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Optimization of an Enzymatic Antibody Drug Conjugation Approach based on CoA Analogs Jan Grunewald, Yunho Jin, Julie Vance, Jessica Read, Xing Wang, Yongqin Wan, Huanfang Zhou, Weijia Ou, Heath E. Klock, Eric C. Peters, Tetsuo Uno, Ansgar Brock, and Bernhard H. Geierstanger Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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Bioconjugate Chemistry

Optimization of an Enzymatic Antibody Drug Conjugation Approach based on CoA Analogs Jan Grünewald,* Yunho Jin, Julie Vance, Jessica Read, Xing Wang, Yongqin Wan, Huanfang Zhou, Weijia Ou, Heath E. Klock, Eric C. Peters, Tetsuo Uno, Ansgar Brock, and Bernhard H. Geierstanger* Genomics Institute of the Novartis Research Foundation (GNF), 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States

*Bernhard H. Geierstanger, Ph.D. E-mail: [email protected]. Phone: (858) 812-1633. Genomics Institute of the Novartis Research Foundation (GNF), 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States *Jan Grünewald, Ph.D. E-mail: [email protected]. Phone: (858) 332-4473. Genomics Institute of the Novartis Research Foundation (GNF), 10675 John-Jay-Hopkins Drive, San Diego, California 92121-1125, United States

ABSTRACT: Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug-conjugates (ADCs) by enzymatically coupling CoA-linker-payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables using peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reducing the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.

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INTRODUCTION Over the last decade antibody-drug conjugates (ADCs) have been extensively pursued as cancer treatments.1-4 With about forty ADCs currently undergoing clinical trials, these biopharmaceuticals combine the favorable safety profiles of monoclonal antibodies with the high efficacy of small-molecule chemotherapeutics. In order to expand the therapeutic window of ADCs, researchers are exploring ways to site-specifically conjugate cytotoxic payloads to monoclonal antibodies as a means of improving ADC homogeneity.5 Besides glycoengineering,6,7 some of the most common methods of preparing site-specific ADCs are based on the incorporation of engineered cysteines8,9 or non-canonical amino acids10,11 into the antibody backbone. In addition, enzyme-based labeling strategies involving formylglycine-generating enzyme,12 transglutaminase,13-15 and sortase A16 have emerged as valuable alternatives for ADC preparation. All these conjugation methods provide control over stoichiometry and attachment site of the cytotoxin resulting in better pharmacokinetic (PK), safety, and efficacy profiles of the conjugates. Challenges to generate linkers that minimize premature systemic release of the cytotoxic agent while ensuring its efficient delivery inside the target cell still persist.17 Undesired drug loss in circulation is attributed to insufficient stability of covalent bonds within the linker or at the antibody attachment point. For instance, deconjugation of maleimide-linked payloads can occur via thioether exchange with serum albumin or thiol-containing metabolites.18 Likewise, the efficacy and safety of ADCs can be impaired by premature chemical or enzymatic degradation of cleavable linkers. Examples include extracellular thiolmediated breakdown of disulfides,19 hydrolytic cleavage of hydrazones in plasma,20 and proteolysis of the valine-citrulline-pamino-benzyloxycarbonyl (VC-PABC) moiety in circulation.21 We previously demonstrated that PPTases can be utilized for preparing homogeneous ADCs.22 CoA modified with maleimidocaproyl-monomethyl auristatin F (mc-MMAF) was enzymatically conjugated to two monoclonal antibodies with 11 or 12-mer peptides inserted into constant region loops. Conjugation reactions proceeded to near completion at more than sixty sites and most of the resulting ADCs were highly potent and exhibited high plasma stability.22 To further optimize the PPTase conjugation platform and to make the PPTase conjugation approach more compatible with ADC manufacturing processes, we explored a two-step strategy (Figure 1) wherein the engineered antibody is first enzymatically activated with a CoA analog that introduces a bioorthogonal group for subsequent payload attachment in the second step. These bioorthogonal CoA analogs are structurally similar to the natural substrate CoA-SH, thus ensuring high enzymatic efficiency independent of payload structure. In addition, the two-step strategy enables enzyme removal using standard purification processes, while antibody activation with a CoA analog that introduces a bioorthogonal functional group facilitates payload

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attachment at a later time. Chemical conjugation in the second step further provides flexibility in the choice of linker length and linker chemistries.

Figure 1. Chemoenzymatic ADC preparation strategy.

RESULTS AND DISCUSSION ADC Design with Variations in Linker Length, Linker Chemistry, Conjugation Site and Tag Size. Our ADC labeling approach exploits the promiscuity of CoA biosynthetic enzymes to accept a variety of structurally distinct substrates.23 To that extent, we synthesized CoA analogs 1–6 (Figure 2) from various pantothenate and pantoate analogs using a previously described chemoenzymatic approach that involves the ATP-dependent biosynthetic enzymes CoaA, CoaD and CoaE (see Supporting Information).23-27 After removal of the recombinant enzymes by ultrafiltration, the identity of the resulting CoA analogs was confirmed by ESI-MS analysis following coupling with auristatins 10, 15 and 16 (Table S1). The CoA analogs are

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equipped with ketone, azide and alkyne groups to allow for selective attachment of cytotoxic payloads via oxime formation, strain-promoted (SPAAC) and copper(I)-catalyzed (CuAAC) alkyne-azide cycloaddition, respectively.

Figure 2. Chemical structures of CoA analogs with different linker lengths and chemical reactivities, as well as auristatin payload compounds.

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The ketone-, alkyne-, and azide-functionalized CoA analogs 1-7 (see synthesis of 7 in Supporting Information) were used for PPTase-catalyzed conjugation of an anti-HER2 IgG1 at three distinct sites in the constant region of the heavy chain. Specifically, PPTase substrate sequences GDSLDMLEWSLM (A1) and DSLEFIASKLA (ybbR) were inserted between CH3 residues E388 and N389 (Eu numbering), generating constructs CH3-A1 and CH3-ybbR, respectively. In addition, CH1 residues S119 to P123 were substituted with GDSLDMLEW (A-3), resulting in construct CH1-A-3. This truncated A1 tag was previously identified as a viable substrate for AcpS, albeit labeling efficiency was inferior to the full-length tag.28 Lastly, the CH1 segments P189 – T195 and S190 – T195 were replaced with GDSLSWL (S-5) and DSLSWL (S-6), giving rise to constructs CH1-S-5 and CH1-S-6, respectively. These tags are shorter than the 8-residue minimal fragment A-4 reported by Walsh and co-workers,28 as well as the 11-12 amino acid tags (S6, ybbR and A1) used in our previous work.22 Subsequent site-specific conjugation of the CoA analogs to CH3-A1, CH3-ybbR, CH1-S-5, CH1-S-6, and CH1-A-3 antibodies (see Supporting Information) was performed in the presence of either B. subtilis Sfp PPTase or E. coli AcpS PPTase containing mutations R26L and C119S (AcpS-L26-S119). Despite the use of peptide tags containing as few as six residues, derivatization of all antibodies was confirmed by mass spectrometry (Table S2) indicating broad substrate promiscuity of all enzymes involved in the chemoenzymatic labeling approach. Chemoselective Drug Attachment via Bioorthogonal Chemistry. The substrate promiscuity of the PPTase labeling enzymes allows functionalization with a diverse set of conjugation handles for subsequent ADC payload attachment. In particular, chemoselective drug attachment via oxime ligation, CuAAC and SPAAC was tested with auristatin compounds 8-11 equipped with aminooxy, azide and bicyclo[6.1.0]nonyne (BCN) groups (see Supporting Information). Prior to chemical derivatization, enzymatically activated antibodies were purified using Protein A affinity chromatography. Chemoselective modification via oxime ligation, CuAAC and SPAAC was performed similar to previously published protocols (see Supporting Information).11,29,30 The degree of labeling with aminooxy- and BCN-functionalized drug molecules varied from 84 to 100%, equivalent to drug-to-antibody ratios (DARs) in the range of 1.7-2.0 as listed in Table 1 (see corresponding linker-payload structures in Table 2). All attempts to label alkyne-functionalized CH1-A-3-4 with azido-linked drug 10 via CuAAC failed. Access of the alkyne group of 4 may be hindered on the protein surface, but 4 and the shorter analog 5 were successfully linked via CuAAC to auristatin 10. Following reaction with 10, the resulting CoA-drug molecules 13 and 14 were subsequently conjugated to CH3-ybbR and CH1-A-3 in the presence of Sfp and AcpS-L26-S119, respectively (see details in Supporting Information). All four ADCs were obtained with a DAR of 2.0 according to ESI-MS analysis (see Tables 1 and S2, Figure S1). The

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positive results with oxime ligation and SPAAC demonstrate that the two-step PPTase-based ADC preparation strategy can accommodate several bioorthogonal labeling methods that give access to a variety of linker architectures. Table 1. ADC conjugation yields and biophysical properties. ADCd CH3-ybbR-13 CH3-ybbR-14 CH3-ybbR-17 CH3-ybbR-18 CH3-ybbR-19 CH3-ybbR-20 CH3-ybbR-21 CH3-ybbR-22 CH3-ybbR-23 CH3-ybbR-24 CH3-ybbR-25 CH3-A1-24 CH1-A-3-13 CH1-A-3-14 CH1-A-3-19 CH1-A-3-21 CH1-A-3-23 CH1-S-5-18 CH1-S-5-20 CH1-S-5-25 CH1-S-6-18 CH1-S-6-23

DAR 2.0c 2.0c 1.8b 1.7b 1.9b 1.8b 1.8b 1.7b 1.9b 1.9b 2.0c 1.9a 2.0c 2.0c 2.0a 2.0c 2.0c 1.8b 1.9b 1.8a 1.8c 1.8c

Tm1/°Ce 68.3 69.1 67.4 67.9 67.7 67.5 67.8 68.2 68.2 68.1 67.1 68.2 63.0 59.7f 62.6 62.2 62.3 68.2 68.1 67.3 68.0 67.9

Monomerg (%) >99 >99 >99 99.9 99.9 99.9 >99 99.8 99.9 99.8 95.5 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99

Yieldh (%) 58 87 49 47 53 56 51 50 56 56 49 68 46 46 37 27 63 56 56 57 55 68

DAR (drug-to-antibody ratio) was calculated from a) RP-HPLC peak areas, b) peak heights (PLRP-S column (4000 Å, 5 µm, 50 x 4.6 mm), Agilent Technologies), or c) ESI-MS peak intensities. d All linker-payload structures are shown in Table 2. e Average of three measurements. Standard deviation was equal or less than ±0.1°C. See Tm2 and Tm3 values in Table S3. f Very weak melting transition. g Analytical size-exclusion chromatography was performed on a Bio SEC-3 column (Agilent Technologies) or a Superdex 200 10/300 GL column (GE Healthcare) using PBS as mobile phase. h Percentage yield of ADC after one (ADCs coupled to 13 or 14) or two (all other ADCs) synthesis steps.

Comparison of One- and Two-Step ADC Preparations. PPTases are known as highly promiscuous enzymes that recognize structurally diverse substrates including CoA analogs featuring bulky drug moieties such as 12.22 However, despite being catalytically promiscuous, PPTases process large analogs of CoA less efficiently than unmodified CoA-SH.31 Similarly, catalytic efficiency of PPTase-mediated phosphopantetheinylation is markedly reduced after truncating the peptide substrate from 12 to 8 residues.28 In the two-step ADC preparation strategy, short CoA analogs can be used to take full advantage of the efficiency of PPTases, while also providing access to truncated peptide tags that cannot be conjugated in a single-step ADC preparation reaction.

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Table 2. In vitro potency and in vivo stability of ADCs with different linker-payload structures at various conjugation sites. Chemis-

Linker-

try

payload

CuAAC

13

CuAAC

Oxime ligation

Oxime ligation

Oxime ligation

Oxime ligation

Oxime ligation

Oxime ligation

Oxime ligation

Oxime ligation

SPAAC

Linker-payload structurea

Labeling site

JIMT1 IC50 (nM)b

DAR retention (sample number)

CH3-ybbR

0.15

0% (n=3)d

CH1-A-3

0.18

33% (n=3)d

CH3-ybbR

0.15

21% (n=3)d

CH1-A-3

0.16

37% (n=3)d

CH3-ybbR

0.28

57 ± 3% (n=3)c

CH3-ybbR

0.41

30 ± 2% (n=6)c

CH1-S-5

1.3

73 ± 3% (n=3)c

CH1-S-6

n. d.

64 ± 4% (n=3)c

CH3-ybbR

0.26

17 ± 2% (n=6)c

CH1-A-3

0.45

97 ± 0% (n=3)c

CH3-ybbR

0.39

0% (n=6)c

CH3-ybbR

0.16

0% (n=3)c

CH1-A-3

0.14

0% (n=3)c

CH3-ybbR

0.16

0% (n=6)c

CH3-ybbR

0.21

0% (n=6)c

CH1-A-3

0.12

0% (n=2)c

CH1-S-6

0.20

0% (n=2)c

CH3-ybbR

0.25

0% (n=6)c

CH3-A1

0.17

0% (n=1)c

CH3-ybbR

0.65

5% (n=6)c

CH1-S-5

1.0

42% (n=3)c

14

17

18

19

20

21

22

23

24

25

a

Linker-payload structure and its covalent connection to the serine residue of the antibody tag. The scissile amide bond within the linker-payload is highlighted. Cytotoxicity data of additional cell lines is summarized in Table S4. Average percentage and standard deviation of covalently retained drug after 504 h in vivo as determined by mass spectrometric analysis of individual plasma samples (the number in brackets corresponds to the number of samples for which ESI-MS peak intensities were high enough to allow determination of DAR retention). d Retained drug percentage after 504 h in vivo as determined by mass spectrometric analysis of pooled plasma samples (number of pooled plasma samples indicated in brackets); n. d., not determined b c

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For example, we have demonstrated that Sfp-catalyzed conjugation of 12 to 7-mer GDSLSWL in CH1-S-5 afforded only negligible amounts of labeled material as shown in Figure 3. In contrast, subjecting the same antibody to a stepwise combination of PPTase catalysis and oxime ligation using 1 and the aminooxy-auristatin 8 resulted in an ADC with a DAR of 1.8 (Table 1 and Figure 3). By taking advantage of this two-step conjugation strategy, we obtained the same conjugate ratio when 6 was enzymatically conjugated to CH1-S-5 antibody followed by SPAAC attachment of the BCN-auristatin 11. Together, these data suggest that a two-step approach of attaching a biorthogonal handle by PPTase-catalyzed labeling followed by chemoselective drug attachment significantly improves conjugation yields over a single-step enzymatic labeling protocol. It thus appears that CoA analogs featuring small bioorthogonal groups are good PPTase substrates, allowing conjugation efficiencies similar to that of unmodified CoA-SH. Moreover, switching from enzymatic to chemical drug attachment allows the usage of shorter peptide substrates (down to six residues), which is desirable because of smaller perturbation to the natural antibody sequence.

Figure 3. Mass spectrometric analysis of one- and two-step procedures for ADC preparation. (A) Low efficiency one-step conjugation of compound 12 to CH1-S-5 in the presence of Sfp PPTase. The same enzyme was used to catalyze the conjugation of 1 (B) or 6 (C) to CH1-S-5. Subsequent conjugation of CH1-S-5-1 with 8 and CH1-S-5-6 with 11 was performed using oxime ligation and SPAAC, respectively, yielding predominantly the desired ADCs. Expected masses of heavy chain species are as follows: CH1-S-5, 49286 Da; CH1-S-5-12, 50551 Da; CH1-S-5-1, 49622 Da; CH1-S-5-18, 50379 Da; CH1-S-5-6, 49635 Da; CH1-S-5-25, 50666 Da.

Biophysical Properties and Potency of ADCs in Correlation to Linker Architecture. To determine the impact of tag insertion and chemoenzymatic drug attachment on antibody scaffold stability, all ADCs were examined by differential scanning fluorimetry and analytical size-exclusion chromatography as summarized in Tables 1 and S3. In agreement with expectations,

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negligible aggregation, and a first melting transition (Tm1) of within 3.0°C of the Tm1 of unmodified anti-HER2 antibody (69.7°C) was observed.22 The only exceptions were the CH1-A-3 based ADCs, whose Tm1 values were 6.7-10.0°C lower than that of the reference. These results are generally in line with our previously reported thermofluorescence data on PPTaseconjugated ADCs.22 In addition, similar observations were reported for “aldehyde-tagged” ADCs, in which both tag incorporation and drug coupling exerted only minor effects on ADC thermostability.12 Thus, our current data add to the notion that site-specific conjugation generally results in biophysical properties resembling those of the parental antibodies. Variations in linker-payload architecture show little effect on the cytotoxic potency of the conjugates as suggested by in vitro cytotoxicity profiling of the ADCs against a panel of five HER2-positive cell lines, as well as two cell lines devoid of HER2 expression (Tables 2 and S4). While HER2-expressing cell lines JIMT-1, HCC1954, NCI-N87, SK-BR-3, and MDA-MB-231 clone 16 were effectively killed at subnanomolar ADC concentrations, no cell killing was observed for the HER2-negative cell lines A-375 and Jurkat at the highest tested ADC concentrations (IC50 >41 nM), thereby confirming a high level of targetdependent cytotoxicity in vitro. Importantly, as shown in Figure 4 and Table 2, no apparent correlation was identified between linker structure or attachment site and ADC cytotoxicity in target-expressing cells. This suggests that linker structure and attachment site have no profound effect on the combined outcome of internalization, intracellular drug release and potency of the resulting metabolite for the auristatin payloads used in the in vitro assays. Assessing the Influence of Linker Design and Payload Attachment Site on ADC Pharmacokinetics. Extracellular stability of the linker-payload is of paramount importance for ensuring ADC efficacy and safety. Premature drug release into the systemic circulation is likely associated with elevated off-target toxicity, while the resulting reduction in ADC drug load correlates with decreased potency against target-expressing tumor cells.32 Previous studies assessing ADC stability in vitro and in vivo have revealed that both attachment site and structure of the linker-payload significantly impact the rate of drug deconjugation.12,18,33 In order to examine the stability of conjugates in vivo, we performed 3-week rodent PK studies in which groups of 3 to 6 male CD-1 mice were intravenously administered a single dose of ADC at 1 mg/kg body weight. Payload deconjugation was monitored over time by comparing the plasma titers of total antibody and antibody-bound drug using anti-IgG and antidrug immunoassays, respectively. In addition, the ADC conjugation state was determined at the end of each PK experiment by affinity-capture LC ESI-MS analysis.34

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Figure 4. In vitro potencies of chemoenzymatically prepared ADCs are not significantly impacted by linker architecture and attachment site. Cytotoxicity profiling against the target-expressing JIMT-1 cell line was performed with ADCs conjugated to payloads 8 (A) and 9 (B). CH1-A-3-23 and CH1-S-6-23 were measured once, while all other data points represent mean values from triplicate (CH3-A1-24) and duplicate measurements (all remaining samples). Curve fitting details are described in the Supporting Information.

PK experiments in mice were conducted with a number of conjugates (Table 2). Surprisingly, and irrespective of the chemical identity of the linker analog (i.e., 1, 2, 3 and 7), all ADCs conjugated with 9 showed rapid deconjugation in vivo. ESI-MS confirmed that extracellular drug release was caused by cleavage of the amide bond at the carboxyl side of the drug’s phenylalanine residue (Figures S2-S9). Consistent with earlier results, no significant cleavage of the phosphodiester linkage was observed.22

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When compound 9 was substituted with 8, the corresponding conjugates displayed markedly increased stability in circulation for many but not all site and linker combinations (Table 2). ESI-MS analysis of ADC samples recovered from terminal-bleed plasma indicated that dependent on linker length and attachment site up to 97% of the initial drug load was still retained after three weeks. The observed mass differences (Figures S10-S15) suggest cleavage occurred at the same bond as in conjugates based on payload 9. Interestingly, the susceptibility to amide bond cleavage in 8 was found to diminish with decreasing chain length of the constituent linker compound. As demonstrated for conjugation site CH3-ybbR, the amount of covalently retained drug was highest for 3 and lowest for 7, while 1 and 2 conferred intermediate stability against drug-linker cleavage (Table 2). A possible explanation of this observation is that shortening of the linker reduces access of serum protease to the scissile amide bond. In order to investigate whether the location of a PPTase-derived linker-payload affects its stability in the mouse circulation, we examined the PK profiles of drug-linkers 18 and 19 at distinct conjugation sites. Mass spectrometric analysis indicated that, after three weeks in vivo, CH1-S-5-18 retained a more than two-fold higher amount of covalently bound drug as compared to CH3-ybbR-18 (Table 2). Likewise, while CH3-ybbR-19 lost more than 80% of its initial drug load in circulation, only 3% drug-linker cleavage was detectable for CH1-A-3-19 under the same conditions (Figure 5A). High in vivo stability of druglinker 19 was further confirmed by immunoassay (Figure 5B). These results highlight the drug-linker instability associated with the CH3-ybbR conjugation site and imply that both CH1 labeling positions confer substantial protection against premature payload loss. Overall, our data confirm that extracellular ADC stability can be modulated by varying the length of the linker moiety and/or its attachment site on the antibody. In order to further demonstrate that both conjugation site and linker length can affect ADC in vivo stability, we performed PK studies with conjugates CH3-ybbR-13, CH3-ybbR-14, CH1-A-3-13, and CH1-A-3-14. As shown by immunoassay and ESIMS, CH3-ybbR-13 underwent complete loss of payload after 3 weeks in circulation (0% DAR retention; Figures 6 and S16S19). MS data confirms amide bond cleavage as the cause. In contrast, compound 13 attached to the CH1-A-3 conjugation site resulted in markedly increased ADC stability with 33% of payload still conjugated to the antibody according to ESI-MS. Similarly, a reduction in linker length by replacing 4 with 5 led to an improved DAR retention of 21% at the CH3-ybbR labeling position. Using compound 14 with the shortened linker at the more protected CH1-A-3 labeling site further reduced drug loss in circulation, resulting in 37% payload retention after 3 weeks. Together, these results underline the ability to rationally optimize ADC plasma stability by fine tuning conjugation site and linker length.

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Figure 5. PK study of CH1-A-3-19 in mice reveals no significant drug loss in circulation indicative of high linker stability. Three CD-1 mice were intravenously administered with a single dose of CH1-A-3-19 at 1 mg/kg per body weight. (A) Representative deconvoluted mass spectrum of affinity-purified terminal-bleed plasma. ESI-MS analysis was performed with unpooled plasma from an individual CD-1 mouse. Expected mass of conjugated heavy chain, 50862 Da (sodium adduct); expected mass of heavy chain after linker cleavage, 50163 Da (sodium adduct); expected mass of light chain, 23443 Da. (B) Plasma titers of total antibody and antibody-bound drug were monitored over a time course of 504 h (3 weeks) using the Gyrolab Bioaffy 200 immunoassay platform (Gyros Protein Technologies AB; see experimental details in the Supporting Information).

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Figure 6. Linker length and conjugation site impact ADC stability. (A) Plasma concentration of antibody-bound drug was monitored over three weeks for four ADCs with variations in linker length and labeling position. Each data point represents the mean plasma concentration in three individually measured CD-1 mice as described in the Supporting Information. Error bars denote standard deviation. (B) DAR retention of ADCs in the same samples as measured by mass spectrometry. For each time point and ADC, plasma samples from three animals were pooled, affinity-purified, and subjected to ESI-MS. See details in Supporting Information.

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In addition, key differences in stability arise from the choice of payload structure as the scissile amide bond in 8 appears to be generally less susceptible to cleavage than the sulfonamide bond in 9, 13 and 14. As demonstrated for CH1-A-3-19 (Table 2, Figure 5) simultaneous optimization of linker length, attachment site and payload structure can yield a highly stable conjugate. Our data indicate that conjugation site, linker length and payload structure contribute to the stability of an ADC in the extracellular space, while having no significant impact on its cell-killing potency. Similar observations have been made for unrelated site-specific ADC formats containing the cleavable VC-PABC linker, which is lysosomally processed by cathepsin B.10,21,33 In these studies, the extent of in-plasma payload deconjugation was tightly controlled by the site of VC-PABC linker attachment, indicating differential steric protection of the proteolytically cleavable bond by the antibody scaffold.

CONCLUSION We have established a two-step chemoenzymatic conjugation platform for the preparation of homogeneous ADCs. The combination of PPTase catalysis and bioorthogonal chemistry ensures site-specific and near quantitative drug conjugation, while offering extensive flexibility in regard to linker architecture. Taking advantage of this flexibility, we have demonstrated that shortening of the linker length correlated with significantly improved conjugate stability in mouse plasma, while not affecting cytotoxic potency of the auristatin payloads in cell killing assays. Since the conjugation platform is compatible with a large variety of labeling sites, extracellular ADC stability can additionally be modulated by choosing the appropriate location for linker-payload attachment. Optimization of linker length and conjugation site affords ADCs that are highly stable in circulation. In addition, the enzymatic conjugation of short bioorthogonal CoA analogs permits reducing the tag length for drug attachment down from eleven22 or eight residues28 to six residues. This substantial reduction in tag length may further minimize perturbations of the protein fold while concomitantly improving ADC biophysical properties. Overall, the flexibility of the PPTasebased conjugation platform may represent a viable alternative to current approaches for producing ADCs and other protein conjugates. Importantly, restrictions imposed by the limited PPTase substrate tolerance toward large molecules are bypassed by using bioorthogonal chemistry for conjugation.

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EXPERIMENTAL PROCEDURES Cloning, expression, and purification of CoA biosynthetic enzymes. The cloning, expression, and purification of CoaA, CoaD, and CoaE have been described previously.27,35-37 Briefly, the gene encoding CoaA was PCR-amplified from S. aureus subsp. aureus Mu50 chromosomal DNA using primers coaA-sau_Fwd and coaA-sau_Rev (oligonucleotide sequences are listed in Table S5). Coding regions of CoaD and CoaE were PCR-amplified from E. coli K-12 MG1655 chromosomal DNA using oligonucleotides coaD-eco_Fwd, coaD-eco_Rev, coaE-eco_Fwd, and coaE-eco_Rev. Utilizing the PIPE method,38 the resulting amplificates were cloned into a pET vector derivative (Novagen) following amplification of the vector backbone with primers vector_Fwd and vector_Rev (Table S5). The expression vector contains an arabinose-inducible araBAD promoter and appends an N-terminal 6-thio-His6-TEV tag to the protein of interest. Next, all three CoA biosynthetic enzymes were expressed in an E. coli GeneHog derivative (Invitrogen). Transformed cells were cultured in kanamycin-containing terrific broth medium (Teknova) at 37°C and 270 rpm using baffled shake flasks (Thomson). Induction was carried out with 0.2% (w/v) arabinose (Sigma-Aldrich) when the optical density at 600 nm reached about 0.5. The cell culture was shaken for an additional 4 h at 37°C, before cells were harvested by centrifugation. Following storage at -20°C, cell pellets were homogenized and sonicated in lysis buffer (40 mM Tris/HCl, 300 mM NaCl, 10% (v/v) glycerol, 10 mM imidazole, 1 mM TCEP, pH 8.0). Cell debris was removed by centrifugation, and the cleared lysate was supplemented with Ni-NTA agarose (Qiagen). After incubation for 1 h at 4°C, the resin was drained, followed by rinsing with 6 column volumes of washing buffer (40 mM Tris/HCl, 300 mM NaCl, 10% (v/v) glycerol, 40 mM imidazole, pH 8.0) and 0.5 column volumes of elution buffer (20 mM Tris/HCl, 300 mM NaCl, 10% (v/v) glycerol, 300 mM imidazole, pH 8.0). Finally, purified enzyme was recovered by addition of 2 column volumes of elution buffer. The purity of the enzyme preparations was verified by SDS-PAGE, and the molecular weights of all three CoA biosynthetic enzymes were confirmed by ESI-MS. Large scale preparations of S. aureus CoaA, E. coli CoaD, and E. coli CoaE were performed using fermentation. For long-term storage, purified enzymes were buffer-exchanged into storage buffer (20 mM Tris/HCl, 190 mM NaCl, 10% (v/v) glycerol, 1 mM TCEP, pH 8.0), flash-frozen in liquid nitrogen, and stored at -80°C. Cloning, expression, and purification of PPTases. The cloning, expression, and purification of Bacillus subtilis Sfp was described previously.39 E. coli AcpS-L26-S119 was expressed using a pET vector derivative (Novagen), in which expression is regulated by an arabinose-inducible araBAD promoter. AcpS mutations R26L and C119S were generated using oligonucleotides AcpS-R26L_Fwd, AcpS-R26L_Rev, AcpS-C119S_Fwd, and AcpS-C119S_Rev (Table S5). The mutated enzyme was expressed without any tag in an E. coli GeneHog derivative (Invitrogen), which was grown in kanamycin-containing terrific

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broth medium (24 g yeast extract (Becton Dickinson), 16 g tryptone (Fisher Scientific), 10 g Bacto™ Casamino Acids (Becton Dickinson), 2% (w/w) glycerol, 100 mM Tris/HCl (pH 7.9 - 8.1) dissolved in water to a total volume of 1 L) at 37°C and 250 rpm using baffled shake flasks (Thomson). When the optical density at 600 nm reached 0.6, the temperature was lowered to 28°C. After shaking for an additional ~20 min, protein production was induced by the addition of arabinose (Sigma-Aldrich) to a final concentration of 0.125% (w/v). Shaking at 28°C was continued for another 5 h, before the bacterial cells were harvested by centrifugation at 4,000 x g for 2 min. Cell pellets were stored at -20°C until further use. Protein purification was initiated by resuspending harvested cells in about 6 mL of lysis buffer (50 mM Tris (pH 7.0), 50 mM NaCl, 5% (v/v) glycerol, 2 mM MgCl2) per gram of pellet. The lysis buffer was further supplemented with cOmplete™, EDTAfree Protease Inhibitor Cocktail (Roche; 1 tablet per 50 mL lysis buffer) and Benzonase® Nuclease (Novagen; 5 µL per 50 mL lysis buffer). After disrupting the cells by sonication, insoluble cell debris was removed by centrifugation (30 min, 32,570 x g). Next, the cleared lysate was loaded onto a RESOURCE S cation-exchange column (GE Healthcare). Cation-exchange chromatography was performed using a gradient from 0% - 100% buffer B over 15 column volumes (buffer A: 50 mM Tris (pH 7.0), 50 mM NaCl, 5% (v/v) glycerol, 2 mM MgCl2; buffer B: 50 mM Tris (pH 7.0), 1 M NaCl, 5% (v/v) glycerol, 2 mM MgCl2). Fractions containing the desired protein were pooled, concentrated, and injected onto a 16/60 Superdex 200 Prep Grade column (GE Healthcare). Size-exclusion chromatography was carried out in 50 mM Tris buffer (pH 7.0) supplemented with 100 mM NaCl, 2 mM MgCl2, and 5% (v/v) glycerol. AcpS-L26-S119 was confirmed by ESI-MS (expected mass, 13862.0 Da (without N-terminal methionine); observed mass, 13863.55 Da), and the purity of the enzyme was assessed by SDS-PAGE. Conjugation of chemoenzymatically prepared CoA analogs to peptide-tagged antibodies. Site-specific antibody labeling reactions with compounds 1, 2, 3, 4, 5, and 6 were catalyzed by either B. subtilis Sfp or E. coli AcpS-L26-S119. Sfp was used for conjugating CoA analogs to CH3-ybbR, CH3-A1 and CH1-S-5, while AcpS-L26-S119 was used for coupling reactions with CH1-A-3 and CH1-S-6. Enzymatic conjugation with Sfp is exemplified for the labeling of CH1-S-5 with 6 (identical conditions were used for the Sfp-catalyzed conjugation of 1 to CH1-S-5). In detail, the reaction mixture containing 6 at a final concentration of 100 µM (assuming quantitative conversion of pre-6 to its CoA analog 6, see section 3 of Supporting Information) was supplemented with CH1-S-5 and Sfp at final concentrations of 2.5 µM and 3 µM, respectively. The labeling reaction was carried out at 23°C for about 16 h in 75 mM Tris buffer (pH 8.0) containing 12.5 mM MgCl2 and 20 mM NaCl. After removing enzyme and excess compound by protein A affinity chromatography, conjugate formation was confirmed by ESI-MS (Table S2).

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AcpS-L26-S119 catalyzed conjugation is exemplified for the labeling of CH1-A-3 with 3. Specifically, the reaction mixture containing 3 at a final concentration of 200 µM (assuming full conversion of pre-3 to its CoA analog 3, see section 3 of Supporting Information) was supplemented with final concentrations of 10 µM CH1-A-3, 40 µM AcpS-L26-S119, and 75 mM HEPES buffer (pH 7). After incubating for 16 h at 37°C, the reaction mixture was purified using protein A affinity chromatography. Conjugate formation was verified by ESI-MS (Table S2). Labeling of compounds 7 and 12 to peptide-tagged antibodies. Sfp-mediated antibody modification with 7 is exemplified for the conjugation of CH1-S-5. Specifically, 2.5 µM CH1-S-5 was reacted with 100 µM 7 in 75 mM Tris buffer (pH 8.0) supplemented with 12.5 mM MgCl2 and 20 mM NaCl. After adding Sfp to a final concentration of 3 µM, the reaction was allowed to proceed for approximately 16 h at 23°C. The resulting antibody conjugate was purified by protein A affinity chromatography. Conjugate formation was confirmed by ESI-MS (Table S2). Conjugation of compound 12 (100 µM) to CH1-S-5 (2.5 µM) was catalyzed by Sfp (3 µM) in 50 mM HEPES buffer (pH 7.5) containing 10 mM MgCl2. The labeling reaction was performed for approximately 16 h at 37°C. Antibody conjugation with compounds 13 and 14. Site-specific antibody reactions with CoA-drug molecules 13 and 14 were carried out in the presence of either B. subtilis Sfp or E. coli AcpS-L26-S119. Sfp was used for coupling reactions with CH3-ybbR, while AcpS-L26-S119 was used for conjugating CH1-A-3. Sfp-catalyzed antibody conjugation is exemplified for compound 14. Specifically, 2.5 µM CH3-ybbR was reacted with 30 µM 14 in the presence of 2 µM Sfp in 100 mM HEPES buffer (pH 8) containing 10 mM MgCl2. After the conjugation reaction was allowed to proceed for 3 days at 23°C, enzyme and excess reagent were removed using protein A affinity chromatography. ADC formation was confirmed by ESI-MS (Table S2). Antibody labeling in the presence of AcpS-L26-S119 is exemplified for 13. In this case, 10 µM CH1-A-3 and 78 µM 13 were reacted in the presence of 38 µM AcpS-L26-S119 in 75 mM HEPES buffer (pH 7) supplemented with 10 mM MgCl2. After incubating for 16 h at 37°C, enzyme and excess reagent were removed by protein A affinity chromatography. Formation of the resulting ADC was verified by ESI-MS (Table S2). Drug attachment to ketone-functionalized antibodies via oxime ligation. Ketone-functionalized antibodies CH3-ybbR-1, CH3-ybbR-2, CH3-ybbR-3, CH3-ybbR-7, CH3-A1-7, CH1-A-3-2, CH1-A-3-3, CH1-S-5-1, CH1-S-5-7, CH1-S-6-1, and CH1-S-6-2 were modified with aminooxy-linked payloads 8 and 9 via oxime ligation. Drug attachment via oxime ligation is exemplified for the conjugation of CH3-A1-7 with 9. In this case, 25 µM CH3-A1-7 was reacted with 500 µM 9 in 100 mM

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sodium acetate buffer (pH 5.0) supplemented with 2.5% (v/v) DMSO. After incubation for 2 days at 23°C, excess payload was removed using size-exclusion chromatography purification. Formation of the oxime-linked ADC was verified by ESI-MS (Table S2). Drug attachment to azide-functionalized antibodies via SPAAC. Azide-functionalized antibodies CH3-ybbR-6 and CH1S-5-6 were conjugated with payload 11 using SPAAC. The copper-free click chemistry reaction is exemplified for the coupling of 11 to CH3-ybbR-6. Specifically, 127 µM CH3-ybbR-6 was reacted with 1270 µM 11 in 100 mM sodium phosphate buffer (pH 7.5) containing 1 M NaCl and 6% (v/v) DMSO. After incubation for approximately 16 h at 23°C, excess payload was removed by protein A affinity chromatography. Formation of CH3-ybbR-25 was confirmed by ESI-MS (Table S2). ADC pharmacokinetic studies in mice. Pharmacokinetic studies were carried out in male CD-1 mice (Envigo; n = 3-6/group) which were intravenously administered with a single dose of site-specific ADC at 1 mg/kg per body weight. Plasma titers of total antibody and antibody-bound drug were monitored over a time course of 504 h (three weeks) using the Gyrolab Bioaffy 200 immunoassay platform (Gyros Protein Technologies AB). Specifically, plasma samples were collected at eight time points (i.e., 1, 7, 24, 72, 168, 240, 336, and 504 h) and assayed using Biotin-SP AffiniPure Goat Anti-Human IgG (Jackson ImmunoResearch Laboratories) as capture antibody. Plasma titers of total IgG were assessed using Alexa Fluor® 647 AffiniPure Donkey Anti-Human IgG (Jackson ImmunoResearch Laboratories) as detection antibody. In addition, an in-house rabbit antiauristatin IgG was used to determine the remaining drug load of the assayed anti-HER2 ADC sample. The quantitation of plasma titers of total antibody and antibody-bound drug is based on standard curves prepared from a 1:3 serial dilution series of the PK dosing solution of the respective ADC sample. All herein described animal studies were carried out in compliance with the Novartis Animal Welfare Policy. Mass spectrometric analysis of ADCs recovered from plasma samples. To determine the remaining drug load of an ADC sample after 504 h in murine circulation, approximately 200 µL of plasma from individual animals was obtained by cardiac puncture. The collected plasma was affinity purified and subjected to mass spectrometry. In order to monitor ADC DAR retention over the course of a 3-week PK study, 20 µL of plasma was collected from each animal after 1, 7, 24, 72, 168, 240, 336, and 504 h. For each time point, plasma samples from three mice were pooled, affinity purified, and analyzed by mass spectrometry. Detailed procedures pertaining to the mass spectrometric characterization of ADCs from plasma samples have been reported earlier.22

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: http://pubs.acs.org/journal/bcches. Additional experimental procedures including antibody preparation, chemical synthesis of linker and payload compounds, assessment of ADC cytotoxicity and thermal stability, ESI-MS based characterization of ADC in vivo stability, supplementary figures and tables, gene and protein sequences (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Phone: (858) 812-1633. *E-mail: [email protected]. Phone: (858) 332-4473.

Notes All authors are employees of the Genomics Institute of the Novartis Research Foundation, a subsidiary of Novartis AG. A patent application pertaining to the chemoenzymatic preparation of ADCs has been filed.

ACKNOWLEDGMENTS We thank David H. Jones, Joseph P. Churchman, and Caitlin J. Steckler for the bioanalytical characterization of ADCs. We further thank Timothy R. Smith and Lang Huynh for carrying out cytotoxicity assays, Donnie Delarosa for performing immunoassays, Daniel McMullan and Hung Tong for purifying ADCs, and Ruben Vasquez for purifying CoA biosynthetic enzymes.

ORCID Jan Grünewald: 0000-0001-7029-3070

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ABBREVIATIONS AcpS, holo-(acyl-carrier-protein) synthase; ADC, antibody-drug-conjugate; BCN; bicyclo[6.1.0]nonyne; CoA-SH, coenzyme A; CuAAC, copper(I)-catalyzed alkyne-azide cycloaddition; DAR, drug-to-antibody ratio; HER2, human epidermal growth factor receptor 2; IC50, half maximal inhibitory concentration; mc-MMAF, maleimidocaproyl-monomethyl auristatin F; PK, pharmacokinetics; PPTase, phosphopantetheine transferase; SPAAC, strain-promoted alkyne-azide cycloaddition; Tm, melting temperature; VC-PABC, valine-citrulline-p-amino-benzyloxycarbonyl

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TABLE OF CONTENTS GRAPHIC

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115x186mm (300 x 300 DPI)



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Figure 2. Chemical structures of CoA analogs with different linker lengths and chemical reactivities, as well as auristatin payload compounds. 111x276mm (300 x 300 DPI)


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Figure 3. Mass spectrometric analysis of one- and two-step procedures for ADC preparation. (A) Low efficiency one-step conjugation of compound 12 to CH1-S-5 in the presence of Sfp PPTase. The same enzyme was used to catalyze the conjugation of 1 (B) or 6 (C) to CH1-S-5. Subsequent conjugation of CH1S-5-1 with 8 and CH1-S-5-6 with 11 was performed using oxime ligation and SPAAC, respectively, yielding predominantly the desired ADCs. Expected masses of heavy chain species are as follows: CH1-S-5, 49286 Da; CH1-S-5-12, 50551 Da; CH1-S-5-1, 49622 Da; CH1-S-5-18, 50379 Da; CH1-S-5-6, 49635 Da; CH1-S5-25, 50666 Da. 214x77mm (300 x 300 DPI)



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Figure 4. In vitro potencies of chemoenzymatically prepared ADCs are not significantly impacted by linker architecture and attachment site. Cytotoxicity profiling against the target-expressing JIMT-1 cell line was performed with ADCs conjugated to payloads 8 (A) and 9 (B). CH1-A-3-23 and CH1-S-6-23 were measured once, while all other data points represent mean values from triplicate (CH3-A1-24) and duplicate measurements (all remaining samples). Curve fitting details are described in the Supporting Information. 191x276mm (300 x 300 DPI)


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Figure 5. PK study of CH1-A-3-19 in mice reveals no significant drug loss in circulation indicative of high linker stability. Three CD-1 mice were intravenously administered with a single dose of CH1-A-3-19 at 1 mg/kg per body weight. (A) Representative deconvoluted mass spectrum of affinity-purified terminal-bleed plasma. ESI-MS analysis was performed with unpooled plasma from an individual CD-1 mouse. Expected mass of conjugated heavy chain, 50862 Da (sodium adduct); expected mass of heavy chain after linker cleavage, 50163 Da (sodium adduct); expected mass of light chain, 23443 Da. (B) Plasma titers of total antibody and antibody-bound drug were monitored over a time course of 504 h (3 weeks) using the Gyrolab Bioaffy 200 immunoassay platform (Gyros Protein Technologies AB; see experimental details in the Supporting Information). 191x224mm (300 x 300 DPI)



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Figure 6. Linker length and conjugation site impact ADC stability. (A) Plasma concentration of antibodybound drug was monitored over three weeks for four ADCs with variations in linker length and labeling position. Each data point represents the mean plasma concentration in three individually measured CD-1 mice as described in the Supporting Information. Error bars denote standard deviation. (B) DAR retention of ADCs in the same samples as measured by mass spectrometry. For each time point and ADC, plasma samples from three animals were pooled, affinity-purified, and subjected to ESI-MS. See details in Supporting Information. 172x277mm (300 x 300 DPI)


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Table of contents graphic 88x44mm (300 x 300 DPI)


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