so-called besides vaccine (4) and chimeric antigen receptor T-cell megakaryocyte-promoting factor (MPF), which is remov- immunotherapy (5), a murine dsFv to mesothelin has been ed by furin cleavage, leaving an approximately 50 kDa fused to the protein synthesis inhibiting (PE38) domain of glycosylphosphatidylinositol-anchored mature mesothe- pseudomonas exotoxin to generate SS1P (CAT-5001; lin on the cell surface (2). Mesothelin-knockout mice exhibit refs. 1, 6). This immunotoxin exhibits marked antitumor no discernible phenotype, thus the normal biologic func- activity against xenografts of mesothelin-transfected tions of MPF and mesothelin are unknown (3). Mesothelin A431 cells, but was not tested in endogenously expressing is also overexpressed in a high percentage of various epi- xenografts due to lack of suitable models. In a phase I trial, SS1P produced only 4 of 34 minor responses in mesothelin-selected cancers, and was dose limited by pleuritis (7), 1 Department of Molecular Biology, Genentech, South San Francisco, likely because PE38 inhibited protein synthesis in the California. 2Department of Translational Oncology, Genentech, South San Francisco, California. 3Department of Pathology, Genentech, South San mesothelin-positive pleura. Immunogenicity prevented Francisco, California. 4Department of Protein Chemistry, Genentech, further cycles of treatment even following lymphocyte South San Francisco, California. 5Department of Biochemical and Cellular Pharmacology, Genentech, South San Francisco, California. 6Department depletion (8), so a deimmunized version (RG7787) is of Antibody Engineering, Genentech, South San Francisco, California. currently under investigation (9). Chimeric SS1 IgG with- Note: Supplementary data for this article are available at Molecular Cancer out PE38 (MORAb-009, amatuximab) exerts modest pre- Therapeutics Online (http://mct.aacrjournals.org/). clinical activity via antibody-dependent cell-mediated Corresponding Author: Suzie J. Scales, Genentech, 1 DNA Way, MS37, cytotoxicity (10), but no clinical activity (11). South San Francisco, CA 94080. Phone: 650-225-1510; Fax 650-467- Recent advances in antibody–drug conjugate (ADC) 8882; E-mail: [email protected] technology suggest that antimitotic ADCs with human doi: 10.1158/1535-7163.MCT-14-0487-T or humanized antibodies to mesothelin could be a 2014 American Association for Cancer Research. more promising approach. ADCs comprise whole IgGs

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covalently linked to cytotoxic drugs that are orders of SV-T2 were not authenticated and cells were not passaged magnitude more potent than traditional chemotherapeu- beyond 2 months. gD-MSLN- PC3, SV-T2, HT1080, and 293 tics (12, 13). ADCs are essentially prodrugs, optimally cells were generated by stable transfection with herpes requiring internalization so the antibody and/or linker is simplex virus glycoprotein D (gD)–tagged MSLN or gD- digested to release the active drug (12, 14). The recent MPF-MSLN (GenBank NM_005823), using the gD signal approval of two microtubule-inhibiting ADCs with dif- sequence in a puromycin-resistant pCMV vector. For effi- ferent linker-drug platforms—anti-CD30–valine–citrul- cacy studies, NIH:OVCAR3 and NCI-H226 cells were line–monomethyl auristatin E (SGN-35; ref. 15) and adapted for in vivo growth by dissociating the best growing anti-HER2-MCC-DM1 (T-DM1; ref. 16)—offers hope for tumor (in the SCID beige mammary fat pad and subcuta- these technologies with other tumor antigens. Indeed, neously in athymic nude mice, respectively) and cultivat- several other ADCs are at various stages of clinical devel- ing the cells in vitro. These x1 cells were retransplanted into opment, including an antimesothelin–DM4 conjugate mice, and resultant tumor growth was improved but (BAY-94-9343) with a reducible SPDB linker (17). Anti- variable, so the entire procedure was repeated (except mesothelin ADCs have three major potential advantages orthotopically in the lung of NCr nudes for H226-x1) to over SS1P: antimitotic activity conferring inherent spec- generate OVCAR3-x2.1 and H226-x2, which grew repro- ificity for rapidly dividing cancerous over nondividing ducible tumors. mesothelial cells; less immunogenicity, permitting multiple dosing cycles; and longer serum half-life to increase Humanization and conjugation therapeutic exposure. 7D9 hybridoma was chimerized onto a human IgG1 Given the association of DM4 with ocular toxicity backbone as reported (22), and then conjugated to mc- (18, 19), we chose to test auristatin-based antimeso- MMAF (3.8 drugs/Ab), mc-vc-PAB-MMAE (3.8/Ab), and thelin ADCs using novel monoclonals to mesothelin. mc-vc-PAB-MMAF (3.9/Ab) via cysteines of the inter- The most efficacious was an internalizing protease- chain disulfide bonds at Seattle Genetics as described sensitive valine–citrulline–linked monomethyl aurista- (23, 24). Negative control ADCs were antiragweed (aRW) tin E (vcMMAE) conjugate. In addition, because SS1P with 2.8, 3.2, and 3.0 drugs/Ab, respectively. See Supple- preclinical activity in highly overexpressing MSLN- mentary Methods for h7D9.v3 humanization. h7D9.v3 A431 xenografts did not translate to the clinic, we was protein A-purified from Chinese Hamster Ovary cells conducted comprehensive IHC of mesothelin expres- as described (22) and conjugated to an average of 3.5 vc- sion levels in human tumors and xenografts to identify PAB-MMAE per antibody at Genentech. The isotype the most representative models for efficacy studies. We control was humanized anti-gD-tag 5B6 (Genentech; aver- used these to demonstrate that a single dose of human- aging 3.3 MMAE/Ab), which doubled as a positive con- ized anti-MSLN-vcMMAE could regress ovarian, pan- trol on gD-MSLN cells. creatic, and mesothelioma xenografts. Internalization Internalization was performed as described (20) except Materials and Methods using 1 mg/mL antimesothelin antibodies and 0.4 mg/mL Antibody production Alexa488-EGF (Molecular Probes). Two human mesothelin antigens were expressed in E. coli 58F3: unizyme His (HQ)-tagged MPF-MSLN propep- FACS tide (aa 34–580 of GenBank NM_005823) and HQ-mature FACS was as published (25). MSLN extracellular domain (aa 296–580). Both were purified on a Ni-NTA column (Qiagen) followed by Superdex qRT-PCR 200 gel filtration in 20 mmol/L MES pH 6.0, 6 mol/L qRT-PCR was as reported (26), using human Guanidine-HCl, and then dialyzed into 1 mmol/L HCl. mesothelin probe set Hs00245879_m1, spanning exons Antibodies were produced as described (20), and 16 to 17 (Applied Biosystems), and RPL19 as the refer- screened by ELISA (21; see Supplementary), then by FACS ence gene. on MSLN-transfected SV-T2 cells. Western blotting Cell lines Cells were lysed [in 1% NP-40, 150 mmol/L NaCl, Cell lines originating from ATCC (acquisition year in 50 mmol/L Tris pH 8.0, 1 mmol/L EDTA, 1 mmol/L brackets) included NCI-H226 (2009), NIH:OVCAR3 phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L sodi- (2006), Caov-3 (2009), HPAC (2010), MSTO-211H (2010), um fluoride, and 1 Complete Protease Inhibitor tablet PC3 (2006), 293 (2007), and SV-T2 (before 2002). All except (Roche)]. Lysates of flash-frozen xenografts were pre- SV-T2 were obtained through in-house repositories, which pared using Tissue Lyzer (Qiagen). Thirty micrograms authenticated the lines by short tandem repeat profiling of lysates (measured by BCA; Pierce) were separated by before banking. MSTO-211H was instead completely SDS-PAGE, transferred to nitrocellulose, and blocked in sequenced by next-generation sequencing. No further 5% w/v nonfat milk, 0.1% Tween-20 in PBS. Primary authentication by our laboratory was performed, but only antibodies were used at 1 to 10 mg/mL overnight, detected

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with horseradish peroxidase–conjugated secondaries and IACUC guidelines, animals were euthanized before ECL (enhanced chemiluminescence) or ECL prime (all tumor volumes reached 3,000 mm3 or ulcerated, or body from Amersham), and exposed to BioMaxMR ﬁlm weight loss exceeded 20%. Primary OVXF1023 and (Kodak). Films were scanned on an HP ScanJet 8200 and PAXF1657 xenograft studies were conducted at Oncot- ﬁgures made with Adobe Photoshop (CS5.1). est GmbH; refs. 30, 31).

Scatchard analysis Scatchard analysis was as described (27) except 125I- Results h7D9.v3 was incubated for 2 hours at room temperature. Antibody, linker, and drug selection We began by generating novel antibodies to mesothe- IHC lin. In contrast with published antimesothelin anti- Formalin-fixed paraffin-embedded (FFPE) human bodies (10, 32–35), our mouse monoclonals were raised tumor microarrays (Supplementary Table S1A) were to unfolded, nonglycosylated mesothelin extracellular stained with 10 mg/mL 19C3 or IgG2b isotype control domain (with and without MPF). Hybridoma super- MPC-11 (BD Pharmingen) at room temperature for 60 natants were screened by ELISA, followed by FACS minutes on a Dako autostainer following TARGET and IHC analysis of cells stably transfected with retrieval (Dako). Detection used horse antimouse bioti- mesothelin to select those capable of detecting native nylated secondary (Vector), ABC complex (ABC Elite Kit; glycosylated and formalin-fixed mature mesothelin, Vector Laboratories) and DAB visualization (Pierce). To respectively (data not shown). avoid mouse-on-mouse issues with xenografts, rabbit The two highest affinity antibodies, 19C3 (0.06 nmol/L monoclonal antihuman mesothelin SP74 (Spring Biosci- by surface plasmon resonance analysis) and 7D9 (0.23 ence; M3744) or isotype control DA1E (Cell Signaling nmol/L), which bound noncompeting epitopes, were Technology; 3900S) were used at 1 mg/mL for 16 minutes further characterized. Both were specific by FACS and at 37C on the Ventana platform, with CC1 digestion, Western blotting, only recognizing transfected and Omnimap detection, and DAB visualization. 19C3 and endogenously mesothelin-expressing cells, but 19C3 was SP74 were first validated for IHC (Supplementary Figs. S1 more sensitive on low expressers (Fig. 1A–C), possibly and S2). reflecting its higher affinity. However, because internal- Mesothelin staining was scored by a Board-certified izing ADCs are generally more efficacious due to greater pathologist, noting the distribution and intensity of stain- drug release from lysosomes (12, 13, 36), we selected 7D9 ing, with a 10% cutoff as follows: 0 (negative) indicates for conjugation because it internalized to lysosomes to a very weak or no staining in >90% of tumor cells; 1þ greater extent in H226, OVCAR3, and MSLN-PC3 cells indicates predominantly weak staining (i.e., >50% of the (Fig. 1D and data not shown), all known to express stained cells are weak); 2þ, predominantly moderate; and mesothelin (34, 37). Even so, internalization was relatively 3þ, predominantly strong. This differs from the published slow, with little uptake before 6 hours and significant 5B2 (Novocastra) scoring system, which typically uses a surface signal remaining after overnight incubation. 1% cutoff and scores percentages of cells only, ignoring Chimeric 7D9 antibody was conjugated via the pro- intensity. tease-sensitive linker maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PAB) to the aur- In vitro efficacy istatin MMAE and the relatively impermeable MMAF, In vitro efficacy was assayed with CellTiterGlo II (Pro- and also to uncleavable maleimidocaproyl-MMAF mega) as described (27), except with 2,000 gD-MSLN- (mc-MMAF; refs. 23, 24). Conjugation did not affect HT1080 cells per well. 7D9 binding, and all three conjugates, but not the naked antibody, exhibited antiproliferative activity in vitro In vivo efficacy against OVCAR3 cells (not shown). By IHC, monoclonal All animal experiments were conducted according to 19C3 specifically stained mesothelin-positive cells, the Guide for the Care and Use of Laboratory Animals including normal mesothelia (Supplementary Fig. S1), (28). Cells or tumor fragments were implanted subcu- and detected mesothelin on OVCAR3 cells at 2þ by both taneously into female mice as summarized in Supple- IHC (Fig. 2A) and FACS (Fig. 1B, red). Serially trans- mentary Table S2, except OVCAR3 were in the mam- planted OVCAR3 tumors exhibited stronger (IHC 3þ) mary fat pad (27). Mice were randomized into groups of apical mesothelin staining (Fig. 2B), similar to many 8 to 11 with tumors averaging approximately 150 mm3. ovarian tumors. In this model, a single 3 mg/kg dose Tumors were measured with UltraCal-IV calipers and of cleavable 7D9-mc-vc-PAB-MMAE was clearly more volumes calculated as 0.5 (length width2). Growth efficacious than both the MMAF conjugates (Fig. 2C and curves were fitted by linear mixed effect modeling and Supplementary Fig. S3A), with robust tumor regressions analyzed using the nlme package of R software (R (138% inhibition) and mean tumor doubling delayed Foundation for Statistical Computing; ref. 29). Percent- from 6.5 (vehicle) to more than 91 days (Supplementary age tumor growth inhibition was calculated relative to Table S2). The relapsed tumors retained their original 0 the vehicle on day 21 or day 22 after dosing. As per levels of mesothelin expression (Fig. 2B , D–G),

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A MSLN-PC3 cells C 19C3 7D9 7 1 8 3 5

19C3 Clone 3 Clone 5 OVCAR3 PC3 vector Clone 3 Clone 5 OVCAR3 PC3 vector Figure 1. 7D9 has lower affinity but internalizes better than 19C3. 98 FACS of MPF-MSLN–transfected 1 10 100 1,000 10,000 64 PC3 stable cells (numbered # Events subclones in different colors, A) or 50 endogenously expressing ovarian 7 1 8 3 5 36 cells (B) OVCAR5 (blue), OVCAR4 (green), Caov-3 (purple), and 7D9 22 OVCAR3 (red), with PC3 vector (gray) as a negative control in all panels, using 1 mg/mL 19C3 (top) D 19C3 7D9 or 7D9 (bottom) detected with Alexa488-antimouse. C, Western blot (enhanced 1 10 100 1,000 10,000 chemiluminescence detection) Mean fluorescence intensity analysis showing specificity of B Ovarian cells 19C3 (left) and 7D9 (right) for mesothelin. Arrowhead, 75 kDa MPF-MSLN; arrow, mature 50 19C3 α Mesothelin kDa mesothelin. D, H226 cells after 20 hours of cointernalization of 1 mg/mL 19C3 or 7D9 (top and red channel in merge) and the lysosomal pathway marker Alexa488-EGF (middle and green 1 10 100 1,000 10,000 channel). 7D9 internalizes to # Events lysosomes (arrows). Insets (50% Alexa488-EGF magnification) show lack of isotype control uptake. Scale bars, 20 mm. 7D9

1 10 100 1,000 10,000 Merge + DAPI Mean fluorescence intensity

suggesting target that downregulation did not account In vitro characterization of aMSLN-MMAE ADC for recurrence and raising the possibility of re-treatment. h7D9.v3 (hereafter named aMSLN) retained high We speculate that the lower efficacy of the uncleavable affinity for mesothelin, ranging from 0.4 to 3 nmol/L mc-MMAF conjugate (74% inhibition) may be due to as measured by Scatchard analysis of endogenous slower diffusion than free MMAE/F into the cytoplasm mesothelin expressed from 0.5þ on MSTO-211H to of its lysosomal metabolite, cys-mc-MMAF (cys being 2þ on HPAC and OVCAR3-x2.1 [in vivo growth- the conjugated cysteine of the antibody that remains adapted OVCAR3 cells (see Materials and Methods), following its lysosomal degradation; ref. 24). The inter- which express equivalent mesothelin levels to the mediate activity of the mc-vc-PAB-MMAF conjugate parental line, Fig. 2H] and 3þ on H226-x2 cells (Fig. (110% inhibition) probably reflects more efficient dif- 3A). MMAE conjugation neither inhibited antigen bind- fusion of free MMAF (than cys-mc-MMAF) following ing, nor introduced nonspecific binding, as was also the val-cit cleavage and PAB self-immolation, but lack of a case for the agDcontrolADC(Fig.3A–C).Toverify bystander effect (the ability to intoxicate neighboring cytotoxic activity, gD-MSLN-HT1080 cells (expressing K cells; ref. 38) compared with free MMAE; MMAF has 130,000 copies of mesothelin; d,0.97nmol/L),which poor membrane permeability unless protonated in low- internalize both aMSLN and agD ADCs (Scales and pH environments such as lysosomes (24). Humanized colleagues, unpublished data), were assessed for via- 7D9 (h7D9.v3) was, therefore, conjugated to mc-vc-PAB- bility following 3 days’ incubation with these ADCs in MMAE, henceforth called aMSLN-MMAE, and charac- vitro. aMSLN-MMAE exhibited more potent killing n ¼ terized in detail. [average IC50 of 0.32 0.1 nmol/L ( 3 independent

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Untreated 7D9-vc-MMAE αRagweed-vc-MMAE ADB F

B¢ EG Figure 2. 7D9 is more efﬁcacious conjugated as MMAE than MMAF conjugates in an OVCAR3 xenograft model. Antimesothelin 19C3 IHC staining of OVCAR3 cells (A) and untreated (B, 10;B0 2.5), C 3,000 7D9-vc-MMAE-treated relapsed (day 91; D and E, tumors from two Vehicle representative animals), or 2,500 aRagweed-vc-MMAE–treated

3 7D9-mc-MMAF (day 74; F and G) OVCAR3 tumors. 7D9-mc-vc-PAB-MMAF Scale bars, 200 mm. C, growth rate 2,000 [calculated by linear mixed effects 7D9-mc-vc-PAB-MMAE (LME) modeling] of OVCAR3 tumor αRW-mc-MMAF transplants in the mammary fat pad 1,500 following one intravenous dose of 3 α RW-mc-vc-PAB-MMAF mg/kg chimeric 7D9 (black) or α 1,000 RW-mc-vc-PAB-MMAE negative control antiragweed (aRW, gray) conjugates. Raw mean tumor volumes are shown in Fitted tumor volume (mm Fitted tumor volume ) 500 Supplementary Fig. S3A. H, FACS with 0.5 mg/mL h7D9.v3 showing ex vivo–derived OVCAR3-x2.1 0 (blue) express similar levels of 0 20 40 60 80 100 mesothelin to parental OVCAR3 Days since dosing (red) cells by FACS. Dashed lines, Alexa488 antihuman alone; gray, 100 H cells alone. 80 60

40 % of max 20

0 11 10 1001,000 10,000 Mean fluorescence intensity

experiments)] than the positive control agD-MMAE cytotoxic activity up to 100 nmol/L on any cell line (IC50 2.2 0.9 nmol/L) despite similar drug loading tested (Fig. 3E and data not shown). (Fig. 3D), likely reflecting its greater binding capacity as their EC50s were similar (Fig. 3B). Cytotoxicity was Mesothelin prevalence and intensity in human specific as there was no detectable killing except tumors at the highest concentration on untransfected HT1080 It is important to demonstrate in vivo efficacy in models cells (with comparable sensitivity with free MMAE: IC50 expressing mesothelin at levels representative of human 0.17 0.01 nmol/L versus 0.13 0.02 nmol/L for tumors; however, typical levels are unknown as most of MSLN-HT1080, Fig. 3E), nor with aTENB2-vc-MMAE, the antimesothelin IHC (5B2 antibody) literature scores whose antigen is absent from these cells (Fig. 3D). As only percentages (not intensities) of positive cells (sum- expected, on lower expressing (40,000 copies) OVCAR3- marized in Supplementary Table S1). We, therefore, x2.1 cells with similar MMAE sensitivity (0.2 nmol/L), scored mesothelin staining by 19C3 in ovarian, pancreatic, a the IC50 of MSLN-MMAE was substantially higher and mesothelioma tissue microarrays (TMA) using inten- (20.3 nmol/L, data not shown). Naked aMSLN had no sity thresholds of no staining (0), weak (1þ), moderate

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A Figure 3. In vitro characterization of 2,500 100 αMSLN αMSLN-MMAE αgD αgD-MMAE aMSLN-MMAE ADC. A, FACS binding of naked aMSLN (black 2,000 80 a bars) and MSLN-MMAE (gray) 60 – m incubated at 0.05 50 g/mL with 1,500 þ þ 40 mesothelin 0.5 MSTO-211H, 2 % of max HPAC, 2.5þ OVCAR3-x2.1, and 20 1,000 3þ H226-x2 cells (detected with 0 Alexa647-antihuman). Negative 1 10 1001,000 10,000 control agD (white) and agD- 500 Mean fluorescence intensity MMAE (striped) were only tested at

Mean fluorescence intensity 0 5 5 5 50 mg/mL, showing minimal 5 50 50 50 50 0.5 0.5 0.5 0.5 0.05 0.05 0.05 background binding. Inset, FACS 0.05

MSTO-211H HPAC OVCAR3-x2.1 H226-x2 μ g/mL histograms of 0.5 mg/mL naked MSLN/cell 136 ± 4 2,600 39,963 216,000 ± 34,400 aMSLN (cells in same order); gray Kd 0.36 ± 0.03 nmol/L 1.04 nmol/L 0.56 nmol/L 3.24 ± 0.59 nmol/L is secondary alone on MSTO- (nmol/L) 211H. Scatchard results for mesothelin copy number and ﬁ B gD-MSLN-HT1080 C af nity are shown below (mean of 3,000 3,000 HT1080 two experiments or mean of three αMSLN αMSLN SD). B, gD-MSLN-HT1080 α αMSLN-MMAE 2,500 MSLN-MMAE 2,500 stable cells were incubated as in A αgD αgD with naked (closed symbols) or α 2,000 αgD-MMAE 2,000 gD-MMAE MMAE-conjugated (open symbols, dashed lines) aMSLN (black triangles), or agD control (grey 1,500 1,500 squares) at concentrations spanning 5 logs. Mean 1,000 1,000 ﬂuorescence intensity versus concentration plots reveal 500 500 sigmoidal dose-dependent binding Mean fluorescence intensity Mean fluorescence intensity with an EC50 of 0.46 0.11 mg/mL 0 0 for aMSLN-MMAE binding versus 0.0001 0.001 0.01 0.1 1 10 100 0.0001 0.001 0.01 0.1 1 10 100 0.5 0.17 for naked h7D9.v3 μg/mL antibody μg/mL antibody aMSLN; and 0.54 0.13 for agD- MMAE versus 0.24 0.04 for agD. D E C, there was no detectable binding 120 120 to untransfected HT1080 cells. D, cell viability of gD-MSLN-HT1080 100 100 (closed symbols) or untransfected HT1080 (open symbols and 80 80 dashed lines) with aMSLN-MMAE (triangles), agD-MMAE (gray 60 squares), or aTenB2-MMAE 60

(circles), normalized to untreated % Viability % Viability cells. E, as D, except with naked 40 40 aMSLN (triangles) and free MMAE αMSLN-MMAE (diamonds). The means and SDs of 20 αgD-MMAE 20 αMSLN three independent duplicate αTENB2-MMAE free MMAE experiments are shown in A–E. 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 (nmol/L) (nmol/L)

(2þ), and strong (3þ; Fig. 4). We used a 10% cutoff (instead adenocarcinomas, 23% of which were 1þ, 44% 2þ, and of the 1% cutoff used in the 5B2 literature), hypothesizing 33% 3þ. Twenty-six of 33 (79%) endometrioid, 5 of 13 clear that tumors would need 10% positive cells for mean- cell, and 1 of 10 mucinous ovarian tumors were also ingful antitumor responses with our bystander-capable positive. These prevalence data are consistent with the ADC, but the actual threshold and intensity remain to be 5B2 literature (Supplementary Table S1B) and further determined. validate 19C3 for IHC. Of 86 unspeciﬁed ovarian adenocarcinomas 85% were In contrast, only 75% of 196 pancreatic ductal adeno- mesothelin positive, with a membranous pattern and carcinomas were mesothelin positive. The staining was diffuse distribution. Staining intensity was mostly mod- usually weaker than in ovarian tumors (36% were 1þ, 27% erate (2þ, 35% of the total) or strong (3þ, 36%), with only 2þ, and only 11% 3þ) and often restricted to the apical 14% weak (1þ, Fig. 4A). In a separate TMA, mesothelin membrane of the ducts (Fig. 4B). This is lower than the prevalence was 75%, including 98% (42 of 43) serous reported 95% prevalence (Supplementary Table S1C),

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A 01+2+3+ 15% 14% 35% 36% Ovarian Figure 4. Representative mesothelin expression levels in human tumors by IHC. Typical anti- B 25% 36% 27% 11% mesothelin 19C3 signal intensities on 86 ovarian adenocarcinomas (A), 196 pancreatic ductal adenocarcinomas (B), and 29 mesotheliomas (C). The percentage of tumors with each IHC score is indicated (0þ is Pancreatic negative or less than 10% cells stained; 1þ is predominantly weak; 2þ is moderate; and 3þ is strong m C 55% 10% 14% 21% staining). Scale bars, 50 m. Mesothelioma

likely because staining is focal in up to 20% cases (39, 40) lated mesothelin in vivo: the 2þ OVCAR3-x2.1 cells and would have missed our 10% cutoff and/or not been (Fig. 2H) increased approximately 6.5-fold at both mRNA captured on the TMA. and protein levels to 3þ expressers in vivo (Fig. 5A–D; as Unexpectedly, only 45% of 29 mesotheliomas (4 of 12 did their parental OVCAR3 cells, Fig. 2A, B and H). pleural, 6 of 13 peritoneal and 3 of 4 pericardial) were Unfortunately, four pancreatic cell lines with 2þ meso- positive, far fewer than the 86% for pleural (95% for thelin by FACS/IHC also upregulated to 3þ in vivo, epithelioid pleural) in the literature (see Supplementary including HPAC cells (9, Fig. 5E–G). We were unable Table S1D and S1E). Staining was mostly diffuse and to identify a suitable 1þ cell line that remained 2þ in membranous, as reported, and was of variable intensity: vivo, thus selected 2þ primary pancreatic PAXF1657 10% 1þ, 14% 2þ, and 21% 3þ (Fig. 4C). Our lower transplants as the most representative pancreatic model prevalence is probably not due to lower 19C3 sensitivity, (Fig. 5H). Two mesothelioma models similarly increased because in a head-to-head comparison 19C3 detected mesothelin expression in xenografts: H226-x2 (Fig. 5I–K) more ovarian samples than 5B2 (not shown). It is possible and MSTO-211H (from 0.5 to 1þ, Fig. 5L–O). Ovarian that this mesothelioma TMA was simply not representa- OVXF1023 (3þ) primary human tumors were identified tive, as histologic subtypes were not specified and sarco- by IHC (Fig. 5P). matous mesotheliomas usually lack mesothelin. These data suggested mesothelin 2-3þ ovarian, 1-2þ pancreatic, aMSLN-MMAE is highly efficacious in multiple and 3þ mesothelioma efficacy models would be the most xenograft models representative of human tumors. To determine the preclinical activity of humanized aMSLN-MMAE ADC, we measured the antitumor ef- Identification of suitable efficacy models fects of single doses from 1 to 20 mg/kg in the identified To identify such xenograft models, we screened cell models compared with the nontargeted agD-MMAE lines for mesothelin expression by FACS and selected ADC. Efficacy parameters for the 10 mg/kg dose used ovarian OVCAR3-x2.1 (2þ), pancreatic HPAC (2þ), and in all models are shown in Supplementary Table S2. The mesothelioma H226-x2 (3þ). Mesothelioma MSTO-211H mesothelin 3þ ovarian OVCAR3-x2.1 model was the (0.5þ, with only 136 copies per cell) was additionally most sensitive to aMSLN-MMAE, with an approximated chosen as a low-expressing comparator (Fig. 3A). IHC IC50 for tumor growth inhibition (TGI) of 2.7 mg/kg and scores for cell pellets were all consistent with FACS, but almost complete regressions (108% TGI) at 10 mg/kg, unexpectedly, all cell lines tested substantially upregu- which increased the mean tumor-doubling time from

2636 Mol Cancer Ther; 13(11) November 2014 Molecular Cancer Therapeutics

Preclinically Efﬁcacious Antimesothelin–MMAE Conjugate

AEILOVCAR3-x2.1 HPAC H226-x2 MSTO-211H 12 20 12 12

16 8 12 8 8

8 4 4 4 4 Relative mesothelin Relative n.a. 0 0 0 0 Cells X1 X2/3 Ave X Cells X1 X2 X3 Ave X Cells X1 Cells X1 X2 Ave X B 2+ F 2+ J 3+ M 0.5+ Cells

C 3+ G 3+ K 3+ N 1+ SP74 staining Xenografts

D 0 H 2+ PAXF1657 P 3+ OVXF1023 O 0

50 μm 1°Xenografts 1°Xenografts or isotype

Figure 5. Efficacy model selection and upregulation of mesothelin in vivo. Top row, expression level of mesothelin in OVCAR3-x2.1 (A), HPAC (E), H226- x2 (I), and MST0-211H (L) by qRT-PCR (black, normalized to RPL19) and Western blotting (gray; Supplementary Fig. S4) of the same samples with rabbit monoclonal antimesothelin SP74 in cells and xenograft tumors (X1, X2, and X3), with cells normalized to 1. Error bars, SD of triplicate PCR reactions for individual samples or SD of the mean of all xenograft samples for "Ave X." n.a., insufficient tumor for Western blotting. Second row, SP74 IHC of cell pellets of OVCAR3-x2.1 (B), HPAC (F), H226-x2 (J), and MST0-211H (M) showing IHC scores consistent with the respective FACS shifts (Fig. 3A). Third row, SP74 IHC of xenografts of OVCAR3-x2.1 (C), HPAC (G), H226-x2 (K), and MST0-211H (N), confirming upregulation of mesothelin compared with parental cell lines. Bottom row, adjacent section isotype control IHC of OVCAR3-x2.1 (D) and MSTO-211H (O; rest in Supplementary Fig. S2) showing no signal; or SP74 IHC of primary xenografts PAXF1657 (H, 3/4 were 2þ) and OVXF1023 (P, 7/7 were 3þ). Scale bar, 50 mm(20, all panels).

4.5 (vehicle) to 83 days (Fig. 6A and Supplementary Fig. TGI), with 9 of 10 complete responses and doubling S3B). Activity was specific because agD-MMAE had delayed beyond 81 days (Fig. 6D and Supplementary little activity at this dose, and naked aMSLN had no Fig. S3E), significant nonspecific activity occurred at this activity at 15 mg/kg in any of the models tested, indi- dose. Similar efficacy was observed in the higher (IHC 3þ) cating the importance of conjugating MMAE to a expressing PAXF736 primary pancreatic model (data not mesothelin-targeting antibody. Despite similar mesothe- shown). lin levels, the primary ovarian model OVXF1023 was less In addition, aMSLN-MMAE inhibited the growth of sensitive, with 10 mg/kg ADC achieving only 53% TGI the 3þ mesothelioma H226-x2 model, albeit only result- after 3 weeks, necessitating a second dose that achieved ing in stasis (102% TGI) for 3 weeks and delaying tumor 78% TGI by 84 days and slowed tumor doubling from doubling to 34 days at 10 mg/kg (Fig. 6E). Although 8 to 45 days (Fig. 6B and Supplementary Fig. S3C). aMSLN-MMAE also caused stasis (96% TGI) in the low aMSLN-MMAE was also active in pancreatic models: (1þ) mesothelin-expressing MSTO-211H xenografts, HPAC xenografts strongly regressed at 10 to 15 mg/kg the duration of the response was shorter and the high (Fig. 6C) with an IC50 of 3.4 mg/kg and a tumor-doubling permeability of these tumors resulted in some activity delay to 74 days. The 2þ primary pancreatic PAXF1657 with agD-MMAE as well (Fig. 6F and data not shown), model was more sensitive than OVXF1023, with tumor suggesting that mesothelin targeting played less of a shrinkage (104% TGI) at 10 mg/kg and doubling delayed role in this model. Pharmacokinetic analysis confirmed to 37 days. Although 20 mg/kg was more effective (107% similar exposure in all models (data not shown).

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AB2,500 OVCAR3-x2.1 1,500 OVXF1023 αMSLN-MMAE: αgD-MMAE 1.0 5 2,000 2.5 10 5.0 15 1,000 10 20 Figure 6. aMSLN-MMAE is highly 1,500 15 efﬁcacious in multiple xenografts 20 of each indication. Xenografts 15 naked (150 mm3, Supplementary Table 1,000 Vehicle S2) of ovarian OVCAR3-x2.1 (A), 500 primary ovarian papillary serous 500 adenomatous carcinoma OVXF1023 (B), HPAC (C), primary pancreatic ductal adenocarcinoma 0 0 PAXF1657 (D), and mesotheliomas 0102030405060 0 102030405060 H226-x2 (E), and MSTO-211H (F) CD2,500 HPAC 3,000 PAXF1657 with 8 to 11 mice per group were dosed on day 0 (and day 21 for ) 3 2,500 OVXF1023 only, arrow). Tumor 2,000 growth curves were ﬁtted using LME modeling. All models were 2,000 1,500 dosed with vehicle (dashed line), aMSLN-MMAE (aMSLN, black), 1,500 and control agD-MMAE (agD, 1,000 gray) at 5 mg/kg (circles) and 10 1,000 mg/kg (triangles, dashed line), and some models also received 1 500 500 mg/kg (crosses), 2.5 mg/kg (open

Fitted tumor volume (mm Fitted tumor volume diamonds, dashed line), 15 mg/kg (squares), and/or 20 mg/kg 0 0 0 102030405060 0 102030405060 (inverted triangles); see B for legend. For clarity, inefﬁcacious 1,500 EFH226-x2 2,000 MSTO-211H 1 and 2.5 mg/kg groups are omitted in F. Naked aMSLN (hatched squares) had no effect at 1,500 15 mg/kg (A, C, and F). Graphs in 1,000 A to D are truncated at day 60 to better reveal the effects of lower doses. Later time points are shown 1,000 in Supplementary Fig. S3 as mean tumor volumes (SEM), with 500 efﬁcacy measures in 500 Supplementary Table S2.

0 0 0 1020304050 0 1020304050 Days since dosing

To summarize, potent dose-dependent antitumor models in which aMSLN-MMAE was highly active activity by a single systemic injection of aMSLN-MMAE expressed clinically relevant levels of mesothelin (2-3þ wasachievedineachofthethreeindications. by tumor IHC). Modest activity was seen in the 1þ mesothelioma MSTO-211H model. Although further work is required to determine the minimum mesothelin Discussion expression level required for activity, the differential We have demonstrated strong and sustained anti- responses of the 3þ models indicate that other factors tumor activity by aMSLN-MMAE ADC in one mesothe- such as internalization rate, MMAE-sensitivity, tumor lioma, two ovarian, and two pancreatic xenograft models, accessibility, and growth rate likely also play a role. including two primary tumor explants. In these ﬁve mod- We took pains to undertake systematic scoring of els, signiﬁcant activity was seen at 5 mg/kg and tumor mesothelin expression intensities in clinical samples to regressions at 10 mg/kg following a single dose. Similar identify relevant xenografts, with the goal of achieving results were obtained in OVCAR3, PAXF736, and H226 better predictive power for clinical activity than over- models with 10 mg/kg antimesothelin–DM4 conjugate expressing models. Notably, the A431-K5 cells used to (17), except with 3 to 6 repeated doses. Importantly, the demonstrate SS1P preclinical activity express 2.5 million

2638 Mol Cancer Ther; 13(11) November 2014 Molecular Cancer Therapeutics

Preclinically Efﬁcacious Antimesothelin–MMAE Conjugate

copies of mesothelin per cell (41), 10-fold more than the colleagues, manuscript in preparation). Furthermore, highest endogenous line (H226-x2) and likely more than in phase I trials (NCT01469793), it (DMOT4039A) is the patients who failed to respond to SS1P (7). In addition, showing activity in both ovarian and pancreatic cancers aMSLN-MMAE internalizes consistently better in trans- (47), suggesting that our preclinical models do indeed fected than endogenous lines, even with matching expres- have predictive value. sion levels (data not shown), supporting the superiority of endogenous models for assessing ADC activity. Nonethe- fl a Disclosure of Potential Con icts of Interest less, in all lines, MSLN accumulation in lysosomes was No potential conflicts of interest were disclosed. far slower than most other ADC targets (25, 36, 42), likely explaining the higher activity with cleavable vcMMAE Authors' Contributions than mc- or vc-MMAF, and also the lower (by an order of Conception and design: S.J. Scales, S. Ross, M.S. Dennis, S.D. Spencer magnitude) in vitro potency on OVCAR3 cells (20 nmol/L) Development of methodology: D.M. French, L. Rangell, Y. Zhang compared with other vcMMAE conjugates to similarly Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.J. Scales, N. Gupta, G. Pacheco, R. Firestein, expressed endogenous targets in other adherent cell lines. D.M. French, H. Koeppen, L. Rangell, V. Barry-Hamilton, E. Luis, J. Chuh, Our efficacy data in OVCAR3 cells and xenografts are Y. Zhang, G.S. Ingle, A. Fourie-O’Donohue, K.R. Kozak, S. Ross similar to those of aMuc16-auristatin conjugates, which Analysis and interpretation of data (e.g., statistical analysis, bio- statistics, computational analysis): S.J. Scales, N. Gupta, R. Firestein, also internalize slowly (27, 43). D.M. French, H. Koeppen, V. Barry-Hamilton, E. Luis, J. Chuh, Unexpectedly, endogenous (but not exogenous) A. Fourie-O’Donohue, K.R. Kozak, S. Ross, M.S. Dennis in vivo Writing, review, and/or revision of the manuscript: S.J. Scales, L. Rangell, mesothelin was upregulated up to 10-fold by an S. Ross, S.D. Spencer unknown mechanism in all the lines tested (including Administrative, technical, or material support (i.e., reporting or orga- others not shown). This enabled robust ADC activity even nizing data, constructing databases): L. Rangell, A. Fourie-O’Donohue, K.R. Kozak in HPAC cells with only 2,600 copies of mesothelin, Study supervision: S.J. Scales, M.S. Dennis suggesting many other tumorigenic cell lines could be Other (conducting animal research, and providing raw data and some potential efficacy models. It is unclear if upregulation is graphed figures that may have been changed): G. Pacheco mediated by the CanScript promoter, as this is reportedly less active in OVCAR3 and MSTO-211H cells (44), which Acknowledgments upregulated as much as the others. Cell–cell interactions The authors thank Seattle Genetics for the ADC technology license and are indebted to Jennifer Batson, Suzy Clark, Eric Cheng, Kathryn Parsons- might play a role, as we observed higher mesothelin in Reponte, Lisa Crocker, Nika Polevaya, Randy Musterer, Gail Colbern, confluent cells (which may mimic the in vivo situation), Klara Totpal, Allison Cordrey, and Inna Balter for xenograft development; but are likely independent of mesothelin-Muc16 binding Leanne Ross, Jenny Bostrom, and Oncotest GmbH for primary xenograft studies; Fred de Sauvage, Paul Polakis, Jay Tibbitts, Michel duPage, (45) because Muc16 is not expressed in MSTO-211H. This Brigitte deVaux, Yvonne Chen, Sreedevi Chalasani, Caroline Meloty- would be consistent with the converse finding that Kapella, Danielle Pasqualone, and Genentech core services for excellent support. mesothelin is lost from patient-derived mesotheliomas The costs of publication of this article were defrayed in part by the cultured in vitro (46). payment of page charges. This article must therefore be hereby marked Overall, aMSLN-MMAE is highly active at inhibiting advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. or even shrinking tumor growth in mesothelin-positive mouse models. It is also well tolerated, and did not Received June 6, 2014; revised August 1, 2014; accepted September 5, exhibit pleuritis in non-human primates (Scales and 2014; published OnlineFirst September 23, 2014.

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2640 Mol Cancer Ther; 13(11) November 2014 Molecular Cancer Therapeutics

An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Suzie J. Scales, Nidhi Gupta, Glenn Pacheco, et al.

Mol Cancer Ther 2014;13:2630-2640. Published OnlineFirst September 23, 2014.

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