Conjugation site modulates the in vivo stability and therapeutic activity of antibody conjugates

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1 Conjugation site modulates the in vivo stability and therapeutic activity of antibody conjugates Ben-Quan Shen*, Keyang Xu*, Luna Liu, Helga Raab, Sunil Bhakta, Margaret Kenrick, Kathryn L. Parsons-Reponte, Janet Tien, Shang-Fan Yu, Elaine Mai, Dongwei Li, Jay Tibbitts, Jakub Baudys, Ola M. Saad, Suzie J. Scales, Paul J. McDonald, Philip E. Hass, Charles Eigenbrot, Trung Nguyen, Willy A. Solis, Reina N. Fuji, Kelly M. Flagella, Darshana Patel, Susan D. Spencer, Leslie A. Khawli, Allen Ebens, Wai Lee Wong, Richard Vandlen, Surinder Kaur, Mark X. Sliwkowski, Richard H. Scheller, Paul Polakis and Jagath R. Junutula Genentech Inc., 1 DNA Way, South San Francisco, CA Supporting Online Material: Supplementary Figure Legends and Tables * Equal contribution by first two authors Address for Correspondence: Jagath R. Junutula, Ph.D. Genentech Inc., 1 DNA Way, South San Francisco, CA jagath@gene.com Phone: (650) ; Fax: (650)

2 Supplementary Figures and Legends Supplementary Figure 1. Screening Thio-Fc variants (Fc-THIOMABs) for site-specific conjugation. Val269, Val275, Val278, Val280, Ser320, Ala333, Ala335, Ser371 and Ser396 residues were selected based on their predicted highest fractional solvent accessibility values and lack of predicted interference with FcRIII, FcRn or Protein A binding (based on structural modeling of antibody-fc domain bound with FcRIII, FcRn or miniz, an IgG binding minimal Protein-A domain). All the selected residues were substituted with Cys to engineer site-specific THIOMABs. The THIOMABs were expressed in 293 cells, purified and conjugated with Biotin-PEO-maleimide as described earlier 1. The amount of conjugated biotin-linker was quantitated by LC-MS. Two biotinlinkers per antibody were depicted as 100% biotinylation. 2

3 Supplementary Figure 2. Functional properties of engineered THIOMABs. All three trastuzumab THIOMAB variants (LC-V205C, HC-A114C and Fc-S396C) showed similar binding to HER2 (a) and internalization in HER2 over-expressing MCF7 cells (b). (a) Quantification by flow cytometric analysis showed that thio-trastuzumab variants bound to MCF7-HER2 cells similarly to trastuzumab. MCF7-HER2 cells were bound with 1 µg/ml Alexa488-anti Fc-labeled trastuzumab or THIOMAB variant on ice, washed, detached and quantitated by FACS. Surface levels are shown as mean fluorescence intensity ± standard deviation. (b) THIOMABs are internalized at the same rate as trastuzumab. MCF7-HER2 cells were bound with 1 µg/ml Alexa488-anti Fc-labeled trastuzumab or THIOMAB variant (LC- V205C, HC-A114C and Fc-S396C) on ice, washed and chased for 0, 1 or 3 h at 37 C. After detachment, half the samples were surface quenched with anti-alexa488 prior to FACS analysis in order to determine the amount of internalized antibody. Results are plotted as % internalization normalized to the amount initially bound (shown in a), with error bars denoting the standard deviation. 3

4 Supplementary Figure 3. Hydrophobic interaction chromatographic (HIC) analyses for LC-V205C (A), HC-A114C (B) and Fc-S396C (C) ADCs. HIC analysis was carried out to analyze drug-to-antibody ratio (DAR) as described previously 1. 4

5 Supplementary Figure 4. Schematic diagram of ELISA-based assay to measure total antibody and ADC concentrations in the plasma. Concentrations of total trastuzumab antibody (conjugated and unconjugated) in the plasma were measured by capturing with HER2 ECD (blue ovals) and detecting with anti-human-fc-hrp secondary antibodies. The amount of trastuzumab-mc-vc-mmae ADC (predominantly consisting of two drugs per antibody) in the plasma was measured by capturing with anti-mmae mouse monoclonal antibody SG3.33 (generously provided by Seattle Genetics, Inc.) and detecting conjugates with biotinylated HER2 ECD and streptavidin-hrp. 5

6 Supplementary Fig. 5 In vitro plasma stability of thio-trastuzumab-mc-vc-mmae ADCs in human plasma. Concentrations of total trastuzumab antibody (conjugated and unconjugated) in human plasma (solid symbols) or ADC buffer control (open symbols) were measured by ELISA by capturing with HER2 ECD and detecting with anti-human-fc-hrp secondary antibodies as depicted in Supplementary Figure 4. The anti-mmae antibody preferentially recognizes DAR2 species (100% recovery) and the recovery of the DAR1 species in the conjugate was less than 10%. 100 LC-V205C-ADC HC-A114C-ADC Fc-S396C-ADC LC-ADC-Buffer HC-ADC-Buffer Fc-ADC-Buffer 80 ADC (DAR2) Remaining (%) Time (h) 6

7 Supplementary Figure 6. In vitro stability of thio-trastuzumab-mc-mmaf ADCs in human plasma. Thio-trastuzumab-MC-MMAF variants showed drug loss over time when incubated in human plasma at 37 o C, but were mostly stable in the vehicle PBS buffer under the same conditions. Each ADC variant was incubated at 100 µg/ml in human plasma or PBS control, then samples were collected at different time points and analyzed by affinity capture LC-MS using the ECD of HER2 receptor. (a) LC-V205C-MC- MMAF, (b) HC-A114C-MC-MMAF, and (c) Fc-S396-MC-MMAF. The order of plasma stability was LC > HC > Fc. a. LC-V205C-MC-MMAF 7

8 b. HC-A114C-MC-MMAF 8

9 c. Fc-S396C-MC-MMAF 9

10 Supplementary Figure 7. In vitro stability of thio-trastuzumab-mc-alexa488 (AFC) conjugates in human plasma. The three AFC variants showed fluorophore loss with time when incubated in human plasma at 37 o C, but were mostly stable in the control buffer under the same conditions (data not shown). Samples were treated and analyzed as in Supplementary Fig. 6. (a) LC-V205C-MC-Alexa488, (b) HC-A114C-MC-Alexa488, and (c) Fc-S396C-MC-Alexa488. The order of stability was likewise LC > HC > Fc. 10

11 Supplementary Figure 8. In vitro stability of thio-trastuzumab-mpeo-dm1 conjugates in human plasma. Thio-trastuzumab-MPEO-DM1 conjugated to the same three cysteine THIOMAB variants also showed drug loss with time when incubated in human plasma at 37 o C but not in PBS buffer (data not shown). Samples were treated and analyzed as in Supplementary Fig. 6. (a) LC-V205C-MPEO-DM1, (b) HC-A114C-MPEO-DM1, and (c) Fc-S396C-MPEO-DM1. The order of stability was LC > HC > Fc. 11

12 Supplementary Figure 9. Consistent mass differences resulting from drug losses in plasma from DAR2 to DAR1 to DAR0 were observed across various HC-A114C ADCs with different antibodies, linkers and drugs (e.g. MC-vc-MMAE and MC-MMAF). ADCs were incubated in human plasma at 37 o C for 96 h and the samples were analyzed by affinity capture LC-MS using their respective extra cellular domains to capture (i.e., HER2, MUC16 and CD22 extra cellular domains). (a) Thio-trastuzumab-MC-MMAF, (b) Thio-anti-MUC16-MC-vc-MMAE, and (c) Thio-anti-CD22-MC-MMAF. Mass shifts of ~1200 Da and ~810 Da were observed for MC-vc-MMAE and MC-MMAF, respectively, whichever antibody was conjugated. The mass shifts could be explained by the loss of MC-vc-MMAE or MC-MMAF from the antibody, respectively, and followed by possible addition of cysteine to the antibody to form a disulfide bond. Supplementary Figure 12

13 10. Chemical structures of MC-vc-MMAE and MC-MMAF linker-drugs. 13

14 Supplementary Figure 11. Maleimide exchange between HC-A114C ADC (thiotrastuzumab-mc-mmaf) and albumin in human plasma. Representative extracted mass spectra obtained by affinity capture LC-MS using an anti-auristatin monoclonal antibody for thio-trastuzumab-mc-mmaf (100 µg/ml) incubated in human plasma at 37 o C with samples collected at selected time points of 0 h (blue) and 96 h (red), respectively. An albumin-mc-mmaf adduct with a molecular mass of approximately 67,370 Da was detected in the 96 h sample, indicating maleimide exchange of the entire linker-drug of MC-MMAF between the ADC and human plasma albumin. 14

15 Supplementary Figure 12. Good correlation was observed between the extent of maleimide exchange, albumin adduct formation and ADC instability. (a) LC, HC and Fc variants of thio-trastuzumab-mc-mmaf were incubated in human plasma at 37 o C for up to 96 h and analyzed by affinity capture LC-MS using the ECD of HER2 receptor. (b) Signals of albumin-mc-mmaf adducts for corresponding ADC variants represented by their peak areas are shown here for the 96 h incubated samples captured by anti-auristatin monoclonal antibody. The Fc variant was also incubated in PBS buffer with 0.5% BSA as control. Data indicated that the greater the maleimide exchange (i.e albumin adduct formation), the less stable the ADC. 15

16 Supplementary Figure 13. Maleimide exchange occurred between the HC-A114C ADC (thio-anti-muc16-mc-vc-mmae) and cysteine or glutathione when the ADC was incubated in human plasma at 37 o C for up to 24 h in the presence of excess cysteine or glutathione, respectively. Samples were prepared by protein precipitation using methanol and analyzed by LC-MS with the single ion monitoring (SIM) mode. (a) Addition of 5 mm cysteine showed the formation of isoforms of the cysteine-mc-vc-mmae adduct after 24 h of incubation in red versus the original signal at 0 h in blue. (b) As in (a) except with 5 mm glutathione. 16

17 Supplementary Figure 14. Succinimide ring hydrolysis inhibited maleimide exchange. (a). Evidence suggested that maleimide exchange was a concentration dependent process. Incubation of spiked non-hydrolyzed human albumin-mc-vc-mmae (measured MW of Da) with excess cysteine (5 mm) in human plasma at 37 o C for to 24 h showed complete conversion to the cysteine-mc-vc-mmae adduct. Samples were analyzed by affinity capture LC-MS using anti-auristatin antibody. Signals are shown in blue for 0 h and in red for 24 h of incubation, respectively. Excess cysteine can therefore force the maleimide exchange from human albumin-mc-vc-mmae to cysteine-mc-vc-mmae. (b) A similar experiment was conducted by incubating a hydrolyzed human albumin- MC-vc-MMAE (an aliquot of the 96 h sample of HC-A114C ADC (Thio-anti-MUC16- MC-vc-MMAE) that had already been incubated in human plasma at 37 o C for another 24 h) at 37 o C with the addition of 5 mm cysteine. Signals before and after the addition of 5 mm cysteine with 24 h of incubation are shown in blue and red, respectively. Left panels describe LC-MS chromatogram for albumin-mc-vc-mmae and corresponding LC-MS chromatogram for cysteine-mc-vc-mmae was shown in right panels. No conversion to cysteine-mc-vc-mmae from human albumin-mc-vc-mmae was observed, indicating that succinimide ring in the linker of human albumin-mc-vc-mmae conjugate was hydrolyzed (measured MW of Da, which was approximately 18 Da (mass of H 2 O) more than that of the fresh albumin-vc-mmae) during the original 96 h of incubation in human plasma. Hydrolysis of the succinimide ring thus prevents any further conversion from human albumin-mc-vc-mmae to cysteine-mc-vc-mmae even in the presence of excess amount of cysteine. 17

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19 Supplementary Figure 15. Hydrolysis of the succinimide ring in the linker was faster with the LC-V205C conjugate than the HC-A114C and Fc-S396 conjugates. LC-V205C, HC-A114C and Fc-S396C ADCs (all thio-trastuzumab-mc-vc-mmae variants) were incubated in Tris-HCl buffer, ph 7.2 (a) or ph 8.0 (b) at 37 o C for up to 120 h. At each time point, an aliquot was reduced with DTT and the resulting antibody fragments were analyzed by LC-MS. The obtained m/z spectra were deconvoluted using Agilent Mass Hunter software to calculate the mass of reduced, conjugated antibody fragments. The results were analyzed for the addition of 18 Da to the mass of the conjugated antibody chain as a result of H 2 O addition to the maleimide ring. Ratios of hydrolyzed to nonhydrolyzed masses were calculated. Note that hydrolysis was faster at ph 8.0 than

20 20

21 Supplementary Figure 16. Maleimide exchange and hydrolysis were observed for HC- A114C ADC (thio-anti-cd22-mc-mmaf) in vivo. Deconvoluted mass spectra at representative time points showed changes in the DAR distribution profile corresponding to the drug losses as a result of maleimide exchange occurring on HC-A114C ADC (thioanti-cd22-mc-mmaf) with time following intravenous dosing at 30 mg/kg in cynomolgus monkeys. Plasma samples were analyzed by affinity capture LC-MS using the CD22 ECD. Mass shifts for DAR1 and DAR2 species were also observed with time in comparison with their individual molecular masses when they were first detected. On day 35 post-dosing, mass shifts of approximately 17 Da and 33 Da were observed for DAR1 and DAR2 species, respectively. By contrast, no clear evidence of a mass shift was observed for the DAR0 species. These mass shifts were confirmed as the result of succinimide ring hydrolysis. 21

22 Supplementary Figure 17. Conjugation site influences the therapeutic activity of ADCs. (a) Thio-anti-CD22-MC-vc-MMAE ADCs are not equally efficacious, as the HC-A114C ADC showed substantially greater activity than the Fc-S396C ADC against a preestablished Granta-519 xenograft model of human mantle-cell lymphoma. Animals were given a single dose intravenously of thio-trastuzumab-mc-vc-mmae (negative control, HC-A114C ADC), thio-anti-cd22-mc-vc-mmae (HC-A114C and Fc-S396 ADCs) at ~4 mg/kg (equivalent to 100 µg conjugated MMAE/m 2 mouse) on the day of randomization (Day 0). Tumor growth was plotted as mean tumor volume ± SEM of each group over time. The difference in time to tumor progression distributions between groups was determined using a nonparametric log-rank test with a p-value of 0.05 being considered significant. Fc-S396-ADC vs. Control ADC: p=0.051 HC-A114C-ADC vs. Control ADC: p < HC-A114C-ADC vs. Fc-S396C-ADC: p <

23 (b). Thio-anti-TMEFF2-MC-MMAF ADCs also show differential in vivo efficacy, with the LC-V205C ADC showing about 2-fold greater activity than the HC-A114C ADC against a LuCaP77 xenograft model of human prostate cancer. Animals were given a single dose intravenously of vehicle, thio- anti-tmeff2-mc-mmaf (HC-A114C and LC-V205C) ADCs at 3 or 6 mg/kg (equivalent to µg or µg of conjugated MMAF/m 2 mouse, respectively) on the day of randomization (Day 0). Tumor growth was plotted as mean tumor volume ± SEM of each group over time. The difference in time to tumor progression distributions between groups was determined using a nonparametric log-rank test with a p-value of 0.05 being considerd significant. HC-A114C-ADC, 3mg/kg vs. LC-V205C-ADC, 3mg/kg: p < HC-A114C-ADC, 6mg/kg vs. LC-V205C-ADC, 6mg/kg: p=0.005 HC-A114C-ADC, 6mg/kg vs. LC-V205C-ADC, 3mg/kg: p=0.38 (not significant) 23

24 Supplementary Figure 18. Fc-S396C-MC-vc-MMAE ADC showed higher elevation in liver transaminases compared to LC-V205C-MC-vc-MMAE and HC-A114C-MC-vc- MMAE ADCs in rats. Serum chemistry analysis revealed the highest increase in aspartate aminotransferase (AST) in rats administered high dose Fc-MMAE ADC (2,635 µg MMAE/m 2 ) relative to controls and the other two conjugates at Day 5 (a). These findings were suggestive of liver toxicity and reversed to control levels by the end of study, day 12 (b). A similar trend was present in alanine aminotransferase (ALT) and bilirubin levels in the high dose Fc-MMAE ADC group (data not shown). Data are plotted as average AST ± SD and the black asterisk indicates statistical significance relative to control, LC-V205C, and HC-A114C at all doses tested (Tukey-Kramer test; p<0.05) Fc-S396C high dose vs. control: p= Fc-S396C high dose vs. LC-V205C low dose: p=0.003 Fc-S396C high dose vs. LC-V205C high dose: p=0.022 Fc-S396C high dose vs. HC-A114C low dose: p= Fc-S396C high dose vs. HC-A114C high dose: p=0.002 Fc-S396C high dose vs. Fc-S396C low dose: p=0.36 (not significant) All other combinations are not statistically significant. 24

25 Supplementary Table 1: List of antibody conjugates and their DAR (drug-antibody ratios) used in this study. Antibody Target Antibody Thiol reactive DAR Conjugate linker 1 LC-V205C Trastuzumab MC-vc-MMAE HC-A114C Trastuzumab MC-vc-MMAE Fc-S396C Trastuzumab MC-vc-MMAE LC-V205C Trastuzumab MC-MMAF HC-A114C Trastuzumab MC-MMAF Fc-S396C Trastuzumab MC-MMAF LC-V205C Trastuzumab mpeo-dm HC-A114C Trastuzumab mpeo-dm Fc-S396C Trastuzumab mpeo-dm LC-V205C Trastuzumab MC-Alexa HC-A114C Trastuzumab MC-Alexa Fc-S396C Trastuzumab MC-Alexa Fc-V278C Trastuzumab MC-vc-MMAE Fc-S371C Trastuzumab MC-vc-MMAE LC-V205C anti-tmeff2 MC-MMAF HC-A114C anti-tmeff2 MC-MMAF Fc-S396C anti-tmeff2 MC-MMAF HC-A114C anti-cd22 MC-vc-MMAE Fc-S396C anti-cd22 MC-vc-MMAE HC-A114C anti-muc16 MC-vc-MMAE

26 Supplementary Table 2 The concentrations of total antibody (Tab) and thio-trastuzumab-mc-vc-mmae ADC (LC-V205C, HC-A114C and Fc-S396C) were determined by HIC as described in Supplementary Fig. 3. The collected plasma samples were from the mouse in vivo study described in Figure 1e. Plasma pharmacokinetic parameters were derived from ADC or total antibody concentrations using a two compartment analysis (Model 8, WinNonlin, v 5.2; Pharsight,, Mountain view, CA) with naïve pooling of individual animal concentrations for each group at each time point. Values are reported as population estimates with a standard error of the estimate. All dosing solutions were within 10% of expected, therefore nominal doses were used in the pharmacokinetic analyses. ADC Variant Total Antibody Clearance (ml/day/kg) ADC Clearance (ml/day/kg) LC-V205C 8.2 ± ± 0.7 HC-A114C 7.4 ± ± 1.2 Fc-S396C 10 ± ±