Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent

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Attachment site cysteine thiol pKa is a key driver for sitedependent stability of THIOMAB antibody-drug conjugates TM

Breanna S Vollmar, Binqing Wei, Rachana Ohri, Jianhui Zhou, Jintang He, Shang-Fan Yu, Douglas Leipold, Ely Cosino, Sharon Yee, Aimee Fourie-O'Donohue, Guangmin Li, Gail D. Lewis Phillips, Katherine Ruth Kozak, Amrita Kamath, Keyang Xu, Genee Lee, Greg A. Lazar, and Hans K. Erickson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00365 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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Bioconjugate Chemistry

Attachment site cysteine thiol pKa is a key driver for site-dependent stability of THIOMABTM antibody-drug conjugates

Breanna S. Vollmar*, Binqing Wei, Rachana Ohri, Jianhui Zhou, Jintang He, Shang-Fan Yu, Douglas Leipold, Ely Cosino, Sharon Yee, Aimee Fourie-O’Donohue, Guangmin Li, Gail L. Phillips, Katherine R. Kozak, Amrita Kamath, Keyang Xu, Genee Lee, Greg A. Lazar, Hans K. Erickson*

Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA

* email: [email protected], phone: +1 650-467-4814 * email: [email protected], phone: +1 650-225-5124

Abstract Incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the

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attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C which reduced the cysteine thiol pKa and thus decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimidebased linker. Taken together, our results provide evidence that the site-dependent stability of cysengineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serve to modulate the side chain thiol pKa. The influence of cysteine thiol pKa on stability & efficacy offers a new parameter for the optimization of ADCs that utilize cysteine-engineering.

Introduction Antibody-drug conjugates (ADC) are designed to deliver lethal concentrations of cytotoxic agents to cancer cells1, 2. FDA approvals of KadcylaTM for HER2+ metastatic breast cancer and AdcetrisTM for Hodgkin’s Lymphoma provide clinical validation for ADCs3-6. Despite these successes, extensive clinical development of ADCs to diverse targets in broad cancer indications has not led to additional approvals. Nearly all these clinical evaluations have been with ADCs that utilize anti-tubulin cytotoxic agents. General disease insensitivity to tubulin agents could possibly be limiting the clinical successes of many of these ADCs2. In addition, most of the ADCs evaluated in the clinic to date utilize conjugation strategies that result in a heterogeneous mixture of antibody-drug conjugates with different molar drug-to-antibody ratios (DARs).

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Conjugation strategies that eliminate or reduce this heterogeneity have been shown to improve both the anti-tumor activity and safety of ADCs in animal models7-9. Thus, efforts to improve the efficacy of ADCs have focused on the development of novel payloads and site-specific conjugation technologies that afford homogeneous products. We have adopted a cysteine-engineering strategy called THIOMABTM antibody technology that allows for the straightforward conjugation of novel payloads to yield homogeneous THIOMABTM antibody-drug conjugates (TDCs)7, 8, 10. Native amino acids in the HC or LC of the antibody are mutated to cysteine for conjugation to small molecules, peptides or proteins using standard thiol reactive coupling chemistries. The homogeneous TDCs produced using an appropriately selected stable attachment site have markedly improved pharmacokinetics, efficacy, and safety profiles in preclinical animal models as compared to the heterogeneous ADCs prepared via conjugation to endogenous lysines and cysteines7-9, 11. Several other cysteine engineering strategies have been reported12-19 as well as other site-specific conjugation strategies20. Candidates utilizing cysteine engineering to produce site-specific conjugates have advanced to clinical evaluation16, 21, 22 highlighting the importance of studies to further the understanding of this technology. Antibody-drug coupling strategies that minimize the release of the drug from the TDC during circulation prior to delivery to the cancer cell are desired. Maleimide-based linkers have been employed for TDCs that offer excellent in vivo stabilities provided the appropriate site is selected9, 15, 16, 23-27. Recently, disulfide-based linkers were described that also provide circulation stability to TDCs with unique advantages over maleimide-based linkers23, 28 such as the ability to decouple circulation stability from drug release. With disulfide-based linkers, the antibody attachment site can provide increased circulation stability that is subsequently lost when the

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antibody is internalized and degraded in the lysosome23, 28. The resulting payload-cysteine disulfide then can be easily reduced, releasing the free payload in the reductive cytosol compartment. Central to the optimization of these disulfide-linked TDCs is selecting an engineered cysteine in the HC or LC that affords sustained stability to the linker in circulation. We recently initiated a large screening campaign to map the most stable sites of trastuzumab for disulfide-linked drugs using our THIOMABTM antibody platform (unpublished results). The basis for this site-dependent stability was hypothesized to be due to steric protection of the disulfide provided by the local antibody environment23. However, we examined the solvent accessibility of the five different sites shown in Figure 1 and found no correlation with stability (Figure S1). Another possible mechanism known to drive the rate of thiol-disulfide exchange is the pKa of the leaving group thiol29. We speculated that differences in the local protein environment surrounding the engineered cysteines at different sites could modulate the side chain thiol pKa values thereby influencing the stabilities of the disulfide bond linkage in the TDCs. To explore this, we first expanded the relationship between the antibody attachment site and TDC stability by evaluating TDCs prepared using HC A118C, HC A140C, LC S121C, LC K149C and LC V205C (EU numbering) trastuzumab mutants. We then investigated the relationship between the cysteine thiol pKa for the different variants and their stabilities as disulfide-linked TDCs. Lastly we explored the correlation between thiol pKa and the stability of maleimide-linked payloads for LC K149C and known attachment sites, HC S239C15 and HC S400C9.

Results & Discussion In vivo efficacy & stability of disulfide-linked PBD THIOMABTM antibody drug conjugates

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Five different sites for engineering cysteines were chosen, covering both the LC and HC of trastuzumab, including several sites previously identified in the literature8, 9, 23. The five different mutants of trastuzumab—LC S121C, LC K149C, LC V205C, HC A118C and HC A140C were prepared and each mutant (Figure 1A) was conjugated to a disulfide-activated PBD referred to as SG3231 as described previously28. Figure 1B depicts the structure of the disulfide-linked SG3231 TDCs. All of the TDCs had a DAR of at least 1.8, with less than 2% aggregate and less than 5% free drug remaining in the final product (Table S1). The in vitro potency of the SG3231 TDCs was evaluated in two different HER2 expressing cell lines, SK-BR-3 and KPL-4. All of the conjugates exhibited similar IC50s in both of the cell lines ranging from 0.020 nM to 0.051 nM for SKBR3 and 0.030 nM to 0.067 nM for KPL4 (Table S2). The efficacies of the anti-HER2 SG3231 TDCs were evaluated in a mouse allograft model of MMTV-HER2 Founder 5 (Fo5)30. Tumor bearing mice were treated with a single 100 µg/m2 dose of the SG3231 TDCs (Figure 1C) and tumor volumes were measured over time. Significant differences in tumor growth inhibition were observed across the different SG3231 TDC site variants. The LC S121C TDC was the least efficacious with little tumor growth inhibition compared to vehicle or control anti-CD33 TDC (HC A140C). SG3231 TDCs utilizing HC A118C and LC V205C were both active with modest tumor growth inhibition observed through Day 10. HC A140C and LC K149C SG3231 TDCs had the highest persistent tumor growth inhibition through day 21 of the study, with the LC K149C TDC reaching tumor stasis through day 31.

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Figure 1. (A) Location of conjugation sites on trastuzumab Fab domain. (B) Structure of SG3231 TDCs. (C) In vivo efficacy of SG3231 TDCs conjugated to different sites in HER2+ Fo5 allograft mouse model. (D) In vivo stability of SG3231 TDCs conjugated to different sites in non-tumor bearing mice.

We next evaluated the in vivo stability of the disulfide linkage and pharmacokinetics of the five SG3231 TDCs in non-tumor bearing mice. As expected, the in vivo stabilities were found to correlate well with the observed efficacy data, with the most stable sites, LC K149C and HC A140C, offering the most efficacious TDCs and the least stable LC S121C yielding the least efficacy (Figure 1D). It is worth noting that historical data obtained in our lab showed no significant differences in stability of TDCs in either tumor bearing or non-tumor bearing mice (unpublished results). The exposures for the antibody components were similar across the

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different THIOMABTM antibody variants suggesting that the engineered attachment site and conjugation of drug did not have an impact on the antibody pharmacokinetics (Figure S2, Table S3). Previous reports have shown that the attachment site can affect the total antibody pharmacokinetics27, 31, although this was not observed with the sites screened in this study. Cysteine and glutathione were found to replace the drug over time consistent with deconjugation via thiol-disulfide exchange between the TDCs and cysteine and glutathione (Figure S3). While albumin has also been implicated in the deconjugation of ADCs9, we did not observe adducts between the antibody and albumin in this study. It is notable that the stability observed for LC K149C TDC can be further increased by modifying the drug to include an additional methyl group adjacent to the disulfide as has been reported elsewhere23, 28.

Estimation of cysteine thiol pKa values using isothermal titration calorimetry We next sought to explore whether differences in the cysteine thiol pKa values associated with the THIOMABTM antibody variants could explain the stability differences associated with the corresponding TDCs. We used ITC to estimate the cysteine thiol pKa values for the five THIOMABTM antibody site variants. Previously, Tajc et al. demonstrated that ITC can be used to estimate thiol pKa values by monitoring iodoacetamide alkylation of thiols in proteins as a function of pH32. Iodoacetamide rapidly alkylates the thiolate but not the thiol form of cysteine, allowing for the proportion of thiolate and the thiol pKa to be estimated. Proteins with multiple reactive cysteines complicate the analysis. Fortunately, the trastuzumab mutants used in the current study have just one engineered cysteine on the HC or LC resulting in two available cysteines with equivalent reactivity. Care must also be taken to ensure iodoacetamide reacts exclusively with the cysteines of interest. In initial ITC experiments using trastuzumab without

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engineered cysteines, we found no heats of reaction with iodoacetamide even at the extreme pH value of 11.0 (data not shown). Further analysis using mass spectrometry of trastuzumab after treatment with iodoacetamide confirmed that no alkylation occurred on either the heavy or light chain of the antibody (Figure S4). An example of the titration of LC S121C with iodoacetamide at multiple pH values is shown in Figure 2A. As the pH increases, the max dQ/dt increases indicating a higher proportion of thiolate to thiol is present. Comparison of the ITC titration curves for the five variants reveals that each of the five sites has markedly different thiol pKa values, ranging from 8.0 for LC S121C to > 10.0 for LC K149C and HC A140C (Figure 2B). Antibody instabilities at extreme alkaline conditions (pH > 11) prevented us from obtaining accurate thiol pKa values for LC K149C and HC A140C.

Figure 2. (A) Overlay of raw ITC traces measuring the heat of iodoacetamide alkylation of aHER2 LC S121C as a function of pH. (B) ITC derived pH titration curves of the five different engineered cysteines on trastuzumab.

The trend observed for measured thiol pKa values correlates well with the observed in vivo stability and mouse efficacy for the corresponding disulfide-linked SG3231 TDC site variants. The THIOMABTM antibody associated with the least stable TDC, LC S121C, had the

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lowest thiol pKa. The THIOMABTM antibodies associated with the most stable sites, LC K149C and HC A140C, had the highest thiol pKa values and the HC A118C and LC V205C yielded intermediate thiol pKa, stability and efficacy. Surprisingly, the measured cysteine thiol pKa values of the THIOMABTM antibody variants mirror predicted characteristics of disulfide exchange kinetics described by the Brønsted relationship29, 33, 34 comparing thiol pKa and rate of exchange. Cysteine and glutathione are the most abundant thiol containing small molecule reductants in circulation35 with thiol pKa values of 8.3 and 9.2, respectively32. The most stable THIOMABTM antibody variants, LC K149C & HC A140C, have thiol pKa values significantly higher than even glutathione, thus as predicted by the Brønsted relationship disulfide exchange is very slow. While the other THIOMABTM antibody variants with moderate and low stability have thiol pKa values closer to the thiol pKa values of cysteine and glutathione. Thus, these sites exhibit a faster rate of disulfide exchange according to the Brønsted relationship and are unstable in circulation. In the above assessment of the relationship between attachment site cysteine thiol pKa and stability in circulation, we assume that conjugation of the linker drug does not change the local environment of the different attachment sites and similar nucleophiles are acting as reducing agents across the sites (i.e., cysteine, glutathione & albumin). Additionally, we acknowledge that other factors in addition to thiol pKa could influence thiol leaving group ability, such as electronegativity of the surrounding region, stability of the transition state and ionizability of the protein thiol. However, these factors are also likely to influence the reactivity of the engineered cysteine thiol with iodoacetamide and thus are probably reflected in our measured thiol pKa values determined.

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Modulating the thiol pKa and stability of LC K149C TDC To further explore the relationship between cysteine thiol pKa and stability, we used homology modeling and an algorithm to engineer several mutants of LC K149C with reduced thiol pKa values and examined their stabilities as TDCs. The algorithm described by Jacob et al.36 and implemented in Cyspka script was applied to predict cysteine thiol pKa values using homology models of the antibodies (see experimental procedures). The algorithm predicts thiol pKa by estimating the electrostatic interactions of the thiolate with nearby backbone and side chains, as well as the solvation energy based on the number of atom neighbors. In addition, it takes into account the Boltzmann energy distribution for the rotational states of cysteine. We first compared the predicted thiol pKa values by the algorithm to the measured thiol pKa values determined by ITC. The predicted thiol pKa values were significantly lower than the measured thiol pKa values (Figure 3A) for the five THIOMABTM antibody variants (Figure 1A). This is not unexpected given that the algorithm was based on a model of measured reaction rates of 5,5dithiobis-(2-nitrobenzoic acid) and not validated against known thiol pKa values in proteins, as noted by the authors36. However, a strong correlation (r2 = 0.94) was observed between the rank order of predicted thiol pKa and the thiol pKa values measured by ITC (Figure 3B), suggesting the algorithm could be used to predict thiol pKa differences across sites.

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Figure 3. (A) Comparison of predicted cysteine thiol pKa values and measured thiol pKa values by ITC for five THIOMABTM antibody variants. (B) Plot of predicted and measure thiol pKa values.

The cysteine side chain of LC K149C was predicted by homology modeling to be part of a beta strand exposed to solvent with two negatively charged side chains, E195 and D151, near the thiol group (Figure 4A). We predicted that these negatively charged carboxylates maybe responsible for the high thiol pKa observed for K149C by hindering the formation of the cysteine thiolate. Therefore, we expected mutations removing the negative charge or the introduction of a positive charge at those positions (especially for E195 which is ~6Å away) to perturb the thiol pKa. All of the mutations described are located on the light chain of the LC K149C THIOMABTM antibody. We prepared three LC K149C mutants by mutating E195 to alanine, glutamine, or lysine. An additional three mutants were prepared by mutating D151; however, we were unable to express those mutants. Modeling studies also suggested that the hydrophobic side

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chain, L154, packs against the thiol and may help shield it from the solvent. Mutation of L154 to lysine was predicted to significantly lower the thiol pKa of LC K149C by stabilizing the thiolate through favorable electrostatic interactions. We also predicted that a double mutant of L154 & E195 mutated to lysine would further reduce the thiol pKa of LC K149C beyond that which was predicted with incorporation of a single lysine at either of the positions. Conversely, incorporation of a glutamate at L154 was predicted to increase the thiol pKa of LC K149C (Figure 5A) and was hypothesized as a way to further increase the stability of LC K149C TDCs. Therefore, we prepared another four mutants where L154 was replaced with lysine, glutamate or alanine, as well as the double mutation of both L154 and E195 to lysine. As a negative control, we mutated a residue far away from LC K149C, E187, with alanine, glutamine or lysine (Figure 4A).

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Figure 4. (A) Model of the structure of aHER2 LC K149C identifying location of mutations. Overlay of pH titration curves aHER2 LC K149C pKa mutants at E195 (B), L154 (C) and E187 (D) generated using the MS-based pKa assay.

We developed a high throughput mass spectrometry-based pKa assay utilizing the same pH dependent iodoacetamide alkylation employed in the ITC experiments to estimate the thiol pKa values associated with the pKa mutants. Suitability of the MS-based pKa assay was tested by measuring the thiol pKa values associated with the five THIOMABTM antibody site variants assessed previously using the ITC-based pKa assay (Figure S5). We found similar thiol pKa values using the MS and ITC-based assays. However, the values from the ITC-based assay are more likely to reflect the accurate thiol pKa values because ITC allows direct observation of the reaction compared to the MS method, which required further processing of the sample prior to data collection (see experimental procedures). Additionally, we only observe about 50%

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alkylation in the MS-based assay due to the dilute antibody concentrations used for the assay (Figure S5A). We also used the MS-based assay to assess if the estimated cysteine thiol pKa values varied across antibodies targeting different antigens. For both HC A118C and LC K149C sites, the cysteine thiol pKa values were consistent across the various antibodies tested (Figure S6). The thiol pKa values for the nine mutants as well as LC K149C and LC S121C were assessed using the MS-based assay. LC S121C was included as a control to determine assay variability. The thiol pKa of LC S121C was calculated to be 8.0 ± 0.1 (Figure S7) across different runs of the assay. The changes in thiol pKa values (from LC K149C) for the different mutants are shown in Figure 5A. The relative thiol pKa values for the mutants agreed well (r2 = 0.83) with the relative values predicted by the Cyspka algorithm (Figure 5B) consistent with the results described in Figure 3. As predicted, mutation of E195 to lysine resulted in the largest decrease in thiol pKa of LC K149C with a change of 1.6 pH units, whereas substitution of E195 with glutamine or alanine resulted in more moderate decreases of 0.6 pH units (Figure 4B). These observations suggest E195 plays a significant role in the unusually high thiol pKa observed for LC K149C. Mutation of L154 to lysine decreased the thiol pKa as predicted, while introduction of a glutamate at L154 did not result in a predicted increase in thiol pKa (Figure 4C & 5A), although the assay limitations may have masked any observed improvement (see previous section). The double mutant, L154K/E195K, did not result in a decrease significantly larger than what was observed with the single mutants (Figure 4C). This was surprising given that the algorithm predicted that there would be a synergistic effect to decrease the thiol pKa two-fold further than each of the individual mutations (Figure 5A), however this was not observed. The lack of significant modulation of the LC K149C thiol pKa with the double mutant

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could be due to flexibility of the antibody in that region that is unaccounted for in the algorithm. As a negative control, we substituted E187 with alanine, glutamine and lysine and observed no decrease in thiol pKa (Figure 4D) as expected since the residue lies well outside the local environment of LC K149C (Figure 4A).

Figure 5. (A) Table of predicted and observed changes to the thiol pKa of aHER2 LC K149C by mutation. (B) Correlation of the calculated effect of mutations on aHER2 LC K149C cysteine thiol pKa values using the Cyspka algorithm with observed thiol pKa values determined using MS-based pKa assay.

Influence of mutations on the in vitro and in vivo stability of LC K149C TDC To assess the impact of modulating the thiol pKa of LC K149C, we prepared disulfide-linked TDCs of the pKa mutants to assess the stability of the disulfide linkage. In a separate study, we found that the relative clearance rates for the disulfide-linked SG3231 TDCs in Figure 1 could be predicted from the release kinetics of the corresponding disulfide-linked MMAE TDCs in the

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presence of cysteine in vitro (Figure S8). The MMAE conjugates utilized the same disulfide linker used for the PBD conjugates in Figure 1. We used this in vitro assay to compare the chemical stabilities of the disulfide-linked TDC mutants. Each mutant was conjugated to a disulfide MMAE derivative23 (Figure 6A) using a high throughput conjugation method and the in vitro stability of each conjugate was evaluated in the presence of cysteine. All of the TDCs had a DAR of at least 1.0 with less than 15% aggregate and less than 5% free drug remaining in the final product (Table S4). The disulfide-linked MMAE HC A118C TDC was prepared as a control. Substitution of E195 with alanine or glutamine decreased the stability with a nearly 2fold reduction in the half-life compared to the LC K149C TDC (Figure 6B). Substitution of E195 with lysine reduced the half-life even more. Control mutations (E187 to alanine, glutamine or lysine) had little detectable impact on TDC stability. Substitution of L154 with alanine, glutamate and lysine all resulted in about a 2-fold decrease in the reduction half-life of the LC K149C TDC. These results indicate that our attempt to increase the stability of LC K149C TDCs by mutating L154 to glutamate was unsuccessful. The lack of improvement with the L154E mutation suggests that either we have reached the maximum obtainable thiol pKa for a surface exposed cysteine or that engineering increases in thiol pKa requires a more extensive approach to account for possible structural flexibility. Nonetheless, a comparison between the measured halflives with the observed thiol pKa for all of the mutants reveals a significant correlation (r2 = 0.70) (Figure 6C), suggesting that E195 and L154 both contribute to the observed high thiol pKa of LC K149C and stability of LC K149C TDCs.

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Figure 6. (A) Structure of disulfide-linked MMAE TDCs. (B) In vitro stability of pKa mutants conjugated to disulfide-linked MMAE as assessed in the cysteine reduction assay. Statistical significance compared to LC K149C is indicated by the asterisks, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and n.s = not significant. (C) Correlation of the thiol pKa values of the different LC K149C pKa mutants with in vitro stability as disulfide-linked MMAE TDC.

We conjugated four of the LC K149C mutants to SG3231 (Figure 4A) and assessed the in vivo circulation stability in mice as described earlier for the THIOMABTM antibody variants in Figure 1 (Figure 7). Consistent with the in vitro data, the mutations that lower the thiol pKa of LC K149C the most (E195K and E195K/L154K) yield the TDCs with the greatest rates of deconjugation in vivo. The L154K mutation leads to a modest decrease in thiol pKa that is reflected in the slightly faster clearance of the TDC as compared to the LC K149C TDC. The L154E mutation that had the least impact on the thiol pKa of LC K149C also had no discernable impact on the clearance of the LC K149C TDC.

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Figure 7. In vivo stability of disulfide-linked PBD, SG3231, conjugated to different trastuzumab LC K149C pKa mutants in mouse.

Correlation of maleimide-based linker stability with cysteine thiol pKa The results presented here demonstrate that cysteines engineered at different sites on the antibody have different thiol pKa values, which correlate with the site-dependent circulation stability observed with disulfide-linked TDCs. We next investigated whether attachment site cysteine thiol pKa is also a driver of the observed site-dependent circulation stability of TDCs using maleimide-based linkers. We compared the in vivo efficacy & circulation stability of a maleimide-linked PBD, SG320328 (Figure 8A), conjugated to two different sites on trastuzumab, LC V205C and LC K149C (Table S1). We observed similar efficacy of the SG3203 TDCs in the HER2+ Fo5 mouse model with tumor growth inhibition through day 21 (Figure 8B). We also observed sustained in vivo stability of SG3203 conjugated to LC K149C and LC V205C in nontumor bearing mice (Figure 8C). The stability of LC V205C TDCs utilizing maleimide-based linkers has previously been described9 in which accelerated hydrolysis of the succinimide observed in vivo was suggested to stabilize the attachment. Thus, sustained circulation stability of SG3203 LC V205C TDC was expected, however LC K149C was not known.

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Figure 8. (A) Structure of maleimide-linked SG3203 TDCs. (B) In vivo efficacy of SG3203 TDCs conjugated to two different sites, LC K149C and LC V205C, on trastuzumab in Fo5 allograft mouse model. (C) In vivo stability of LC K149C and LC V205C SG3203 TDCs in nontumor bearing mice.

We investigated the succinimide hydrolysis in vitro of maleimide-linked MMAE (Figure 9A), utilizing the same linker as SG3203, conjugated to LC K149C and LC V205C trastuzumab (Table S3). In substituting MMAE for SG3203, we are assuming that different payloads attached with the same linker do not impact the rate of succinimide hydrolysis. We were surprised to see that over the time course of 21 days at pH 7.4 at 37°C there was no evidence of succinimide hydrolysis on LC K149C (Figure 9B), however we observed complete succinimide hydrolysis after 7 days on LC V205C. Recently, the disconnect between succinimide hydrolysis and stability was reported in the literature by Tumey et al. at two different sites27, HC K392C and HC K334C, suggesting that our observations are not specific to LC K149C. While succinimide hydrolysis has been established as a key driver of circulation stability of maleimide-based TDCs9, 37, the observations from our study and Tumey et al.27 suggest there are additional

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mechanisms contributing to stability. Baldwin & Kiick38 have shown that succinimide thioethers can undergo a retro Michael-type addition resulting in deconjugation and reformation of the maleimide. The rate of deconjugation via retro Michael-type addition was found to be directly proportional to the pKa of the thiol in the succinimide thioether linkage. We propose that TDCs utilizing succinimide thioether attachment at LC K149C are stable due to the elevated cysteine thiol pKa that we described earlier (Figure 2B). In contrast, the sustained circulation stability of maleimide-linked TDCs at LC V205C is a balance between two opposing forces, deconjugation via retro Michael-type addition versus succinimide hydrolysis to stabilize the attachment. Thus, the observed stability of LC V205C TDCs suggests that succinimide hydrolysis is faster than deconjugation in circulation. For LC K149C TDCs, the high cysteine thiol pKa is likely a driver of the observed sustained stability in circulation for both disulfide and maleimide-based coupling chemistries.

Figure 9. (A) Structure of maleimide-linked MMAE TDCs. (B) In vitro assessment of succinimide hydrolysis of MMAE TDCs in PBS at 37°C.

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Furthermore looking at two previously characterized sites in the literature, HC S400C9 and HC S239C15, we have observed a similar correlation between connection stability and attachment site cysteine thiol pKa. We have found that the unstable site, HC S400C, in fact has a low cysteine thiol pKa of 8.2 and that the stable site, HC S239C, has a high apparent cysteine thiol pKa of ≥ 10.1 (Figure S9). The poor circulation stability of maleimide-linked TDCs at HC S400C is likely to be a combination of the lack of succinimide hydrolysis9 and the low thiol pKa. Similar to what we described for LC K149C TDCs, we also observed that HC S239C does not undergo rapid succinimide hydrolysis (Figure 9B). This suggests that elevated thiol pKa maybe the driver for the observed sustained circulation stability of HC S239C TDCs using maleimidebased linkers. Though this is limited set of observations, the data suggests that cysteine thiol pKa is a driver of site-dependent stability that can be extended to both maleimide and disulfide-based attachment chemistries.

Conclusions The stability of disulfide-linked TDCs in circulation varies depending on the site in the antibody HC or LC selected for cysteine engineering. For TDCs, this stability is lost once the TDC is degraded23 thereby allowing for high circulation stability without compromising intracellular payload release. This represents a significant advantage over other disulfide-linked ADC designs such as those described for disulfide-linked maytansinoid conjugates in which the disulfide stability is retained following intracellular degradation23, 39. It is especially important if traceless release of the drug is desired23 to avoid any potential reduction in potency of the released payload due to the presence of the remaining linker. Steric protection of the disulfide by the antibody provided a reasonable explanation for the site-dependent stability observed for the

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TDCs. However, the results of this study suggest a more important driver is the modulation of the cysteine thiol pKa by the local protein environment. We established a strong correlation between thiol pKa and TDC stability for five different sites selected from across the antibody LC and HC of trastuzumab as well as several pKa mutants of the LC K149C TDC. We identified two residues, E195 & L154, which significantly contribute to the elevated cysteine thiol pKa of LC K149C. Differences in cysteine thiol pKa values may not be restricted to explaining sitedependent stability of disulfide-linked TDCs. It is likely that thiol pKa is also a significant contributing factor to the site-dependent stability of maleimide linked TDCs as well. In addition, we show that the thiol pKa differences measured across the different variants were similar to the corresponding differences calculated using an algorithm described by Jacob et al36. These results suggest that cysteine thiol pKa could be used as a guiding principle to select stable sites for cysteine coupling or to engineer a stable site into a desired location on the antibody or protein using a computational approach.

Experimental Procedures Generation of THIOMABTM antibodies & conjugates All THIOMAB TM antibodies and THIOMAB TM antibody pKa mutants were derived from trastuzumab and sequence numbering is reported using EU system. Briefly, THIOMAB TM antibody pKa mutants of the anti-HER2 hu4D5 THIOMABTM antibody were obtained either by gene-synthesis (GeneWiz) or overlapping PCR with site-specific mutagenic primers. LC variants were cloned as EcoRV/HindIII fragments into expression vector pRK5 humAb4D5–8 LC and HC variants were cloned as EcoRV/BsrG1 fragments into expression vector pRK5 humAb4D5– 8 HC 40 with DANG mutations (DANG mutations remove the glycosylation site for ease of

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analysis by mass spectrometry). Protein expression was carried out by cotransfection of the LC plasmid and HC plasmid each carrying either a LC variant/LC wild-type or a HC variant/wildtype gene into Expi293 cell (ThermoFisher Scientific) in 30 ml culture. Antibody expression was carried out for 7 days at 37°C with vigorous shaking. The culture supernatants were collected and incubated with 300 ml MabSelect SuRe resin (GE Healthcare) with vigorous shaking overnight. The resin was then transferred to filter plates and washed 3 times, 1 ml each with PBS. The bound IgG was then eluted with 50 mM phosphoric acid pH 3.0 and neutralized (1:20) with 20x PBS pH 11.0. The IgG protein was then sterilized by filtration through a 0.22 mm filter. THIOMABTM antibody drug conjugates with SG3231 were conjugated, purified and characterized as previously described28. The conjugates were formulated by dialysis with 20 mM histidine acetate pH 5.5 and 240 mM sucrose, and 0.02% polysorbate-20 was added to the formulated conjugate. The DAR of the conjugates was assessed by limited digestion with LysC and analyzed using reverse phase LC/MS with a PLRP-S column utilizing a water/acetonitrile/TFA gradient and Agilent ESI-TOF instrument. The amount of unconjugated linker drug was analyzed using reverse phase LC/MS and calculated based on a standard curve of the unconjugated linker drug. Aggregation of the conjugates was assessed using analytical SEC HPLC equipped with a Shodex Protein KW 802.5 column with 0.2 M potassium phosphate pH 6.2, 0.25 M KCl and 15% isopropanol. Endotoxin levels in formulated conjugates were assessed using LAL assay by Charles River Laboratories. All conjugates used for in vivo studies had DAR values > 1.8, < 5% unconjugated linker drug, < 2% aggregation and endotoxin levels < 0.05 EU/mg antibody. THIOMABTM antibody drug conjugates of pKa mutants were prepared using pyridyl disulfide-MMAE23 as previously described using a high-throughput partial reduction-reoxidation

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and conjugation protocol (unpublished results). All conjugates had DAR values of at least 1.0 and less than 15% aggregation.

In vitro cell potency Cells were plated in black-walled 96-well plates (4, 000 cells for SK-BR-3 and 1300 for KPL4 cells) and allowed to adhere overnight at 37˚C in a humidified atmosphere of 5% CO2. Medium (Ham’s F-12: high glucose DMEM [50:50] supplemented with 10% heatinactivated fetal bovine serum and 2 mmol/L L-glutamine) (Invitrogen Corp.) was then removed and replaced by 100 µl fresh culture medium containing various concentrations of each conjugate. Cell Titer-Glo (Promega Corp.) was added to the wells at 5 days after drug administration and the luminescent signal was measured using EnVision Multilabel Plate Reader (PerkinElmer).

In vivo efficacy All animal studies were carried out in compliance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Genentech, Inc. The efficacy of the anti-HER2 antibody-drug conjugates was investigated in a mouse allograft model of MMTV-HER2 Founder #5 (murine mammary tumor). The MMTV-HER2 Founder #5 (Fo5) model (developed at Genentech) is a transgenic mouse model in which the human HER2 gene, under transcriptional regulation of the murine mammary tumor virus promoter (MMTV-HER2), is overexpressed in mammary epithelium. The overexpression causes spontaneous development of mammary tumors that overexpress the human HER2 receptor. The mammary tumor from one of the founder animals (founder #5, Fo5)

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was surgically implanted into the thoracic mammary fat pad of female nu/nu mice (Charles River Laboratories) as fragments of approximately 15-30 mm3 in size. When tumors reached the desired volume, the mice were divided into groups of n = 8 each with similar mean tumor size and received a single intravenous injection of antibody drug conjugates through the tail vein (referred to as Day 0). The treatment information is not blinded during tumor measurement. Tumors were measured in two dimensions (length and width) using calipers and the tumor volume was calculated using the formula: Tumor size (mm3) = 0.5 x (length x width x width). The results were plotted as mean tumor volume ± SEM of each group over time.

Total Antibody ELISA analysis Plasma samples were collected from non-tumor bearing mice at 10 min, 60 min, 6 hr, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days and 21 days post dose. Samples were analyzed by ELISA for total antibody concentrations, as previously described7. Nunc® MaxiSorp™ 384-well plates (Nalge Nunc International, Rochester, NY) were coated with 1 µg/mL sheep anti-human IgG antibody (Binding Site, San Diego, CA, USA) diluted in coat buffer (0.05 M carbonate/bicarbonate buffer pH 9.6) and incubated overnight at 4oC. The plates were washed 3 times with wash buffer (0.5% Tween-20 in PBS buffer, pH 7.4) and treated with block buffer (PBS/0.5% BSA/15 ppm Proclin, pH 7.4) for 1 to 2 hours. The plates were again washed 3 times with wash buffer and then samples diluted in sample diluent (PBS/0.5% BSA/0.05% Tween 20/5mM EDTA/0.25% CHAPS/ 0.35M NaCl/15 ppm Proclin, pH 7.4) were added to the wells and incubated overnight at 4oC. The next day, the plates were brought to room temperature and then washed 6 times with wash buffer. A detection antibody, goat anti-human antibodyhorseradish peroxidase (HRP) (Bethyl Laboratories, Inc., Montgomery, TX, USA), diluted to 50

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ng/mL in assay buffer (PBS/0.5% BSA/15 ppm Proclin/0.05% Tween 20, pH7.4) was added to the wells and incubated on a shaker for 1 hour at 4oC. The plates were washed 6 times with wash buffer and developed using TMB peroxidase substrate (Moss Inc., Pasadena, Maryland) for 15 minutes followed by 1 M Phosphoric acid to stop the reaction. Absorbance was measured at 450 nm against a reference wavelength of 620 nm. The concentration of the samples was extrapolated from a 4-parameter fit of the standard curve. The assay standard curve ranged from 0.39 – 50 ng/mL and tolerated a 1/100 minimum dilution of serum. Therefore, the minimum quantifiable concentration in neat serum was 40 ng/mL.

Affinity capture LC-MS To determine the in vivo stability of TDCs, affinity capture LC-MS was performed as described previously9, 41. Briefly, human HER2 extracellular domain (ECD) was biotinylated and immobilized onto Dynabeads M280 streptavidin (Invitrogen) in a 96-well plate, and then the ECD-bead system was used to capture SG3231 TDCs by incubating with approximately 30-100 µL of mouse plasma samples for 2 hours at room temperature. The captured TDCs were then washed with buffer and deglycosylated using N-Glycanase (Prozyme) at 37 °C overnight. Following extensive washes, the TDC analytes were eluted using 50 µL of 30% acetonitrile in water with 1% formic acid. A KingFisher 96 magnetic particle processor (Thermo Electron) was used to mix, wash, gather, and transfer the paramagnetic beads in the above steps. A volume of 5 µL of the eluent was analyzed by LC-MS using a TripleTOF 5600 mass spectrometer (AB Sciex). Chromatographic separation of TDCs was performed on a nanoACQUITY UPLC® system (Waters Corporation) equipped with a PS-DVB monolithic column (500 µm i.d.

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X 50 mm) (Thermo Scientific). TOF-MS spectra were extracted from the total ion chromatograms (TICs) and deconvoluted using the Bayesian Protein Reconstruct algorithm incorporated in BioPharmaViewTM 2.0 software (AB Sciex), and the average DAR was calculated based on the peak areas of different DAR species (i.e, DAR0, DAR1 and DAR2).

Pharmacokinetic Analysis Plasma samples were collected from non-tumor bearing mice at 10 min, 60 min, 6 hr, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days and 21 days post dose. Samples were analyzed for total antibody by ELISA and DAR assessment by affinity capture LC-MS. Total antibody pharmacokinetic analysis performed using WinNonlin® (Pharsight) version 6.3, Pharsight Corporation and graphs for both total antibody and DAR assessment were completed using Prism® Graph Pad software.

Isothermal Titration Calorimetry Method was adapted from published protocols by Tajc et al. for determining cysteine thiol pKa in proteins32. THIOMABTM antibody variants were buffer exchanged into 50 mM sodium phosphate, 50 mM sodium pyrophosphate, 50 mM AMPSO (ITC Triple Buffer) at the desired pH using Zeba 7K MWCO desalting column and diluted to a concentration of 75 µM. Solutions of 45 mM iodoacetamide were freshly prepared with ITC Triple Buffer at the desired pH. THIOMABTM antibody variants in pH 6 to pH 11 buffer were titrated with 4-fold excess iodoacetamide over antibody concentration using a MicroCal ITC200. The titrations were conducted at 30°C and data was collected for 50 min post injection of iodoacetamide into the

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THIOMABTM antibody solution. ITC data was processed in Origin using the MicroCal ITC module and fit with a Boltzmann distribution as previously described32.

Cysteine thiol pKa assessment by LC/MS THIOMABTM antibody variants & THIOMABTM antibody pKa mutants were buffer exchanged into 10 mM succinate pH 5.0 prior to analysis. Antibodies were diluted to 0.5 mg/mL in ITC triple buffer at pH 6 to 11. Four-fold molar excess over antibody of freshly prepared iodoacetamide was added to the antibody solution and incubated at room temperature in the dark for 1 hour. Reactions were quenched with 20 mM DTT and incubated at 37°C for 30 min to reduce antibodies, the pH of the reactions was adjusted using 10% acetic acid prior to analysis using LC/MS. LC/MS spectra were analyzed using MassHunter Bioconfirm software. Fraction of antibody alkylated by iodoacetamide was plotted as a function of pH and fit using Origin software as described for ITC data.

Cysteine reduction assay TDCs composed of THIOMABTM antibody pKa mutants conjugated to disulfide MMAE23 derivative were diluted to 1 mg/mL in 100 mM HEPES pH 7.4 and sufficient molar excess of cysteine was added for pseudo first order kinetics conditions. Samples were quenched with 5% acetic acid at the desired time point and analyzed using LC/MS as described above for cysteine thiol pKa assay. LC/MS spectra were analyzed using MassHunter Bioconfirm software and deconvoluted spectra were analyzed to calculate drug-to-antibody ratio (DAR) using Agilent DAR Calculator software. DAR values were plotted as a function of time and analyzed using non-linear regression first order decay algorithm in Prism 6.

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Succinimide hydrolysis assay Succinimide hydrolysis assay was conducted as previously described9 in phosphate buffered saline at 37°C and monitored over the course of 21 days using LC/MS.

Homology modeling of anti-HER2 4D5 antibody and site-specific mutants Protein sequence of the 4D5 antibody was searched against Protein Data Bank (PBD) to identify homologous proteins with known crystallographic structure. Besides the degree of sequence homology, the resolution and interaction context of the crystallographic complex were also taken into account in the selection of structural template for homology modeling. PDB entries 3P0Y (resolution 1.8 Å) and 1OQO (resolution 2.3 Å) were chosen as the structural templates for Fab and Fc domain, respectively. Sequence alignment and modeling were performed with MOE software (v2013.08, Chemical Computing Group) using Amber10/EHT force field, Reaction Field solvation model. Site-specific mutations were subsequently modeled with MOE Residue Scan tool using the same parameter setting.

Cysteine thiol pKa prediction The algorithm described by Jacob et al.36 and implemented in Cyspka script (http://www.pymolwiki.org/index.php/Cyspka) was applied in PyMol (version 1.8, Schrodinger, LLC) to predict cysteine pKa using homology models of the antibodies. Of the two models of thiol pKa calculation that Cyspka script offers, “Model 2” takes into account of solvation energy in addition to electrostatic interactions of cysteinate anion with protein backbone and sidechains. Cysteine thiol pKa for the initial set of 5 THIOMABTM antibodies (LC S121C, HC A118C, LC

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V205C, HC A140C) as well as a subsequent round of mutants of THIOMABTM LC K149C antibody were all predicted using Cyspka Model 2.

Acknowledgements We would like to thank Jagath Junutula, Sunil Bhatka and Josefa Chuh for their efforts in the initial screen leading to identification of the five THIOMABTM antibody variants, Erin Deuber for guidance with ITC instrumentation and analysis, and Carl Ng and Brandon Latifi for helping in preparation and analysis of materials described in the study.

Supporting information Conjugate characterization, total antibody PK, cysteine thiol pKa determination, cysteine reduction assay and MS data.

Abbreviations ADC

antibody-drug conjugate

HC

heavy chain

LC

light chain

PBD

pyrrolobenzodiazepine

DAR

drug to antibody ratio

ITC

isothermal titration calorimetry

TDC

THIOMABTM antibody-drug conjugates

MMAE

monomethyl auristatin E

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Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent

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