Nature Biotechnology: doi: /nbt Supplementary Figure 1

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1 Supplementary Figure 1 Generation of homogeneous 8-load or heterogeneous 4-load cysteine-conjugated ADCs from IgG1s. (a) The number of reactive cysteine residues available for drug conjugation is determined by the extent of disulfide reduction using the reducing agent TCEP. Conventional cysteine conjugates with an average DAR of 4 are made by reduction with about 2 equivalents of TCEP, resulting in a heterogeneous mixture of reduced antibody products (major products are shown), with an average of 2 reduced disulfides, and hence 4 reactive thiols 4. By reducing with a molar excess of TCEP (> 5 equivalents), all of the accessible disulfides are reduced resulting in a homogeneous antibody with 8 thiols available for conjugation. Provided that an excess of drug-linker is added in the subsequent conjugation step, this will result in an ADC with highly homogeneous drug loading. Compared to chromatographic fractionation of heterogeneous ADCs as reported by others 19, this methodology is simpler and produces a more homogeneous product profile. (b) ydrophobic interaction chromatography of DAR 8 (left) and DAR 4 (right) formats of h1f6-1 separates the individual ADC species on the basis of drug loading, revealing the difference in heterogeneity between the two constructs. (c) Whole-antibody mass spectrometry provides a second methodology for assessing drug loading heterogeneity 20. ature Biotechnology: doi: /nbt.3212

2 Supplementary Figure 2 epatic uptake of h1f6 antibody or h1f6-1 ADC visualized by IC. Prior biodistribution studies with MMAF ADCs revlealed significant concentations of auristatin in the liver by mass spectrometry, but specific sites of uptake were not known 21, 22. In the current study, Sprague-Dawley rats were dosed at 6 mg/kg and sacrificed at the times indicated. The livers were perfused and stained with anti-human Fc detection antibody as described in methods. Antibody uptake is high in areas stained dark brown. a) Rats were dosed with either h1f6 parent antibody (left) or homogeneous DAR 8 h1f6 1 (right) and livers were harvested after 1 hour. Intense, punctate staining of the sindusoidal endothelium and Kupffer cells was observed for the ADC, but not for the unconjugated antibody (bar = 50 μm). (b) A kinetic study to follow the timing of this distribution pattern revealed that the ADC uptake was very rapid, with similarly intense staining observed from 15 minutes to 6 hours, and detectable ADC within the hepatic sinusoids for 48 hours. ature Biotechnology: doi: /nbt.3212

3 Supplementary Figure 3 Effect of disulfide reduction on antibody pharmacokinetics in rats. (a) The four interchain disulfides of h1f6 were reduced with excess tris-carboxyethylphosphine (TCEP) and the resulting 8 thiols were conjugated with the hydrophilic capping agent -(2-aminoethyl)-maleimide (AEM). The pharmacokinetics of reduced and capped h1f6 were compared to unmodified h1f6 in Sprague-Dawley rats at 3 mg/kg. (b) Pharmacokinetic parameters for three separate antibodies evaluated by this process: h1f6 (anti-cd70), hbu12 (anti-cd19), and cac10 (anti-cd30). In all cases reduction of the disulfides and capping with AEM has minimal impact on pharmacokinetics. Error bars represent the standard deviation from groups of three animals. ature Biotechnology: doi: /nbt.3212

4 Supplementary Figure 4 Antitumor activities of ADCs prepared with drug-linkers 1 3. (a) Relative in vitro activities of DAR 4 h1f6 1 and DAR 8 h1f6 3 against 786- cells indicates that h1f6 1 has greater intrinsic potency, despite its inferior antitumor activity in vivo. Error bars represent standard deviation from measurements in triplicate. (b) Relative in vivo activities of cac10 1, 2, and 3 in the L540cy model of odgkin lymphoma. ADCs were prepared at their optimum DARs of 4 (cac10 1 and 2) or 8 (cac10 3) and dosed as indicated. ature Biotechnology: doi: /nbt.3212

5 Supplementary Figure 5 Effect of ADC PEGylation on antigen binding. Binding of unconjugated cac10 antibody or ADCs with drug-linkers 4, 5, and 6 to CD30 + L540cy cells was determined by competition with fluorescently labeled cac10. nly negligible differences between the ADCs or unconjugated antibody were observed. ature Biotechnology: doi: /nbt.3212

6 Supplementary Figure 6 epatic uptake of cac10-4, -5, and -6 ADCs visualized by IC. Sprague-Dawley rats were dosed at 6 mg/kg and sacrificed at 1 hour post-dose. The livers were perfused and stained with anti-human Fc detection antibody as described in methods. Antibody uptake is high in areas stained dark brown. Substantial staining is observed in reticuloendothelial cells (including sinusoidal endothelium and Kupffer cells) for the non-pegylated ADC cac10 4 (a) as well as for cac10 5, in which the drug payload is positioned on the end of a PEG tether (b). In contrast, minimal hepatic staining was observed for cac10 6 with the branched geometry of the PEGylated drug-linker (c). ature Biotechnology: doi: /nbt.3212

7 Supplementary Figure 7 Stability of cac10-4 and -6 ADCs in rat plasma, evaluated by reversed-phase PLC analysis. Loss of drug-linker over time due to maleimide elimination is nearly identical for the PEGylated and non-pegylated drug-linkers. The plateau effect in drug loss observed after 3 days is due to hydrolysis of the drug-antibody thiosuccinimide linkage, which has a stabilizing effect 18. ature Biotechnology: doi: /nbt.3212

8 Supplementary Figure 8 Intratumoral concentrations of MMAE delivered by cac10 ADCs. L540cy xenografts were grown to 200 mm 3 prior to dosing with the indicated cac10 ADCs at 1 mg/kg.after 3 days, tumors were harvested and MMAE concentrations within the tumor homogenate were determined by LC-MS/MS. bserved concentrations followed the same trend as ADC pharmacokinetics, with PEG in the branched configuration (cac10-6) resulting in slightly greater MMAE delivery than the non-pegylated ADC (cac10-4), while PEG-tethering (cac10-5) substantially diminished drug concentrations in the tumor. ature Biotechnology: doi: /nbt.3212

9 Supplementary Figure 9 Effect of drug loading on the antitumor activity of cac10-6. Karpas-299 xenografts were grown to 100 mm 3 then animals dosed with 0.15 mg/kg of cac10 6 at a DAR of 4 or 8. The DAR 8 ADC produced cures in 5 of the 6 animals in the group, while the DAR 4 ADC produced no cures and only a modest delay in tumor outgrowth. This result contrasts sharply with what has previously been reported 2 for cac10 conjugates of val-cit-pab-mmae in the same xenograft model, where the DAR 4 ADC was superior to the DAR 8. ature Biotechnology: doi: /nbt.3212

10 Synthesis of drug-linkers Unless otherwise noted, materials used for synthesis were obtained from commercial sources in the highest grade purity available and used without further purifications. Anhydrous DMF and C 2 Cl 2 were purchased from Aldrich. Fmoc-Dap- and Dov-Val-Dil- were custom synthesized by Albany Molecular Research, Inc. Fmoc protected amino acids and 2-Cl-Tritylchloride resin (substitution 1 mmol/g, mesh, 1% DVB) were purchased from ovabiochem. Fmoc-L-Dpr(Alloc)- was acquired from Chem-Impex International. MDpr was prepared as described previously 18. Solid phase synthesis was performed in plastic syringes (ational Scientific Company) fitted with a filter cut out of fritware PE medium grade porous sheet (Scienceware). A Burrell wrist action shaker (Burrell Scientific) was used for agitation. All solid-phase synthesis yields reported are based upon the initial substitution level of the resin and constitute a mass balance of isolated pure material, unless otherwise stated. PEG reagents were obtained from Quanta BioDesign. Radial chromatography was performed on a Chromatotron apparatus (arris Research). Column chromatography was performed on a Biotage Isolera ne flash purification system. Preparative PLC purification of solid phase synthesis products was performed on Varian instrument equipped with C12 Phenomenex Synergy MAX-RP 4µm reversed phase column, mm, eluting with 0.05% TFA in a gradient acetonitrile from 10 to 80 % in 60 min with monitoring at 220 nm. Preparative PLC purification of solution phase synthesis products was carried out on a ProStar 210 solvent delivery system configured with a ProStar 330 PDA detector (Varian). Products were purified over a C12 Synergi 10.0 x 250 mm, 4 μm, 80 Å reverse phase column (Phenomenex) eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The purification method consisted of the following gradient of solvent A to solvent B: 90:10 from 0 to 5 min; 90:10 to 10:90 from 5 min to 80 min; followed by isocratic 10:90 for 5 min. The flow rate was 4.6 ml/min with monitoring at 254 nm. Analytical thin layer chromatography was performed on silica gel 60 F254 aluminum sheets (EMD Chemicals, Gibbstown, J). Analytical PLC was performed on a ProStar 210 solvent delivery system configured with a ProStar 330 PDA detector. Samples were eluted over a C12 Synergi 2.0 x 150 mm, 4 μm, 80 Ǻ reverse-phase column. The acidic mobile phase consisted of acetonitrile and water both containing 0.1% formic acid. Compounds were eluted with a linear gradient of acidic acetonitrile from 5% at 1 min post injection, to 95% at 11 min, followed by isocratic 95% acetonitrile to 15 min (flow rate = 1.0 ml/min). LC-MS was performed on three different systems. LC-MS system 1 consisted of a ZMD Micromass mass spectrometer (Waters) interfaced to an P Agilent 1100 PLC instrument equipped with a C12 Synergi 2.0 x 150 mm, 4 μm, 80 Å reverse phase column. The acidic eluent consisted of a linear gradient of acetonitrile from 5% to 95% in 0.1% aqueous formic acid over 10 min, followed by isocratic 95% acetonitrile for 5 min (flow rate = 0.4 ml/min). LC-MS system 2 consisted of a Xevo G2 Tof mass spectrometer (Waters) interfaced to a 2695 Separations Module with a 2996 Photodiode Array Detector (Waters); the column, mobile phases, gradient, and flow rate were same as for LC-MS system 1. LC-MS system 3 consisted of an Acquity UPLC-SQ MS system (Waters) equipped with an Acquity UPLC BE C µm, 2.1 x 50 mm reverse-phase column. The eluent consisted of a linear gradient of acetonitrile from 3% to 97% in 0.1% aqueous formic acid over 2 min, followed by isocratic 97% 1 ature Biotechnology: doi: /nbt.3212

11 acetonitrile for 1 min at flow rate 0.5 ml/min. MR spectral data were collected on a Varian Mercury 400 Mz spectrometer. Coupling constants (J) are reported in hertz. Scheme 1. Fmoc Cl i 20% piperidine/dmf ii Fmoc-Glu (tbu) - ATU DIPEA, DMF Fmoc Cl i 20% piperidine/dmf ii Fmoc-Thr (tbu) - ATU DIPEA, DMF Fmoc Cl i 20% piperidine/dmf ii Fmoc-Dap- ATU DIPEA, DMF Fmoc Cl i 20% piperidine/dmf ii Dov-Val-Dil- ATU DIPEA, DMF Cl i PhSi 3,Pd(PPh 3)4, C 2Cl 2 ii MDpr (Boc) - CMU, 2,6-lutidine, DMF Boc 2 Cl 30% TFA/C 2Cl 2 AT-glu-Dpr-(mDpr) (3) was prepared on solid phase by the following procedure: Resin loading. In a 20 ml solid phase reaction vessel (plastic syringe with PET frit) was added 1 g of 2-Cl- Tritylchloride resin (1 mmol/g loading, 1 mmol) followed by a solution of Fmoc-Dpr(Alloc)- (1.5 mmol, 1.5 equiv) and DIPEA (172 µl, 1 mmol, 1 equiv) in 10 ml of dry C 2 Cl 2 /DMF, 1/1. The vessel was shaken for 5 min, then more DIPEA (258 µl, 1.5 mmol, 1.5 equiv) was added, and the vessel was shaken for additional 2 hours. Unreacted resin was quenched by adding Me (2.5 ml) for at least 30 min. Resin was then washed with DMF (5 10 ml), C 2 Cl 2 (5 10 ml), ethyl ether (5 10 ml), and dried in vacuo. Amino acid loading on the resin was determined as described in ovabiochem Peptide Synthesis Catalogue. Typical loading mmol/g. Standard Fmoc removal procedure. Resin containing Fmoc-protected peptide was treated with 20% piperidine in DMF (10 ml per 1 g of resin) for 2 h at room temperature. Then the resin was washed with DMF (5 10 ml), C 2 Cl 2 (5 10 ml), ethyl ether (5 10 ml), and dried in vacuo. Standard coupling procedure. To the resin containing deprotected -terminus amino acid (1 equiv), a solution of Fmoc-AA- or Dov-Val-Dil- tripeptide (2 equiv), ATU (2 equiv), and DIPEA (4 equiv) in DMF (10 ml) was added. The reaction vessel was agitated for 2 h. The resin was washed with DMF (5 10 ml), C 2 Cl 2 (5 10 ml), ethyl ether (5 10 ml), and dried in vacuo. 2 ature Biotechnology: doi: /nbt.3212

12 Dov-Val-Dil-Dap-Thr(tBu)-Glu(tBu)-Dpr(Alloc)-2-chlorotrityl resin. The peptide was prepared by sequential coupling/fmoc deprotection steps starting from Dpr(Alloc)-2-chlorotrityl resin using of Fmoc- Glu(tBu)-, Fmoc-Thr(tBu)-, Fmoc-Dap-, and Dov-Val-Dil- tripeptide. Maleimide coupling. To remove Alloc protecting group, phenylsilane (PhSi 3,10 equiv) was dissolved in dry C 2 Cl 2 (1 ml per 0.1g resin, degassed by slow bubbling of Ar), and the solution was charged into the syringe containing Dov-Val-Dil-Dap-Thr(tBu)-Glu(tBu)-Dpr(Alloc)-2-chlorotrityl resin. The syringe was shaken for 5 min, the solvent was removed and tetrakis(triphenylphosphine)palladium (0) (Pd(PPh 3 ) 4, 0.3 equiv) was added. The mixture was shaken for 30 min at RT, after with the resin was washed with C 2 Cl 2 (3 10 ml). Treatment with PhSi 3 and Pd(PPh 3 ) 4 was repeated. After the final wash, the resin was dried in vacuo. Solution of mdpr(boc)- (2 equiv), CMU (2 equiv), and 2,6-lutidine (4 equiv) in DMF (10 ml) was added. The reaction vessel was agitated for 2 h. Then the resin was washed with DMF (5 10 ml), C 2 Cl 2 (5 10 ml), ethyl ether (5 10 ml), and dried in vacuo. AT-glu-Dpr-(mDpr) (3). The resin above was treated with 30%TFA/C 2 Cl 2 (10 ml per 1 g of resin) for 2 h at RT for cleavage and final deprotection. Water, couple of drops, was added, and the volatiles were removed on Rotavap. Residue was suspended in DMS, and the product was purified by reverse phase preparative PLC. Yield 0.6 g (84%) of white solid. PLC-based purity (215 nm) was 98 %. 1 MR (D 2 ) δ (ppm): 0.8 (m, 3), (m, 15), (2d, 6), (bm, 2), (m, ), (m, ), 2.95 (2s, 6), 3.15 (s, 3), 3.3 (s, 3), (m, ), 3.45 (s, 3), (m, ), 3.75 (d, ), (m, ), (m, ), 4.3 (m, ), (m, ), (m, ), 5.05 (m, ), 6.97 (s, 2). LC- MS system 3: t R 2.31 min, m/z (ES + ) found (M+) +, calculated exact mass Scheme 2. Ac Ac C 2 Me Ac Fmoc Me A Me Me C 3 ( com pound 8a in US 2008/ A1) Li 87% C 2 2 Me Mal-PEG 24 -Su, DIPEA Me Me C 3 B 55% C 2 Me Me 5 Me C 3 2 -glucuronide-mmae (B): To a flask containing the known (compound 8a in US 2008/ A1) glucuronide-mmae intermediate A (40 mg, 26.8 µmol) was added 0.9 ml methanol and 0.9 ml tetrahydrofuran. The solution was then cooled in an ice bath and lithium hydroxide monohydrate (6.8 mg, 161 µmol) was added drop wise in as a solution in 0.9 ml water. The reaction was then stirred on 3 ature Biotechnology: doi: /nbt.3212

13 ice for 1.5 h, at which time LC/MS revealed complete conversion to product. Glacial acetic acid (9.2 µl, 161 µmol) was then added and the reaction was concentrated to dryness. Preparative PLC afforded the fully deprotected glucuronide-mmae linker intermediate B (26 mg, 87%) as an oily residue. Analytical PLC: t R 9.3 min. LC-MS system 1: t R min, m/z (ES + ) found (M+) +, m/z (ES - ) found (M-) -, calculated exact mass MP-PEG 24 -glucuronide-mmae (5): To a flask containing the deprotected glucuronide-mmae intermediate B (26 mg, 23 µmol) dissolved in anhydrous DMF (0.94 ml) was added maleimido-peg 24 - S ester (32 mg, 23 µmol) as a solution in dimethylacetamide (200 mg/ml). Diisopropylethylamine (20 µl, 115 µmol) was added and the reaction was stirred under nitrogen at an ambient temperature for 6 h, at which time LC/MS revealed conversion to the desired product. The reaction was purified by preparative PLC to provide the linear maleimido-peg 24 -glucuronide-mmae linker 5 (31 mg, 55%) as an oily residue. 1 MR (CD 3 D) δ (ppm) 0.92 (m, 16), 1.15 (m, 6), 1.42 (m, 2), 1.60 (m, 2), 1.91 (m, 4), 2.20 (m, 3), 2.48 (m, 6), 2.66 (m, 3), 2.96 (m, 4), 3.10 (s, 2), 3.27 (s, 2), 3.31 (s, 8), 3.38 (m, 5), 3.44 (m, 2), 3.57 (m, 6), 3.62 (m, 79), 3.77 (m, 5), 3.87 (t, J = 9.6 z, 2), 4.05 (m, 1), 4.21 (m, 3), 4.53 (m, 2), 4.61 (m, 2), 4.80 (m, 2), 5.14 (m, 3), 6.82 (s, 2), 7.10 (m, 2), 7.21 (m, 2), 7.35 (m, 2), 7.39 (m, 2), 7.74 (d, J = 8.8 z, 1), 7.94 (m, 2), 8.10 (m, 1), 8.27 (m, 2). Analytical PLC: t R 9.9 min. LC-MS system 1: t R min, m/z (ES + ) found (M+2) 2+. LC-MS system 2: t R min, m/z (ES + ) found (M+) +, calculated exact mass Scheme 3. Fmoc C 2 Me-PEG 24 -Su DIPEA 53% Fmoc R D R = + B DIPEA 50% (two steps) S, DIC E R = succinimide C 2 Me Me Me C 3 piperidine 87% F R = Fmoc G R = R MC-Su, DIPEA 90% C 2 Me Me 6 Me C 3 4 ature Biotechnology: doi: /nbt.3212

14 Fmoc-Lys(PEG 24 )- (D): To a flask containing α -Fmoc-lysine C (30 mg, 81.5 µmol) was added 1.6 ml anhydrous dichloromethane, followed by methoxy-peg 24 -Su (100 mg, 81.5 µmol). DIPEA (71 µl, 408 µmol) was then added and the reaction was stirred under nitrogen at room temperature and followed by TLC and LC/MS. After 2 h, LC/MS revealed conversion to product. The reaction solution was diluted in dichloromethane and loaded directly on 1 mm chromatotron plate for purification. The plate was eluted with dichloromethane with increasing amounts of methanol (0% to 15%) to provide the desired product D (63 mg, 53%). TLC: R f = 0.17, 10% Me in C 2 Cl 2. 1 MR (CDCl 3 ) δ (ppm) 1.48 (m, 6), 2.47 (m, 5), 3.20 (m, 2), 3.38 (s, 3), 3.63 (m, 86), 4.16 (m, 2), 4.36 (m, 1), 7.26 (m, 3), 7.35 (m, 2), 7.60 (m, 2), 7.71 (m, 3). Analytical PLC: t R 10.8 min. LC-MS system 1: t R min, m/z (ES + ) found (M+) +, m/z (ES - ) found (M-) -, calculated exact mass Fmoc-Lys(PEG 24 )-Su (E): A flask was charged with α -Fmoc-lysine(PEG 24 )- D (63 mg, 43 µmol) and 0.43 ml anhydrous tetrahydrofuran. -hydroxysuccinimide (5.5 mg, 47 µmol) was added, followed by diisopropylcarbodiimide (7.3 µl, 47 µmol). The reaction was sealed under nitrogen and stirred overnight. After 18 h, additional -hydroxysuccinimide (5.5 mg, 47 µmol) and diisopropylcarbodiimide (7.3 µl, 47 µmol) were added and stirring continued for an additional 4 hours, at which time LC/MS revealed complete conversion to product. The crude reaction was diluted in dichloromethane and purified by radial chromatography on a 1 mm plate eluted with dichloromethane with increasing amounts of methanol (0% to 10%) to provide the desired activated ester E (36 mg). The material was carried forward without further characterization. TLC: R f = 0.43, 10% Me in C 2 Cl 2. Analytical PLC: t R 11.4 min. LC-MS system 2: t R min, m/z (ES + ) found (M+) +, calculated exact mass Fmoc-Lys(PEG 24 )-glucuronide-mmae (F): Deprotected glucuronide-mmae linker intermediate B (26 mg, 23 µmol) was dissolved in anhydrous dimethylformamide (0.58 ml) and added to a flask containing α- Fmoc-lysine(PEG)-Su E (36 mg, 23 µmol). Diisopropylethylamine (20 µl, 115 µmol) was then added, the reaction was then stirred under nitrogen at room temperature. After 4.5 h, LC-MS revealed conversion to product. The product was purified by preparative PLC to provide Fmoc-Lys(PEG 24 )- glucuronide-mmae intermediate F (30 mg, 50% over two steps) as an oily residue. Analytical PLC: t R 11.4 min. LC-MS system 1: t R min, m/z (ES + ) found (M+2) 2+. LC-MS system 2: t R min, m/z (ES + ) found (M+) +, calculated exact mass Lys(PEG 24 )-glucuronide-mmae (G): Fmoc-Lys(PEG 24 )-glucuronide-mmae intermediate F (30 mg, 12 µmol) was dissolved in 0.46 ml anhydrous dimethylformamide, followed by addition of 0.12 ml of piperidine. The reaction was stirred under nitrogen for 3 hours and then concentrated to dryness. The product was purified by preparative PLC to provide -Lys(PEG 24 )-glucuronide-mmae intermediate G (24 mg, 87%) as an oily residue. 1 MR (CDCl 3 ) δ (ppm) 0.92 (m, 14), 1.14 (m, 6), 1.42 (m, 5), 1.79 (m, 8), 2.22 (m, 3), 2.42 (t, J = 6.4 z, 2), 2.47 (m, 2), 2.65 (m, 2), 2.76 (m, 2), 2.95 (m, 3), 3.10 (m, 3), 3.31 (m, 8), 3.35 (m, 6), 3.54 (m, 5), 3.63 (s, 70), 3.72 (t, J = 6.0 z, 3), 3.85 (m, 2), 4.07 (m, 1), 4.22 (m, 3), 4.52 (d, J = 7.2 z, 1), 4.61 (d, J = 6.4 z, 1), 4.71 (m, 2), 5.11 (m, 3), 7.12 (m, 1), 7.21 (m, 1), 7.31 (m, 3), 7.37 (m, 2), 7.75 (d, J = 8.8 z, 1), 7.89 (d, J = 8.8 z, 1), 7.95 (d, J = 8.8 z, 1), 8.26 (m, 2). Analytical PLC: t R 8.9 min. LC-MS system 1: t R min, m/z (ES + ) found 5 ature Biotechnology: doi: /nbt.3212

15 (M+2) 2+. LC-MS system 2: t R 9.50 min, m/z (ES + ) found (M+) +, calculated exact mass mc-lys(peg 24 )-glucuronide-mmae (6): Maleimidocaproic acid S ester (4.2 mg, 14 µmol) was dissolved in 0.6 ml anhydrous dimethylformamide and transferred to a flask containing -Lys(PEG 24 )- glucuronide-mmae intermediate G (24 mg, 10 µmol). Diisopropylethylamine (10 µl, 58 µmol) was then added, the reaction was then stirred under nitrogen at room temperature overnight. The reaction mixture was purified directly by preparative PLC to provide mc-lys(peg 24 )-glucuronide-mmae linker 6 (23 mg, 90%) as an oily residue. 1 MR (CD 3 D) δ (ppm) 0.87 (m, 13), 1.12 (t, J = 7.6 z, 2), 1.17 (d, J = 6.8 z, 2), 1.24 (m, 2), 1.48 (m, 9), 1.80 (m, 5), 2.19 (m, 4), 2.42 (t, J = 6.4 z, 2), 2.48 (m, 2), 2.64 (m, 2), 2.96 (m, 3), 3.10 (s, 1), 3.12 (m, 2), 3.15 (s, 1), 3.27 (s, 6), 3.35 (m, 3), 3.43 (m, 3), 3.54 (m, 3), 3.58 (m, 2), 3.63 (m, 64), 3.70 (m, 4), 3.92 (m, 2), 4.22 (m, 4), 4.54 (m, 1), 4.61 (t, J = 6.4 z, 1), 4.83 (m, 1), 5.13 (m, 3), 6.80 (s, 2), 7.10 (m, 1), 7.20 (m, 2), 7.29 (m, 2), 7.38 (m, 2), 7.74 (d, J = 8.8 z, 1), 7.90 (m, 3), 8.08 (s, 1), 8.26 (m, 2). Analytical PLC: t R 10.6 min. LC-MS system 1: t R min, m/z (ES + ) found (M+2) 2+. LC-MS system 2: t R min, m/z (ES + ) found (M+) +, calculated exact mass ature Biotechnology: doi: /nbt.3212

16 Supplementary References 18. Lyon, R.P. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. at Biotechnol 32, (2014). 19. Adem, Y.T. et al. Auristatin antibody drug conjugate physical instability and the role of drug payload. Bioconjug Chem 25, (2014). 20. Valliere-Douglass, J.F., McFee, W.A. & Salas-Solano,. ative intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues. Anal Chem 84, (2012). 21. Kim, K.M. et al. Anti-CD30 diabody-drug conjugates with potent antitumor activity. Mol Cancer Ther 7, (2008). 22. Alley, S.C. et al. The pharmacologic basis for antibody-auristatin conjugate activity. J Pharmacol Exp Ther 330, (2009). 7 ature Biotechnology: doi: /nbt.3212