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Site-Specific Trastuzumab Maytansinoid Antibody−Drug Conjugates with Improved Therapeutic Activity through Linker and Antibody Engineering Thomas H. Pillow,*,† Janet Tien,† Kathryn L. Parsons-Reponte, Sunil Bhakta, Hao Li, Leanna R. Staben, Guangmin Li, Josefa Chuh, Aimee Fourie-O’Donohue, Martine Darwish, Victor Yip, Luna Liu, Douglas D. Leipold, Dian Su, Elmer Wu, Susan D. Spencer, Ben-Quan Shen, Keyang Xu, Katherine R. Kozak, Helga Raab, Richard Vandlen, Gail D. Lewis Phillips, Richard H. Scheller, Paul Polakis, Mark X. Sliwkowski, John A. Flygare, and Jagath R. Junutula*,‡ Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: Antibody−drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumabMCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker−drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker−drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.

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2 targeting antibody.15 The conjugate is formed by attachment of the linker and drug to the reactive lysines on the surface of the antibody. Since trastuzumab contains 90 lysines,16 the resulting conjugate is a heterogeneous mixture comprising varying drug load (0−8 drugs per antibody) conjugated at a number of reactive lysines.17 To reduce heterogeneity, THIOMAB technology was developed, where cysteine residues were engineered into the antibody at specific sites. The resulting site-specific ADCs allow uniform drug loads of two drugs per antibody, with the site of conjugation specified by the placement of the engineered cysteine.18 Connecting DM1 to an engineered thio-trastuzumab gave a site-specific ADC (ThioTmab-MPEO-DM1) with an improved preclinical therapeutic index over Tmab-MCC-DM1.17 One of the key components to an ADC is the linker connecting the cytotoxic drug to the antibody.4−11,19 TmabMCC-DM1 utilizes a noncleavable linker that enabled the conjugate to retain efficacy while improving safety over conjugates with disulfide cleavable linkers due to improved plasma stability.15 Tmab-MCC-DM1 and thioTmab-MPEODM1 both utilize a maleimide to connect the drug to the linker

INTRODUCTION Natural products have had a profound impact on the treatment of cancer. Their ability to eradicate cancer cells through a variety of mechanisms has resulted in many successful drugs.1,2 Yet the capacity to select for cancer cells in the presence of normal cells has been and will continue to be a significant challenge. This limited selectivity is the cause of many side effects associated with cancer chemotherapy as well as the inability to eliminate the tumor at tolerated doses in many cases. Conversely, monoclonal antibodies are able to exquisitely differentiate between cancer cells and normal cells by binding to tumor-associated antigens that are nonexistent or present only at low levels on normal cells. While several monoclonal antibodies have been approved for the treatment of cancer,3 many others lack therapeutic activity. Connecting a cytotoxic natural product to a monoclonal antibody can potentially synergize the strengths of both while mitigating the potential liabilities. The resulting conjugates, known as antibody−drug conjugates (ADCs),4−11 have been validated clinically,12−14 and ado-trastuzumab emtansine (also known as Tmab-MCC-DM1 or T-DM1), the first ADC for the treatment of solid tumors, has recently been granted approval by the FDA. Tmab-MCC-DM1 consists of a maytansinoid natural product microtubule inhibitor linked to trastuzumab, a HER© XXXX American Chemical Society

Received: December 21, 2013

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and in the case of thioTmab-MPEO-DM1, another maleimide to also connect the linker to the antibody. Maleimides are incorporated into the linker primarily for ease of synthesis of the linker−drug and to facilitate conjugation of linker−drug to the antibody. Yet the product of the reaction between a maleimide and a thiol is a thioether succinimide, a potentially unstable functional group. The thioether succinimide, connecting antibody to linker and linker to drug, can revert through a retro-Michael reaction to the starting material thiol and maleimide, whereupon the maleimide can be trapped by other reactive thiols. The reversibility of this connection between antibody and linker has been demonstrated with several ADCs and has been shown to result in a decrease in efficacy of the corresponding drug conjugate.20,21 Here we investigate the impact of the lability of the functional group connecting the maytansinoid drug and the linker in trastuzumab−drug conjugates. We show that through semisynthesis from the natural product analog maytansinol, a maytansine linker−drug lacking this reversible functional group can be prepared. The resulting drug conjugates have improved efficacies over both the conventional ADC (TmabMCC-DM1) and the recently reported site-specific ADC (thioTmab-MPEO-DM1). In addition, we show that increasing the drug to antibody ratio (DAR) from 2 to 4 by engineering two additional cysteines into the previously reported trastuzumab THIOMAB improves the therapeutic activity.

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RESULTS Rationale. Tmab-MCC-DM1, despite having a noncleavable linker between antibody and drug, showed an appreciable level of deconjugation (plasma: total antibody vs plasma: ADC) and overall instability (buffer: ADC vs plasma: ADC) of ADC in plasma as shown in Figure 1A. Another observation indicating deconjugation of Tmab-MCC-DM1 was the detection of increasing unconjugated (or free) DM1 levels over the incubation time with an in vitro plasma stability study, as shown in Figure 1B. Significantly higher concentrations of unconjugated DM1 were measured in mouse plasma than in buffer. Deconjugation is further supported by the difference in PK between the total antibody and Tmab-MCC-DM1 ADC, which has been observed both preclinically22,23 and clinically,24,25 and by LC−MS analysis of Tmab-MCC-DM1 in human plasma.26 We have reported previously that conjugation of DM1 to an engineered thio-HC-A114C-trastuzumab via a BMPEO (bismaleimidopolyethylene oxide) linker gave a sitespecific ADC (ThioTmab-MPEO-DM1).17 In addition to having a potentially labile bond between linker and drug, thioTmab-MPEO-DM1 also has a labile bond between antibody and linker resulting in deconjugation as described previously (Figure 1C).21 Since a THIOMAB conjugate between LC-V205C-trastuzumab and MPEO-DM1 did not show significant levels of deconjugation between linker−drug and antibody,21 this THIOMAB antibody was chosen for the subsequent studies aimed at improving the stability of the sitespecific ADC. Synthesis of Maytansinoid Linker−Drugs. To enable the lysine-conjugated linker−drug in Tmab-MCC-DM1 to be conjugated to a cysteine-containing THIOMAB, a new bifunctional linker was previously selected (Figure 2).17 This linker, designated BMPEO, utilizes one maleimide to connect to the thiol of the maytansinoid drug DM1, to generate the linker−drug MPEO-DM1. The second maleimide reacts with the cysteine of the antibody. The thioether succinimide

Figure 1. (A) In vitro plasma stability analysis of Tmab-MCC-DM1 showed an appreciable level of deconjugation and overall instability of the ADC in mouse plasma as compared to the buffer control up to 96 h. Concentrations of total and conjugated antibodies were determined using ELISA and the average of three replicates plotted. (B) Formation of unconjugated (or free) DM1, a product of deconjugation of Tmab-MCC-DM1, was seen increasing with time up to 168 h and in mouse plasma vs buffer control measured by LC−MS/MS for the in vitro plasma stability study. (C) Reversibility of the connection between a thiol-containing antibody or drug and a maleimide.

connection formed between the maleimide and the sulfur atom of the drug (labeled in red) is reversible. To investigate the impact of the reversibility of this connection between drug and linker, the maleimide and sulfur atom were removed. These linker−drugs were prepared employing a semisynthesis strategy starting with maytansinol (6) (Scheme 1), a maytansine analog easily obtained from the reduction of the C3-ansamitocin esters produced by microbial fermentation.27 Maytansinol (6) reacts with the N-carboxyanhydride of N-methyl-L-alanine (7) in the presence of zinc triflate to provide amine 3 without epimerization of the alanine side chain. This amine was then coupled with carboxylic acids (5, 9) to generate stable maytansinoid linker−drugs. In one example (10, MPA-May), the MPEO unit was kept and connected by an amide to adipic acid (A). This keeps the same number of atoms between the antibody and the maytansinoid core (May) while removing the reversible connection between linker and drug. Additionally, in a second compound (11, MC-May), the PEO group was removed entirely and the antibody was connected directly to B

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Figure 2. Structures of new maytansinoid linker−drugs and their connection to an antibody.

Scheme 1. Synthetic Route for Stable Maytansinoid Linker−Drugs

antibody (Tmab). The effect of the new linker−drugs on potency was explored in a cell proliferation assay (Figure 3B). ThioTmab-MPA-May and thioTmab-MC-May inhibited growth of SK-BR-3 cells in a 5-day assay with an IC50 of 10.9 and 11.8 ng/mL, respectively. Tmab-MCC-DM1 was twice as potent (IC50 = 5.0 ng/mL), which can be explained by the fact that it had twice the drug load (DAR of 3.5 vs 1.8). In Vitro Plasma Stability of the Engineered Trastuzumab Site-Specific ADCs. To investigate whether the chemical modifications to the linker have resulted in ADCs with

the maytansioid core through a maleimidocaproyl (MC) spacer, a common conjugation spacer for ADCs. In Vitro Binding and the Effect on Cell Proliferation of 2-DAR Site-Specific ADCs. To ensure that conjugation of new linker−drugs did not impact the ability of the antibody to bind to its target antigen, an in vitro binding assay was performed using SK-BR-3 cells (Figure 3A). FACS analysis showed that the two new stably linked maytansinoid ADCs, thioTmab-MPA-May and thioTmab-MC-May, were indistinguishable from Tmab-MCC-DM1 and the unconjugated C

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Figure 3. 2-DAR site-specific Tmab ADCs with stable linker−drugs (ThioTmab-MPA-May and ThioTmab-MC-May) showed (A) similar HER2 binding to and (B) reduced in vitro cell killing compared to Tmab-MCC-DM1 (3.5 DAR) in the HER2-amplified SK-BR-3 breast cancer cell line, with the cell proliferation activity correlating with drug load.

Figure 4. In vitro stability of (A) thioTmab-MPA-May and (B) thioTmab-MC-May in mouse plasma and in (C, D) human plasma. In vitro plasma stability analysis of (A) thioTmab-MPA-May and (B) thioTmab-MC-May ADCs showed low levels of deconjugation and overall stability of the ADC in mouse plasma as compared to the buffer control. Concentrations of total and conjugated antibodies were determined using ELISA, and the average of three replicates was plotted. Similar in vitro plasma stability studies were conducted for thioTmab-MPA-May (C) and thioTmab-MC-May (D) in human plasma, and the samples were analyzed using an LC−MS method.

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improved stability, an in vitro plasma stability study was performed in mouse serum for the period of 0−96 h (Figure 4). ELISA was used to monitor both the total antibody and ADC over time as described in the Experimental Section. Unlike Tmab-MCC-DM1 (shown in Figure 1A), less deconjugation and improved stability was observed with thioTmab-MPA-May and thioTmab-MC-May ADCs, where the labile bond between linker and drug has been removed Figure 4A,B). Similar results were observed upon conducting plasma stability studies in human plasma measured by an independent LC−MS assay (Figure 4C,D). In addition, another in vitro stability study was performed in mouse plasma vs buffer control up to 168 h with thioTmab-LC-MPEO-DM1 and thioTmab-MPA-May, and unconjugated (or free) DM1 was measured using LC−MS/ MS (shown in Figure 5). The unconjugated DM1 level

Figure 5. Concentrations of unconjugated (or free) DM1 released upon incubation of thioTmab-LC-MPEO-DM1 and thioTmab-MPAMay in mouse plasma and PBS buffer (control) up to 168 h and measured by LC−MS/MS. Please note that no detectable DM1 was observed in plasma or buffer control for thioTmab-MPA-May.

appeared to initially increase with time and then plateau indicating drug deconjugation. The DM1 concentration in buffer control also rose more slowly with time but ended up at a similar concentration by 168 h. Concentrations of unconjugated DM1 were significantly lower in thioTmabMPEO-DM1 than that in Tmab-MCC-DM1. In comparison, no detectable unconjugated DM1 (lower limit of quantitation of 2.0 nM) was observed for thioTmab-MPA-May. In Vivo Efficacy of Engineered Trastuzumab 2-DAR Site-Specific ADCs. The impact of linker−drug stability on efficacy was studied in a HER2+ Fo5 transplant tumor model. A single dose of ADC was given at day 0, and tumor growth was measured over time, as compared with a vehicle control. At 10 mg/kg and 2-DAR, thioTmab-MPA-May showed improved efficacy over thioTmab-LC-MPEO-DM1 (10mg/kg, 284 μg drug/m2), where thioTmab-MPA-May (10 mg/kg, 300 μg drug/m2) resulted in a greater %TGI at 93% compared with 79% of the MPEO-DM1 linker drug. This increase in antitumor activity is also reflected in the time to tumor progression (TTP 2×) where tumors in the thioTmab-MPA-May group took a median time of 18 days to progress compared with the shorter time of 8 days in the thioTmab-LC-MPEO-DM1 group. This result is consistent with the improved stability between the linker and drug with MPA-May (Figure 6A). When thioTmabMPA-May and Tmab-MCC-DM1 are compared at the same dose, both antibody drug conjugates had similar efficacy despite the higher DM1 drug load with Tmab-MCC-DM1 (10 mg/kg, 578 μg drug/m2) as shown in Figure 6A (93% vs 92% TGI, and 18 vs 16 days TTP 2×, respectively) and in Figure 6B (103% vs

Figure 6. In vivo efficacy of 2-DAR site-specific Tmab ADCs vs TmabMCC-DM1 in a HER2+ Fo5 mammary tumor transplant mouse model. The MMTV-HER2 Fo5 transgenic mammary tumor was surgically transplanted into the number 2/3 mammary fat pads of nu/ nu mice in sections that measured approximately 2 mm × 2 mm. The tumor-bearing animals were randomized to a mean tumor volume range of 126−152 mm3 (A) and 149−162 mm3 (B) of 10 mice/group and then dosed iv once (day 0) with vehicle, thio gD-MPA-May or thio gD-MC-May controls, thioTmab-LC-MPEO-DM1(10 mg/kg, 284 μg drug/m2), thioTmab-MPA-May (10 mg/kg, 300 μg drug/m2), or Tmab-MCC-DM1 (10 mg/kg, 578 μg drug/m2). Fitted tumor volumes are plotted over time (days post dose). (A) At the same antibody dose, thioTmab-MPA-May showed improved efficacy over thioTmab-LC-MPEO-DM1. (A, B) thioTmab-MPA-May and TmabMCC-DM1 had similar efficacy at 10 mg/kg despite the former having a lower drug load.

103% TGI, and 17 vs 15 days TTP 2×, respectively). This similarity in efficacy between thioTmab-MPA-May and TmabMCC-DM1 at the same antibody dose can be attributed to a combination of differences in stability and drug loading. TmabMCC-DM1 has less stability between linker and drug but a higher drug load than thioTmab-MPA-May. To obtain a better understanding of the efficacy improvement associated with the E

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new stable MPA-May linker−drug, we set out to increase the drug load of the site-specific ADC so that it is similar to the DAR of Tmab-MCC-DM1. In Vitro Binding and the Effect on Cell Proliferation of 4-DAR Site-Specific ADCs. Despite improving the stability of the linker−drug, a direct comparison cannot be made between the improved THIOMAB conjugate (ThioTmab-MPA-May) and Tmab-MCC-DM1 because of differences in drug loading. To address this, a thioTmab containing engineered cysteines in both the heavy (HC-A114C, Kabat numbering) and light chain (LC-V205C) was prepared and conjugated with the new stable maytansinoid linkers (thioTmab-MPA-May, thioTmab-MCMay) to give a DAR of 4. LC−MS quantification analysis determined that the DAR of conjugated Tmab is 3.9. A FACS analysis with SK-BR-3 cells indicated that the 4-DAR sitespecific ADCs showed binding similar to Tmab-MCC-DM1 and unconjugated Tmab (Figure 7A). In an in vitro cell proliferation assay, the 4-DAR ADCs were twice as potent as the 2-DAR site-specific ADCs and equipotent with TmabMCC-DM1 (Figure 3B and Figure 7B). In Vivo Efficacy of Engineered Trastuzumab 4-DAR Site-Specific ADCs. We next analyzed the in vivo efficacy of 4DAR ADCs in a Fo5 transplant model as described above. As shown in Figure 6, the 2-DAR thioTmab-MPA-May was as efficacious as Tmab-MCC-DM1 at 10 mg/kg. The 4-DAR was tested at 5 and 10 mg/kg. As expected, 5 mg/kg of the 4-DAR ADCs (5 mg/kg, 326 μg drug/m2) resulted in less efficacy than Tmab-MCC-DM1 (10 mg/kg, 578 μg drug/m2), likely due to halving the drug dose. The slight difference in activity between thioTmab-MC-May and -MPA-May is likely due to tumor growth variability. However, gratifyingly, the 4-DAR ADC thioTmab-MC-May and thioTmab-MPA-May (10 mg/kg, 652 μg drug/m2) demonstrated improved efficacy over TmabMCC-DM1, with tumor stasis being observed with a single dose of 10 mg/kg (Figure 7C). This improved activity is most clearly observed in the time to tumor progression where TmabMCC-DM1-treated tumors took a median time of 16 days to progress while the MPA-May and MC-May-treated tumors took about twice as long (32.5 and 28.5 days, respectively).

Figure 7. 4-DAR site specific Tmab ADCs with stable linker−drugs (thioTmab-MPA-May and thioTmab-MC-May) showed (A) similar HER2 binding to and (B) equivalent in vitro cell killing compared to Tmab-MCC-DM1 (3.5 DAR) in the HER2-amplified SK-BR-3 breast cancer cell line. (C) In vivo efficacy in a HER2+ Fo5 mammary tumor transplant mouse model. The MMTV-HER2 Fo5 transgenic mammary tumor was surgically transplanted into the number 2/3 mammary fat pads of nu/nu mice in sections that measured approximately 2 mm × 2 mm. The tumor-bearing animals were randomized to a mean tumor volume range of 149−162 mm3 of 10 mice/group and then dosed iv once (day 0) with vehicle or thio gDMC-May controls, thioTmab-MC-May, thioTmab-MPA-May, or Tmab-MCC-DM1 at the doses indicated. Fitted tumor volumes are plotted over time (days post dose). 4-DAR site-specific ADCs at 5 mg/kg (326 μg drug/m2) resulted in less efficacy than Tmab-MCCDM1 at 10 mg/kg (578 μg drug/m2). However, as expected, 10 mg/ kg (652 μg drug/m2) of site-specific 4-DAR ADCs demonstrated superior efficacy over Tmab-MCC-DM1 at the same antibody dose.

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DISCUSSION Tmab-MCC-DM1 or T-DM1 was recently approved by the FDA for the treatment of HER2-positive metastatic breast cancer. It is the first ADC approved for the treatment of solid tumors and therefore sets the stage for the next generation of ADCs. The phase III clinical data demonstrate that TmabMCC-DM1 has a very favorable safety profile combined with a significant improvement in progression-free and overall survival over capecitabine plus lapatinib in patients with HER2-positive MBC previously treated with trastuzumab and a taxane.14 However, Tmab-MCC-DM1 has a relatively narrow therapeutic window with a DLT observed at 4.8 mg/kg compared to the clinically efficacious dose of 3.6 mg/kg. Therefore, a second generation ADC with an improved therapeutic index might be desirable. Hence an attempt was made to design site-specific maytansinoid conjugates with improved relative linker stability and therapeutic activity. Our approach toward addressing this challenge was to improve efficacy through modulating the linker and increasing the drug load. Through rational design, we aimed to create a linker with a high level of stability. Previous studies have shown that maleimide exchange has an impact on the pharmacokinetics of an ADC. This exchange results in loss of drug from

the antibody and a corresponding reduction in efficacy of the ADC.21 While the maleimide in Tmab-MCC-DM1 was installed to facilitate the connection of the linker and drug, we envisioned a different synthetic connection between the linker and drug, allowing the assembly of a more stable ADC. To improve stability, two linker−drugs (MPA-May, MCMay) were synthesized that lacked a reversible thioether succinimide connection between the drug and linker and these were conjugated at the LC-V205C site of trastuzumab to minimize the maleimide exchange.21 This change in the linker− drug had no impact on the ability of the resulting conjugates to bind to their target, and the conjugates had in vitro potency half that of Tmab-MCC-DM1, correlating with half the drug load. The new conjugates had increased in vitro stability in plasma F

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tert-Butyl 2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethylcarbamate (2). To a solution of diamine 1 (5.00 g, 0.0260 mol) in THF (525 mL) was added 4-dimethylaminopyridine (320 mg, 0.0026 mol). To this was added a solution of di-tert-butyl dicarbonate (5.68 g, 0.0260 mol) in THF (100 mL) over a period of 1 h, using an addition funnel, all at room temperature. The mixture initially became cloudy but then cleared. The mixture was stirred an additional 2 h and then concentrated and purified by ISCO (0−20% MeOH/DCM) to provide mono-Boc diamine 2 as a light yellow oil (2.70 g, 36%). MS [M + H]+ 293.3. 1H NMR (400 MHz, CDCl3): δ 5.79 (s, 1H), 3.69− 3.57 (m, 8H), 3.56−3.47 (m, 4H), 3.32−3.23 (m, 2H), 2.84 (t, J = 5.1 Hz, 2H), 1.44 (s, 9H). Boc-aminopolyethelene Glycol Adipic Acid (3). To a flask containing amine 2 (1.219 g, 4.169 mmol) and hexanedioic acid (adipic acid, 3.046 g, 20.84 mmol) in tetrahydrofuran (100 mL) was added N,N′-dicyclohexylcarbodiimide (1.23 g, 5.98 mmol). The mixture was stirred at room temperature and became cloudy. After 2 h, the solution was cooled to 0 °C and the byproduct N,N′dicyclohexylurea was removed by filtration. The mixture was concentrated onto silica gel and purified via ISCO (40 g column, 0−10% MeOH/DCM). Concentration provided amide 3 as a clear oil (1.28 g, 73%). MS [M + H]+ 421.4. 1H NMR (400 MHz, CDCl3): δ 10.55 (s, 1H), 6.65 (s, 1H), 5.27 (s, 1H), 3.67−3.62 (m, 8H), 3.60− 3.52 (m, 4H), 3.48−3.41 (m, 2H), 3.35−3.23 (m, 2H), 2.35 (t, J = 6.3 Hz, 3H), 2.25 (t, J = 6.5 Hz, 2H), 1.44 (s, 9H). Maleimidopolyethylene Glycol Adipic Acid (MPA, 5). To a vial containing Boc-protected amine 3 (682.3 mg, 1.623 mmol) was added 4 mL of 4 M hydrogen chloride in 1,4-dioxane. The mixture was stirred for 30 min and then concentrated. A saturated aqueous solution of sodium bicarbonate (5.1 mL) was added. The solution was cooled to 0 °C and stirred for 10 min, and then N-methoxycarbonylmaleimide (4) (251.7 mg, 1.622 mmol) was added. The mixture was stirred for 20 min more at 0 °C and then warmed to room temperature. The solution was diluted with DMF, quenched with 5 drops of formic acid, filtered, and purified by RP-HPLC to provide maleimide 5 as a clear oil (297 mg, 46%). MS [M + H]+ 401.3. 1H NMR (400 MHz, CDCl3): δ 8.66 (s, 1H), 6.56 (t, J = 4.8 Hz, 1H), 3.73 (t, J = 5.6 Hz, 2H), 3.66− 3.60 (m, 9H), 3.56 (t, J = 5.0 Hz, 2H), 3.47−3.41 (m, 2H), 2.35 (t, J = 6.7 Hz, 2H), 2.24 (t, J = 6.9 Hz, 2H), 1.77−1.58 (m, 4H). 3-(S-(N-Methylalaninyl)maytansinol (8). To a solution of maytansinol (6) (50.0 mg, 0.0885 mmol) in DMF (1.12 mL, 14.5 mmol) and THF (380 μL, 4.6 mmol) were added N,Ndiisopropylethylamine (62 μL, 0.35 mmol), zinc triflate (129 mg, 0.354 mmol), and the N-carboxyanhydride of N-methyl-L-alanine (7) (80.0 mg, 0.619 mmol). The mixture was stirred for 24 h, and ethyl acetate (2 mL) was added. Then over 5 min, 2 mL of a saturated 1:1 sodium bicarbonate (aq)/sodium chloride (aq) solution was added. The solution was stirred for 30 min, and the salts were filtered and rinsed with ethyl acetate. The two phases were separated, and the aqueous phase was extracted with 3 × 2 mL of ethyl acetate. The combined organic phases were concentrated to 0.25 mL. Then 2 mL of ethyl acetate was added, and the solution was again reduced to 0.25 mL. This dilution and concentration were done once more. Finally, ethyl acetate was added to give around 2 mL of solution, and salts that had precipitated were filtered through a 0.45 μm syringe filter to give ester 8. Maleimidopolyethylene Glycol Amidoadipic Acid Maytansinoid (MPA-May, 10). To a solution of amine 8 was added acid 5 (65.5 mg, 0.164 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (31.4 mg, 0.164 mmol), and N,N-diisopropylethylamine (7.71 μL, 0.0442 mmol). The mixture was stirred for 2 h, and the mixture was filtered and purified on RP-HPLC to provide MPA-May (10) as a clear oil (48.8 mg, 53%). MS [M + H]+ 1032.7. 1 H NMR (400 MHz, CDCl3): δ 6.83 (s, 1H), 6.71 (s, 2H), 6.70−6.64 (m, 2H), 6.47−6.37 (m, 2H), 6.27 (t, J = 4.8 Hz, 1H), 5.67 (dd, J = 15.3, 9.1 Hz, 1H), 5.35 (q, J = 6.7 Hz, 1H), 4.78 (dd, J = 12.0, 2.8 Hz, 1H), 4.29 (t, J = 10.8 Hz, 1H), 3.98 (s, 3H), 3.72 (t, J = 5.7 Hz, 2H), 3.67−3.56 (m, 12H), 3.54−3.48 (m, 3H), 3.44−3.38 (m, 2H), 3.36 (s, 3H), 3.19 (s, 3H), 3.11 (d, J = 12.7 Hz, 1H), 3.01 (d, J = 9.6 Hz, 1H), 2.84 (s, 3H), 2.60 (dd, J = 14.1, 12.4 Hz, 1H), 2.48−2.38 (m, 1H),

relative to Tmab-MCC-DM1 over time, which can be caused by both a reduction in the deconjugation and an increased stability of the engineered ADC by eliminating higher drugloaded species. It is interesting to note that the stability of the antibody itself may be compromised in plasma at 37 °C for the Tmab-MCC-DM1 ADC, perhaps because of aggregation. In contrast, antibodies of engineered ADCs were stable in plasma. A direct evidence of enhanced stability was that no released drug, i.e., DM1, was detected in thioTmab-MPA-May vs up to approximately 180 nM DM1 was released in Tmab-MCC-DM1 incubated in mouse plasma for 168 h. This translated to improved efficacy, with the stabilized conjugates matching the efficacy of Tmab-MCC-DM1 with half the amount of drug while showing improved efficacy over the previously reported site-specific THIOMAB ADC (ThioTmab-MPEO-DM1) with the same drug load. To improve efficacy even further, four cysteine residues were engineered into the trastuzumab antibody. The resulting 4-DAR site-specific ADCs (ThioTmab-MPA-May, ThioTmab-MC-May) matched the in vitro potency of Tmab-MCC-DM1 while showing superior efficacy. It was important that the change in linker−drug not have an impact on the mechanism of action of the drug or the drug’s metabolism. While an ester at C3 on the maytansine core is required for activity, the nature of that ester is not important.28 Since the only changes to the linker−drug were in modifications of the C3 ester, the stabilized linker−drugs did not impact the potency of the maytansinoid payload as evidenced by their potent inhibition of cell proliferation. It has also been demonstrated that while changes to the linker of maytansinoid ADCs can result in different pharmacokinetics, they do not impact the processing of the conjugates to their respective catabolites.22 As evidenced by the increased plasma stability and efficacy of the stabilized linker−drugs, the linker changes are likely to improve the pharmacokinetic properties. On the basis of historical data, there seems to be a good correlation between drug load and safety.23 Site-specific ADCs with a DAR of 2 showed a 2-fold improvement in safety over Tmab-MCC-DM117 Therefore, it is reasonable to expect that doubling the drug load may make the 4-DAR site-specific ADC more toxic than the 2-DAR site-specific ADC. However, more importantly, doubling the drug load did increase the efficacy of the site-specific ADC. It is believed that increasing the efficacy is likely an impactful way of improving clinical activity for trastuzumab maytansinoid ADCs, given the unpredictable thrombocytopenia that was not seen in preclinical safety studies but is a dose-limiting factor in humans. The newly engineered trastuzumab site-specific ADCs with improved therapeutic activity can therefore serve as a potential nextgeneration ADC for HER2+ breast cancer patients.

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All solvents and reagents were used as obtained. 1H NMR spectra were recorded with a Bruker Avance DPX400 spectrometer or a Varian Inova 400 NMR spectrometer and referenced to tetramethylsilane. Chemical shifts are expressed as δ units using tetramethylsilane as the internal standard (in NMR description, s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak). All coupling constants (J) are reported in hertz. Mass spectra were measured with a Finnigan SSQ710C spectrometer using an ESI source coupled to a Waters 600MS HPLC system operating in reverse mode with an X-bridge phenyl column of dimensions 150 mm by 2.6 mm, with 5 μm sized particles. For all compounds evaluated biologically, a purity of >95% was confirmed by HPLC. G

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ELISA Analysis. Plasma samples from the stability study were analyzed by ELISA for total antibody (conjugated plus unconjugated antibody) and conjugate (conjugated antibody) concentrations. For the total antibody ELISA-based assay, microtiter plates (384 wells) (Nunc, Rochester, NY, USA) were coated with human Her2 extracellular domain (Genentech, Inc.) at 0.4 μg/mL in coat buffer (0.05 M carbonate/bicarbonate buffer, pH 9.6). After an overnight incubation at 4 °C, assay plates were washed and treated with blocking buffer (PBS buffer, pH 7.4, containing 0.05% Tween 20, 0.05% Proclin 300) for 1−2 h before a 2 h incubation with DM1-conjugated standards and samples diluted in sample diluent (PBS buffer, pH 7.4, containing 0.5% BSA, 10 ppm Proclin, 0.05% Tween 20, 0.25% CHAPS, 5 mM EDTA, and 0.35 M NaCl). Assay plates were first washed 6 times with wash buffer (PBS buffer, pH 7.4, containing 0.05% Tween 20) and then incubated for 1 h with AffiniPure F(ab′)2 fragment goat anti-human Fc, Fcγ fragment specific HRP conjugated (GxhFc-HRP) (Jackson Immuno-Research Laboratory) diluted to 40 ng/mL. After washing (6 times), the detection step was done using tetramethylbenzidine (TMB) substrate (Moss, Pasadena, MD, USA). Absorbance was measured at 450 nm against a reference wavelength of 620 nm. The limit of quantitation (LOQ) was 3 ng/mL. For the DM1conjugated antibody ELISA-based assay, microtiter plates (384-well) (Nunc) were coated with 0.5 μg/mL anti-DM1 antibody (clone 2G9.5.7, produced at Genentech, South San Francisco, CA) in coat buffer. After an overnight incubation at 4 °C, assay plates were washed and treated with block buffer for 1−2 h before 2 h incubation with DM1-conjugate standards and samples diluted in sample diluent. Assay plates were washed 6 times with wash buffer and incubated for 1 h with goat anti-human IgG conjugated to HRP (GxhIgG-HRP) diluted to 25 ng/mL in assay buffer. After washing (6 times), the detection step was carried out using TMB substrate. Absorbance was measured at 450 nm against a reference wavelength of 620 nm. The limit of quantitation (LOQ) was 7 ng/mL. Quantification of Unconjugated (or Free) DM1 by LC−MS/ MS. Calibration standards ranging from 2.00 to 500 nM DM1 (SICOR Pharmaceuticals Inc., Irvine, CA) were prepared in mouse plasma or PBS buffer. An aliquot of 30 μL of in vitro incubation samples, standards, or matrix blank was reduced with 1.25 μL of 26 mM tris(2-carboxyethyl)phosphine (TCEP) (neutral pH) (Thermo Scientific, Rockford, IL) by incubation at 37 °C for 15 min. Samples were treated by protein precipitation with 120 μL of internal standard (7.5 nM maytansine from Immunogen, Inc., Waltham, MA, in 80% acetonitrile). Derivatization was conducted by addition of 6.32 μL of 25 mM N-ethylmaleimide (NEM) (Sigma-Aldrich, Saint Louis, MO) to 80 μL of supernatant and incubation at 37 °C for 45 min. NEMderivatized samples were subjected to separation using a reverse phase analytical column (Phenomenex Synergi MAX-RP 80A, C12, 4 μm, 50 mm × 2.00 mm) (Torrance, CA) heated to 50 °C run on a Shimadzu SCL-10A VP system (Columbia, MD). Separation was performed at a flow rate of 0.5 mL/min with a linear gradient using mobile phase A (5 mM ammonium acetate with 0.1% formic acid) and mobile phase B (5 mM ammonium acetate in 95% acetonitrile with 0.1% formic acid). Mass spectra were recorded on a 4000 QTRAP system (AB Sciex, Redwood City, CA) in the multiple reaction monitoring (MRM) mode. Transition m/z 845.7/485.3 was monitored for DM1-NEM, m/ z 738.5/547.3 for DM1, and m/z 692.6/547.2 for maytansine. Data analysis was done with Analyst 1.6 software from AB Sciex. The lower limit of quantitation (LLOQ) of DM1-NEM is 2.0 nM. Affinity Capture LC−MS. Details of the affinity capture LC−MS procedure were described previously.29 Briefly, an aliquot (typically 50 μL) of plasma or serum was added to a 96-well plate containing the HBS-EP buffer followed by addition of streptavidin paramagnetic beads coupled with the biotinylated HER2 extracellular domain (ECD) and incubated at room temperature for 2 h. The ADC analytes captured by the beads were isolated, washed, and subsequently deglycosylated by PNGase F overnight at 37 °C. The beads were extensively washed to remove nonspecifically bound proteins. The ADC was eluted by 30% acetonitrile in water with 1% formic acid and injected onto an LC−MS instrument for analysis. The ADC was analyzed by a Q-STAR XL quadrupole time-of-flight mass

2.30−2.13 (m, 4H), 1.75−1.58 (m, 8H), 1.52−1.40 (m, 1H), 1.29 (t, J = 6.0 Hz, 6H), 1.22 (d, J = 12.9 Hz, 1H), 0.80 (s, 3H). Maleimidocaproyl Maytansinoid (MC-May, 11). To the solution of amine 8 were added 9 (34.6 mg, 0.164 mmol), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (31.4 mg, 0.164 mmol), and N,N-diisopropylethylamine (7.71 μL, 0.0442 mmol). The mixture was stirred for 2 h, and the mixture was filtered and purified on RP-HPLC to provide MC-May (11) as a clear oil (30.7 mg, 41%). MS [M + H]+ 843.5. 1H NMR (400 MHz, CD3OD): δ 7.11 (s, 1H), 6.76 (s, 2H), 6.72−6.65 (m, 2H), 6.60 (dd, J = 14.7, 11.4 Hz, 1H), 5.69 (dd, J = 14.9, 9.1 Hz, 1H), 5.49 (q, J = 6.7 Hz, 1H), 4.65 (dd, J = 11.9, 2.1 Hz, 1H), 4.19 (td, J = 10.3, 4.1 Hz, 1H), 3.97 (s, 3H), 3.62−3.55 (m, 2H), 3.41−3.34 (m, 5H), 3.23 (d, J = 12.7 Hz, 1H), 3.20 (s, 3H), 2.94 (d, J = 9.6 Hz, 1H), 2.84 (s, 3H), 2.72−2.62 (m, 1H), 2.56−2.45 (m, 1H), 2.33−2.23 (m, 1H), 2.14 (dd, J = 14.1, 1.8 Hz, 1H), 1.68 (s, 3H), 1.65−1.42 (m, 7H), 1.29 (d, J = 6.8 Hz, 3H), 1.28−1.25 (m, 2H), 1.23 (d, J = 6.3 Hz, 3H), 0.84 (s, 3H). Antibody−Drug Conjugates. Tmab-MCC-DM1 and thioTmabLC-MPEO-DM1 were prepared as described earlier.15−17 Construction and production of the THIOMAB variants of trastuzumab (HCA114C and LC-V205C, Kabat numbering) were previously reported.17,18,21 The drug to antibody ratios (DARs) of the ADCs were determined by liquid chromatography/mass spectrometry (LC/ MS) analysis as previously described.18 The LC-V205C THIOMAB resulted in 2-DAR ADCs upon conjugation with thiol reactive maleimide linker drugs. To construct a 4-DAR engineered ADC, we have used HC-A114C and LC-V205C constructs co-transfected to generate a THIOMAB with four engineered cysteines. Site-specific 4DAR ADCs were produced using the same method as described earlier for 2-DAR site specific ADCs.17 The resulting thioTMab-MPA-May and thioTMab-MC-May site-specific ADCs had DAR of 3.9 with less than 3% aggregation as characterized by LC−MS and size exclusion chromatography analysis, respectively. Flow Cytometric Analysis and Cell Viability Assays. The SKBR-3 cell line used in this study, a cell line with high human epidermal growth factor receptor 2 (HER2) expression with ∼2 million copies per cell, was obtained from the American Type Culture Collection, and cells were cultured in Ham’s F-12: high glucose (50:50) supplemented with 10% heat-inactivated fetal bovine serum and 2 mmol/L L-glutamine (all from Invitrogen Corp.). SK-BR-3 cells (100,000 cells per sample) were incubated on ice with trastuzumab, Tmab-MCC-DM1, thioTmab-MPA-May, or thioTmab-MC-May at various concentrations in the range of 0−1600 ng/mL in fluorescenceactivated cell sorting (FACS) buffer (PBS buffer containing 1% bovine serum albumin) for 1 h. Cells were washed with FACS buffer and incubated with phycoerythrin-labeled goat anti-human Fc secondary antibody (1:3000 dilution) on ice for 1 h. All samples were washed using FACS buffer and fixed with 2% paraformaldehyde followed by FACS analysis using a BD FACS Calibur system (BD Biosciences). Cell viability assays in the presence of Tmab-MCC-DM1, thioTmabMPA-May, and thioTmab-MC-May (0−10 μg/mL) were carried out in SK-BR-3 cells in a 96-well format as previously described.15 In Vitro Plasma Stability Studies. Plasma (lithium heparin) samples (mouse and human) were purchased from Bioreclamation. Plasma was thawed, spun at 157g (Eppendorf centrifuge 5810R) for 5 min followed by filtration through a 0.22 μm filter (Pall Corp.) into sterile polypropylene tubes and kept on ice. PBS buffer was filtered and kept on ice. Conjugate stock solutions were added to selected plasma samples at a final concentration of 100 μg/mL. PBS with 0.5% BSA was used as a control. Aliquots of 100 μL from each mixture were transferred into sialylated microcentrifuge tubes and incubated at 37 °C in a CO2 incubator to maintain plasma pH levels close to the physiological pH of 7.2 throughout the incubation period. To terminate the reaction, samples were transferred to a −80 °C freezer at predetermined time points (0, 24, and 96 h). Additional time points (6, 48, and 168 h) were collected for the measurement of unconjugated drug (DM1). The 0 h collection was placed on dry ice within the first minute following the addition of the conjugate. Samples were stored in a −80 °C freezer until further analysis. H

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spectrometer (AB Sciex) equipped with a turbo ionspray source and coupled with a reversed phase PLRP-S capillary column (Varian, 0.3 mm × 50 mm, 5 μm, 4000 Å). The chromatographic separation was conducted under a gradient condition at a flow rate of 15 μL/min. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. Positive time-of-flight (TOF) MS scan was acquired. In Vivo Efficacy Studies. The Fo5 mouse mammary tumor model was employed to evaluate the in vivo efficacy of Tmab-MCC-DM1, thioTmab-LC-MPEO-DM1, thioTmab-MPA-May, and thioTmabMC-May (single-dose iv injections) as described previously.15 The Fo5 model is a transgenic mouse model in which the human HER2 gene is overexpressed in mammary epithelium under transcriptional regulation of the murine mammary tumor virus promoter (MMTVHER2). The HER2 overexpression causes spontaneous development of a mammary tumor. The mammary tumor of one of these founder animals [founder 5 (Fo5)] has been propagated in subsequent generations of FVB mice by serial transplantation of tumor fragments (∼2 mm × 2 mm in size). All treatment groups consisted of 10 animals per group, and tumor size was monitored twice weekly using caliper measurement. The tumor volume (mm3) was calculated using the following formula: length × width2 × 0.5. The log 2 (tumor volume) growth traces were fitted to each treatment group with restricted cubic splines for the fixed time effect in each group. To analyze the repeated measurement of tumor volumes from the same animals over time, a mixed-modeling approach was used. Fitting was done as previously described30 via a linear mixed effects model, using the R package “nlme”, version 3.1-108,31,32 in R, version 2.15.2 (R Development Core Team 2012; R Foundation for Statistical Computing; Vienna, Austria). Tumor growth inhibition (TGI) as a percentage of control was calculated as the percentage of the area under the fitted tumor volume−time curve (AUC) per day reduction compared to control based on the fitted curves for only those days where the control group still has animals present, using the following formula: %TGI = 100 × [1 − (AUCdose per day ÷ AUCvehicle per day)]. A TGI value of 100% indicates tumor stasis. A TGI value of >1% but 100% indicates tumor regression. Time to tumor progression of 2× (TTP 2×) is defined as the median time (in days) for a group’s tumors to progress to twice the median tumor volume of the vehicle group at time 0. Plotting was performed using Prism 5 (GraphPad software, San Diego, CA). All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

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ABBREVIATIONS USED ADC, antibody−drug conjugate; MCC, 4-[Nmaleimidomethyl]cyclohexane-1-carboxylate; DM1, an analog of the natural product maytansine; Tmab, trastuzumab

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*T.H.P.: phone, 650-225-1652; e-mail, [email protected]. *J.R.J.: phone, 650-703-2498; e-mail, [email protected]. Present Address ‡

J.R.J.: Cellerant Therapeutics, 1561 Industrial Road, San Carlos, CA, 94070. Author Contributions †

T.H.P. and J.T. contributed equally.

Notes

The authors declare the following competing financial interest(s): All authors are (or were) full time employees of Genentech, Inc. at the time this work was conducted.

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ACKNOWLEDGMENTS We thank our Genentech colleagues from the Early Stage Cell Culture and Protein Chemistry Departments for their help with large-scale production of trastuzumab and its THIOMAB variants. We also thank the core facilities: oligonucleotide synthesis, DNA sequencing, and DNA purification. I

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(32) R Development Core Team 2012. R Package, version 3.1-110; R Foundation for Statistical Computing: Vienna, Austria, 2012.

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