Development, optimization and structural characterization of an

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Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies Jack Sadowsky, Nicholas Vance, Neelie Zacharias, Mark Ultsch, Guangmin Li, Aimee Fourie, Peter Liu, Julien Lafrance-Vanasse, James Andrew Ernst, Wendy Sandoval, Katherine Ruth Kozak, Gail D. Lewis-Phillips, and Weiru Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00809 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Bioconjugate Chemistry

Development, optimization and structural characterization of an efficient peptide-based photoaffinity crosslinking reaction for generation of homogeneous conjugates from wild-type antibodies Nicholas Vance,†,a Neelie Zacharias,† Mark Ultsch, Guangmin Li, Aimee Fourie, Peter Liu, Julien LaFrance-Vanasse, James A. Ernst, Wendy Sandoval, Katherine R. Kozak, Gail Phillips, Weiru Wang, Jack Sadowsky* Research & Early Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080 a. Present address: Imperial College London, Faculty of Medicine, Level 2 Commonwealth Building, Hammersmith Campus, Du Cane Road, London, England W12 0NN † These authors contributed equally to this work. * Corresponding author: [email protected]

Abstract Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein, we report the development and detailed characterization of a robust photoaffinity crosslinking method for site-specific conjugation to fully-glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously-described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. First, we describe the initial discovery of an effective Bpa mutant peptide and optimization of reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage

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of the antibody. Second, we assessed the scope of the photoconjugation reaction across different human and non-human antibodies and antibody mutants. Third, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and show that the resulting conjugate is both stable in plasma and as potent as a conventional antibody drug conjugate in cells, portending well for future biological applications.

Introduction Antibody-drug conjugates (ADCs) are a well-validated class of targeted therapeutic agents, with demonstrated pre-clinical and clinical activity against hyperproliferative diseases and other indications.1-3 The archetypal ADC is composed of an antibody, which binds to a specific antigen on the targeted cell type, a pharmacologically active small molecule (often referred to as the “drug” or “payload”), and a linker to connect the two. Successful ADC development often requires optimization of several factors, including antibody affinity and internalization, antibody-drug linker stability, and cytotoxic drug potency.4 One challenge of ADC engineering is the formation of homogeneous conjugates with a defined drug-to-antibody ratio (DAR).5 Simple conjugation to endogenous antibody lysine or cysteine residues usually results in heterogeneous product mixtures, complicating analytical methods to monitor purity, stability, and pharmacokinetics. More importantly, heterogeneous ADCs can result in increased toxicity and lower therapeutic index relative to homogeneous ADCs, likely due to small amounts of high-DAR side products in the former.6 Because of the limitations posed by heterogeneous ADCs, several site-specific antibody conjugation strategies have been developed.5 Most of these methods require engineering and expression of an antibody variants that incorporate a conjugatable amino acid or peptide tag that can be selectively modified. Conjugation methods that enable robust site-specific modification of wild-type antibodies “off-the-shelf” would be attractive alternatives to methods involving engineered antibodies. Such methods could, for example, enable the generation of homogeneous conjugates from hybridoma antibodies or, when combined with engineering approaches, the construction of dual-labeled ADCs. Chemical reactions targeting specific lysine residues,7-8 the interchain Cys residues,9-11 the so-called “nucleotide binding site” (NBS)12 or other sites13-14 have all been described for the generation of homogeneous ADCs from wild-type antibodies although each method has limitations in terms of reaction efficiency and/or degree of site-specificity. Biocatalytic methods (e.g., employing transglutaminase or glycosyltransferases) have been somewhat more successful for generating exquisitely homogeneous conjugates from wild-type antibodies,15-18 but nevertheless entail use of one or more enzymes, can be inefficient and, in all reported cases, require removal or modification of the native glycans, which can negatively affect conjugate stability and diminish biological activity.19-20


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Several promising methods for site-specific conjugation to wild-type antibodies have employed a photoaffinity crosslinking reaction between the antibody and an antibodybinding protein or peptide incorporating the non-natural amino acid 4benzoylphenylalanine (Bpa).21-27 Following irradiation at 365 nm, the benzophenone side chain of Bpa is converted to a diradical that undergoes a C-H insertion reaction with nearby surface-exposed residues on the antibody surface.28 The reported photoaffinity approaches to wild-type IgG conjugation are powerful in that they employ ligands (e.g., domains from proteins A or G) to direct chemical reaction at a specific location and do not require modifications to the endogenous glycans. A disadvantage of the reported photoaffinity approaches, however, is that they involve conjugation of fairly large Bpacontaining proteins or peptides (>60 residues) to the antibody. Recombinant generation of Bpa-containing proteins requires specialized bacterial expression systems capable of genetically encoding the non-natural Bpa residue, limiting options for optimal placement of the benzophenone moiety or derivatization of the conjugate with bioactive payloads. Furthermore, large bacterial peptides or proteins like proteins A and G are immunogenic, limiting their use for in vivo studies.29 Lastly, most photoaffinity approaches employed to date are inefficient and no one has yet reported a thorough analysis of the reaction products in terms of the specific antibody residue being modified or effects of UV irradiation on antibody integrity or function. Recently, Park and co-workers reported a photoaffinity conjugation method involving fusion proteins of a 13-residue peptide termed Fc-III, which binds to the Fc domain.30-31 Herein, we report the development of a photoconjugation method based on the 13-residue Fc-III peptide alone. We describe a systematic optimization of the conjugation chemistry and characterize structurally at highresolution the reaction product. We also demonstrate that the Fc-III-derived Bpa peptide can be used to install a unique conjugation handle onto a wild-type antibody that can then be site-specifically modified with a payload of choice.

Results and Discussion Development of photoconjugation method We first prepared a series of Bpa mutants of the 13-residue cyclic peptide, Fc-III, discovered previously by phage display to bind to the human Fc domain with nanomolar affinity (Fig. 1).30 We hypothesized that positioning a Bpa residue in Fc-III that upon complexation would be nearby a suitably reactive residue on the Fc domain would enable efficient and site-specific peptide/antibody conjugation upon UV irradiation. Since the reactive radius of benzophenone has been estimated to be >10 angstroms,32 we took a conservative design approach and mutated almost every residue in Fc-III to Bpa, sparing only Trp-4 and Gly-7, which projected away from the Fc, and the two cysteines, which form an intramolecular disulfide bridge shown to be critical for tight binding to the Fc.33 All peptides were synthesized by standard solid-phase peptide synthesis and purified by reverse-phase HPLC prior to conjugation evaluation. Our first attempt at conjugation involved reacting the panel of Fc-III peptides 1a-9a with the human monoclonal antibody Trastuzumab (hereafter referred to as TMab) in PBS in



micro-centrifuge tubes on ice under a hand-held 365 nm lamp for one hour. Upon monitoring by LCMS, a very small peak corresponding to the desired product was observed only in the reaction with peptide 7a giving a drug-to-antibody ratio (DAR) of ~0.04 (data not shown). This initially promising finding led us to react the Bpa-containing peptides in a 96-well plate directly under the 365 nm lamp with minimal space between plate and lamp at room temperature. The new setup resulted in a vastly-improved DAR of ~1.7 for peptide 7a after 4.5 hours of radiation. Conjugation was also observed for peptides 3a and 4a (DAR = 0.2 and 1.2, respectively). These results suggested that duration and/or extent of exposure to the UV source has a strong impact on conjugation efficiency. Thus, subsequent experiments were conducted in 96-well plates within a specialized UV photoreaction chamber to ensure even exposure of conjugation reaction mixtures to light. Using the irradiation chamber, we observed efficient photoconjugation of 7a to the Fc domain of TMab (DAR = 1.8; Fig. 2a left). However, we also observed that the peak for the Fab’2 region of the antibody was broadened by ~5.4-fold relative to that of unreacted antibody (Fig. 2a, right). Irradiation of antibodies with UV light is known to cause radicalmediated oxidation of tryptophan and methionine residues.34 Such effects are not ideal as they result in product heterogeneity and may lead to reduction in performance in vitro or in vivo (e.g., due to reduced binding to antigen). We attempted to minimize negative effects of photoconjugation of 7a by optimizing various parameters of the reaction. Cooling the 96-well reaction plate to ~4 C during irradiation, for example, significantly reduced the relative Fab’2 peak width to 1.4, while maintaining DAR of 1.5 (Fig. 2b). Switching buffer from PBS at pH 7.4 to histidine-acetate at pH 5.5 further reduced Fab’2 peak width ratio to 1.2 while increasing DAR to 1.8 (Fig. 2c). Including in the reaction mixture 5-hydroxyindole, an agent known to protect antibodies from UV-induced damage,35 reduced Fab’2 heterogeneity essentially completely although reduced conjugation efficiency (DAR = 1.4; Fig. 2d). Conjugation efficiency could, however, be restored by raising the concentration of peptide 7a in the reaction and extending the reaction time. Concentrations of peptide 10-fold higher than antibody and UV irradiation for 6 hours were sufficient to achieve DAR of 1.9 with minimal Fab modification (peak width ratio = 1.1; Fig. 2e; Fig. S1). Photoconjugation with these optimized conditions was repeated for all Bpa peptides 1a-9a and confirmed that only 7a reacted efficiently with TMab (Table 1). Using optimized conditions for conjugation of peptide 7a, we next evaluated conjugation of Fc-III peptides incorporating residues with a diazirine photocrosslinking group instead of Bpa. Like benzophenone-based photoaffinity ligands, diazirine-bearing ligands can react with amino acid side chains on bound receptors upon UV irradiation. However, diazirines form carbenes instead of diradicals and have shown different reactivity trends across amino acid side chains relative to benzophenone photocrosslinkers.36-37 Thus, peptide series 1b-9b and 1c-9c, in which either “photo-Leu” or “Tdf”, respectively, was placed at various positions (Fig. 1b) were synthesized and evaluated for conjugation to TMab using reaction conditions optimized for conjugation of Bpa peptide 7a.


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While some diazirine peptides demonstrated detectable conjugation, none reacted nearly as efficiently as Bpa peptide 7a (Table S1). Peptides incorporating photo-Leu were more efficiently conjugated to TMab than those with Tdf although attempted optimization of reaction conditions (e.g., pH, irradiation time) failed to result in DAR above 0.4 for either series. Furthermore, we found that photo-Leu peptides (1b-9b) were not stable as stock solutions in DMF, gradually converting over time to a species with a mass 28 Da lower than that for the starting peptide (data not shown). This byproduct may result from premature conversion to the carbene followed by intramolecular cyclization (effectively, loss of N2). Peptides with Tdf and Bpa were comparably more stable in DMF. Biophysical and structural characterization of Bpa peptide binding and conjugation The affinity of the Bpa peptides 1a-9a and the parent peptide Fc-III for TMab was measured by surface plasmon resonance (SPR). The Fc-III peptide had a dissociation constant (KD) of 17 ± 0.2 nM in our hands (Fig. 3a), consistent with values reported previously for this peptide.30, 33 In all cases, substitution of amino acids in Fc-III for the bulkier Bpa residue to generate peptides 1a-9a resulted in reduced binding affinity from ~27- to >4000-fold (Fig. 3b and Table 1). The solvent accessible surface area of the substituted amino acid in the Fc-III peptide, measured from the published structure,30 was a reasonably strong predictor of loss in binding affinity for the associated Bpa mutant (Fig. S2a). However, there appeared to be no correlation between noncovalent binding affinity of peptides 1a-9a and conjugation efficiency (Fig. S2b). Indeed, peptide 7a bound to TMab ~150-fold less tightly than did 9a (KD = 70 M versus 0.47 M, respectively) and yet 7a photoconjugated efficiently to the antibody (DAR = 1.9) whereas peptide 9a did not (DAR = 0.0). A peptide variant of Fc-III containing an extra disulfide bridge was previously reported to have a significantly improved binding affinity to human IgG (KD = 2.5 nM for the analog versus 70 nM for Fc-III itself in the same publication).38 We generated the analogous doubly-cyclized version of Bpa peptide 7a (peptide 10, Fig. 1b) and measured its affinity for and evaluated for conjugation to TMab. Interestingly, peptide 10 displayed improved binding affinity versus peptide 7a (KD = 11.4 versus 70 µM) as expected, but photoconjugation efficiency was decreased relative to 7a (DAR = 1.2 versus 1.9; Table 1). These results suggest that the photoconjugation reaction between Fc-III Bpa variants and TMab is not driven by the noncovalent affinity of the peptide/antibody complex per se, but rather by the precise positioning of the Bpa moiety, suggesting a highly specific reaction with a residue in the antibody. We first attempted to characterize the conjugation site of peptide 7a on TMab via tryptic peptide mapping of the covalent complex using tandem mass spectrometry. Given that benzophenone radicals are known to preferentially react with methionines over other amino acids,28, 39 we hypothesized that either Met-252 or Met-428 in the Fc-III peptide binding pocket reacts with the Bpa residue of 7a. Indeed, we were able to detect >90% reduction in peak intensity for the tryptic peptide encompassing Met-252, indicative of a reaction with this peptide, but we could not observe directly the conjugated product,



possibly because of poor ionization. Peak intensity for the peptide containing Met-428 was, by comparison, much less affected (Fig. S3). To clarify at higher-resolution the site of conjugation of 7a, we solved a crystal structure of this peptide covalently conjugated to the human Fc domain derived from TMab at 2.6 Å (Fig. 4 and Table S2). The electron-density omit map encompassing the Bpa residue of 7a shows unambiguously that the carbon between the two phenyl rings of the Bpa side chain is tetrahedral, with a (S) stereochemical configuration, and is covalently connected to the epsilon carbon of the Met-252 side chain on the Fc domain (Fig. 4a). These results are consistent generally with previous studies indicating the preference for benzophenone labels to react at methionine residues.39 Studies with free methionine indicate either gamma or epsilon carbons flanking the sulfur atom are approximately equally susceptible to modification by benzophenone-derived radicals.39-40 By contrast, the particular geometry of the complex between 7a and the Fc domain seems to drive a highly specific regio- and stereoselective reaction between the two. The overlay of the original Fc-III peptide bound to the Fc domain onto the 7a/Fc domain structure shows that the original binding pose of the peptide is largely preserved in the photoconjugate (RMSD less than 0.3 Å for both peptides; Fig. 4b).30 On the Fc domain, however, the side chain of Met-428 must move more than 5.0 Å to accommodate the terminal phenyl ring of the Bpa amino acid introduced in place of Val-10 on the peptide (Fig. 4c). This conformation of Met-428 has not been observed in any of the reported structures of the human Fc domain, even in complex with proteins that bind to the same general locale as does Fc-III (e.g., protein A). These results suggest that the conformation of the Met-428 side chain adopted in the 7a/Fc complex may be intrinsically unfavorable, but that the energetic penalty paid to adopt this conformation is offset by the covalent bond formed between 7a and Met-252. Hydrophobic packing or favorable pithioether interactions between the Bpa phenyl groups and the Met-428 side chain may also help to stabilize the Met-428 conformation.41 Influence of Met-252 oxidation or mutations on photocrosslinking Methionine 252 in the Fc domain of human IgGs is conserved in all human IgG antibody subclasses (IgG1, IgG2, IgG3 and IgG4) and in several antibodies from other species (e.g., rabbit IgG, murine IgG2 and rat IgG2c), although conservation is not universal in IgGs (Table S2). Importantly, modification of Met-252 can impact circulating antibody half-life in vivo: oxidation to the sulfoxide reduces half-life due to reduced FcRn binding whereas mutation of Met-252 and other residues can lead to increased half-life due to increased FcRn binding (e.g., the so-called “YTE” mutant, which includes the three mutations Met-252Tyr, Ser-254Thr, and Thr-256Glu).42-43 With representative human and non-human monoclonal antibodies, we assessed the impact of mutational or oxidative changes to Met-252 in the Fc on efficiency of photocrosslinking to 7a. Whereas conjugation of 7a to another human IgG1 antibody, Rituximab, and a human IgG4 antibody were both highly effective (DAR = 2.0 in both cases), the peptide did not react detectably with a human IgG4 “YTE” mutant (DAR = 0.0; Table 2). We observed


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significant crosslinking to a rabbit IgG (DAR = 1.2; antibody “C” in Table 2), but no conjugation to a mouse IgG1 antibody (DAR = 0; antibody “D”). These results are consistent with the conclusion that Met-252 is required for effective photoconjugation of peptide 7a to the Fc since none of the antibodies without this residue conjugated effectively. Interestingly, although rabbit IgG has a methionine at position 252 and the residues surrounding it (within 6 Å) are identical to those in human IgG1, conjugation to a rabbit IgG did not proceed to completion. We confirmed that Met-252 was unoxidized in our experiments by mass spectrometry (data not shown). It is possible that there are subtle conformational differences between human and rabbit mAbs that explains differential binding and/or photoconjugation to peptide 7a. Further optimization of peptide affinity, Bpa positioning and/or reaction conditions may enable more efficient photoconjugation to rabbit antibodies. We next assessed impact of oxidation of Met-252 in TMab on conjugation to peptide 7a. Both Met-252 and Met-428 are susceptible to oxidation under certain stress conditions (e.g., elevated temperatures, chemical oxidants, exposure to UV light), which converts the thioether side chain of these residues to a sulfoxide.44-46 To induce methionine oxidation in the Fc, samples were treated with the oxidant 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) at 37oC for up to 123 hours.45 Oxidation of Met-252 by mass spectrometry of the tryptic peptide covering this residue was monitored over time in antibody samples purified from the AAPH reaction and photocrosslinking to 7a was attempted. We observed a clear negative correlation between extent of Met-252 oxidation on TMab and extent of crosslinking to peptide 7a (Table S3). Since AAPH is a nonspecific oxidant of both Met and Trp, we considered the possibility that lack of 7a conjugation to AAPHtreated Trastuzumab is not due to Met-252 oxidation alone. It has been shown previously that the addition of free methionine in excess can selectively prevent AAPH-induced oxidation of Met-252 and other methionines in antibodies.45, 47 Indeed, in the presence of excess free methionine, TMab treated with 5% AAPH for 24 hours was less oxidized and, correspondingly, conjugation to 7a was largely restored (Table S3). These results suggest that oxidation of Met-252 in the Fc domain ablates photoconjugation of peptide 7a. Overall, based on the results discussed above, Bpa peptide 7a is highly selective for conjugation to the terminal epsilon carbon of the side chain of Met-252 in the Fc domain of antibodies that bear this residue. Both relatively small modifications of Met-252 (oxidation) and larger modifications (e.g., mutation to Tyr) prevent photoconjugation to 7a. These data are consistent with findings that benzophenone-based photoaffinity probes preferentially react with methionine residues on their targets.39 We add to this consensus view that, at least in the case of photoconjugation of 7a to the human Fc domain, the reaction is abrogated by oxidation of the thioether side chain of Met. We note, however, that we did not evaluate effects of Met-252 oxidation or mutation on noncovalent binding to peptide 7a; decreases in binding affinity could have accounted for losses in conjugation efficiency observed. Construction of site-specific ADCs from wild-type antibodies



To explore the applicability of the photoconjugation reaction to generation of antibody drug conjugates (ADCs), we synthesized a variant of peptide 7a bearing a protected thiol to enable, after photoconjugation and deprotection, attachment of thiol-reactive payloads. We evaluated both N-succinimidyl S-acetylthioacetate (SATA) and a PEG-containing SATA variant (SATA-PEG) as the group bearing the protected thiol (attached to the Nterminus) to give SATA-7a and SATA-PEG-7a, respectively (Fig. 5a). Both of these peptides were photoconjugated to TMab, the conjugates were purified, the SATA acetyl groups were removed with hydroxylamine and the conjugates were stored as free thiols available for conjugation to payloads. Whereas both SATA-7a and SATA-PEG-7a antibody conjugates were formed and deprotected efficiently, as indicated by LCMS (Fig. 5b), the SATA-7a/TMab conjugate aggregated significantly upon extended storage at 4 degrees C, as indicated by sizeexclusion chromatography (SEC; Fig. S4). These results are consistent with previous reports highlighting the solubility-enhancing effects of PEG groups on ADCs.48-50 While freezing either conjugate at -80 degrees C prevented aggregation, we decided to proceed with further study and use of the SATA-PEG-7a conjugate due to its superior stability characteristics. The free thiols of TMab/SATA-PEG-7a were reacted with the well-known cytotoxic payload -maleimido-caproyl-valine-citrulline-para-aminobenzyl-monomethyl auristatin E (mc-vc-PAB-MMAE) and the conjugate was purified. The resulting ADC had a final DAR of 1.9, corresponding to final number of MMAE moieties attached to the antibody (Fig. 5b), and was 94.7% monomeric by SEC (Fig. 5c). We assessed the cytotoxicity of the TMab/SATA-PEG-7a/MMAE conjugate and a more traditional THIOMAB™ antibody drug conjugate (TDC) bearing the same payload (DAR = 1.9) in Her2-expressing cell lines KPL-4 and SK-BR3 (Fig. 6). Potency as measured by IC50 value for the photoconjugate was essentially identical to that of the TDC (e.g., 1.7 versus 2.0 ng/mL in Sk-BR-3 cells) indicating that binding, internalization and release of the cytotoxic MMAE payload was likely unaffected by the photoconjugation format versus the more conventional TDC format. To gauge potential utility in in vivo studies, we next assessed the stability of the TMab/SATA-PEG-7a/MMAE conjugate in plasma from rats, cynomolgus monkeys and humans (Fig. 7). Over 96 hours of incubation, we observed minimal degradation or deconjugation of the payload from the photoconjugate. Interestingly, the stability of the photoconjugate was comparable to that of a THIOMAB™ antibody/MMAE conjugate employing the LC K149C conjugation site, which we have shown previously gives rise to highly stable thiosuccinimide-linked TDCs in vivo.51 Binding to FcRn is important for maintaining high circulating half-life of antibodies in vivo, a feature which is usually, but not always desired in therapeutic or imaging applications of antibodies.52 Most previous disclosures of photoconjugation or other site-specific methods targeting the Fc of antibodies have omitted analysis or even acknowledgment of possible effects on FcRn binding.


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Using a competition binding SPR assay, we observed a decrease in FcRn binding to TMab upon increasing concentrations of Fc-III (IC50 ~ 75 nM; Fig. 8). This result is consistent with the reported crystal structures of the Fc domain bound to Fc-III or FcRn, which show that Fc-III and FcRn occupy overlapping sites on the Fc domain.30, 53 Given that Bpa peptide analog 7a occupies the same site as Fc-III, it is highly likely that FcRn binding would be disrupted in the photoconjugate as well. We further predict that all previously-reported photoconjugation strategies that employ protein A-derived Z domain peptides,22-24, 27 the Fc-binding domain of protein G21, 25-26 and especially Fc-III itself31 would also disrupt FcRn binding given that the noncovalently-associating protein or peptide in each case does.54-55

Conclusions In this work we have designed, optimized and thoroughly characterized an improved peptide-based photoconjugation method for the site-specific attachment of payloads to the Fc domain of wild-type human and other IgGs. Our method is based upon a pbenzoylphenylalanine (Bpa)-containing site mutant of a previously-reported, 13-residue peptide Fc-III.30 Initial conjugation attempts with a series of Fc-III-derived peptides revealed effective UV-induced conjugation to the human Fc domain of only one Bpa mutant, peptide 7a, indicative of a highly-specific crosslink. However, these initial experiments, conducted with PBS buffer at room temperature, also resulted in extensive heterogeneity in the Fab region as indicated by mass spectrometry likely due to UVinduced radical-mediated oxidation of methionines, tryptophans or other oxidation-prone residues.56 Given the importance of the Fab domains of the antibody (specifically the complementarity-determining regions, or CDRs, therein) for antigen-binding, we subsequently optimized the photoconjugation reaction conditions to retain high conjugation efficiency while minimizing photodamage to the Fab. We found that reducing the pH, lowering the temperature and including the preservative 5-hydroxyindole were all beneficial changes to the initial method. Previously-reported conjugation methods involving photoaffinity crosslinking employed conditions very similar to those we used initially (e.g., PBS buffer, no additives) and none of the associated reports described mass spectrometry-based or other high-resolution analyses of the conjugated products. We consider the work reported here, therefore, to be the first to highlight the oxidative damage caused by UV-induced photoconjugation to antibodies and to offer practical strategies to overcome it. We characterized at atomic-level resolution the photoconjugated product formed from the human Fc domain and Bpa-containing peptide 7a, including a 2.6-angstrom crystal structure of the complex. The structure showed unambiguously a stereo and regiochemically-defined carbon-carbon bond between the Bpa side chain on 7a and the side chain of Met-252 in the Fc domain. To our knowledge, the 7a/Fc structure is only the second crystal structure of a photocrosslinked ligand/protein complex to be described (the first exhibiting a Bpa-glutamic acid crosslink57) and thus reveals for the first time fine details of a Bpa-Met crosslink in a protein-ligand complex.



Relative to other methods for site-specific antibody conjugation, the photocrosslinking method we described here has advantages and disadvantages. Whereas most methods for site-specific antibody conjugation require recombinant incorporation of a conjugation handle (e.g., Cys or peptide tag) or removal/remodeling of the glycans, our method can be performed on wild-type antibodies with intact glycans. However, we observed a strict dependence of conjugation on the presence of Met-252, which is present in some, but not all antibody species and isotypes. Notably, all antibodies and ADCs approved or in clinical trials employ human IgG antibodies (mostly IgG1),58 for which Met-252 is 100% conserved, but many commercial antibodies (e.g., for research or diagnostic use) are from host species for which the corresponding residue is not methionine (e.g., mouse or rat IgG1). Interestingly, a redox method recently reported to enable site-specific at methionine residues was successful with Fab fragments bearing engineered methionines, but was not applied to an intact antibody.59 It can therefore not be discerned from that report whether the redox method would have caused modification of one or both of the two endogenous methionines, Met-252 and/or Met-423, in the Fc domain of a full-length antibody. Our method has significant advantages relative to other photoaffinity methods reported for site-specific antibody modification to date. Most previous methods employ domains from proteins A or protein G. To our knowledge, the shortest peptide heretofore used for antibody photoconjugation, a peptide derived from a G domain, is 56 residues in length.27 A recent report highlighted the use of the shorter Fc-III peptide sequence containing a Bpa residue to perform antibody photoconjugation.31 However, this study involved recombinantly-expressed Fc-III fusion proteins incorporating the non-natural Bpa residue; the authors did not demonstrate that the Fc-III peptide alone could be used as an efficient photocrosslinking reagent. The Fc-III-derived photoconjugation peptide we describe (13 residues) offers the advantage of straightforward chemical synthesis allowing incorporating of a variety of reactive handles for subsequent attachment of, in principle, any payload. It is likely also possible with our approach to incorporate multiple orthogonal handles into the Fc-III-derived photoaffinity peptide to enable subsequent attachment of two or more different payloads. Finally, our photoconjugation approach may result in lower immunogenicity in vivo relative to approaches based on proteins A or protein G, both of which are bacterial in origin. We expect that the improved and optimized photoconjugation method we describe will enable the more facile generation of homogeneous antibody conjugates for various biological applications. As a prelude to such studies, we demonstrated functional activity in cells of a cytotoxic ADC generated from the photoconjugation method and showed that in plasma the photoconjugate was stable for at least 5 days, a finding that portends well for stability in vivo. Of greatest consequence for in vivo applications is our finding that Fc-III (and likely photoconjugated Bpa variants thereof) competes with FcRn for binding to the Fc domain. Since the Fc/FcRn interaction is important for maintaining high circulating antibody half-lives, a conjugate in which this interaction is disrupted is likely to clear rapidly in vivo. However, only one of the two FcRn binding sites on the Fc domain of an antibody may be required for efficient FcRn-mediated recycling, as is suggested by studies


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with minimized Fc domain fragments.60 Thus, a conjugate in which only one Fc-III Bpa peptide is attached to the Fc domain (DAR1), which could be obtained, for example, by purification from an intentionally-generated mixture of DAR0, DAR1 and DAR2 species, may have sufficiently long half-lives in circulation for certain applications. The utility of DAR=2 conjugates, by comparison, is likely limited to applications that do not require FcRn-mediated recycling and, thus, long circulating half-lives. Potential applications include radioactivity-based immunotherapy or imaging for which long circulating half-lives can, in both cases, increase radiationinduced toxicity and, in the latter case, reduce image contrast.61 For ocular applications of antibody therapeutics, FcRn binding can be detrimental as it drives clearance from the eye,62 providing another potential area where DAR2 photoconjugates may be employed. Finally, the photoconjugation method we described in this report may be useful in a variety of in vitro applications that would benefit from site-specific conjugation to wild-type antibodies. For example, since we have developed the photoconjugation reaction in 96-well plates with relatively small antibody amounts (~0.4 mg), it may be possible to generate libraries of homogeneously-labeled antibody conjugates from hybridomas provided the host species produces antibodies with Met-252 (e.g., rabbit). This capability could be useful for enabling more robust comparison of antibody clones for binding, internalization or potency studies, a process that would otherwise involve individual expression and purification of antibody mutants for conjugation.51, 63-65

Experimental section Peptide synthesis Peptides Fc-III, 1a-9a, 1b-9b, 1c-9c and 10 were synthesized via standard Fmoc solidphase peptide synthesis methods, purified to >90% by reverse-phase HPLC and lyophilized prior to use in conjugation reactions (Elim Biopharmaceuticals). Analytical data for each of these peptides is available as Supporting Information. For synthesis of SATA-7a, approximately 10 mg of des-acetyl 7a (600 µL; 10 mM in DMSO) was reacted with N-succinimidyl S-acetylthioacetate (SATA, ThermoFisher) (600 µL; 10 mM in DMSO) and N,N-diisopropylethylamine (DIEA) (300 µL; 20 mM in DMSO) at room temperature for 2 hours. The resulting SATA-7a peptide was purified by preparative reverse-phase HPLC using a C18 column with a gradient of buffer B (0.1% TFA in acetonitrile) in buffer A (0.1% TFA in water). Fractions were pooled and assessed for presence of the product and purity by LC-MS. Pooled fractions were lyophilized to obtain ~1.8 mg of the final product. Preparation of SATA-PEG-7a from 10 mg of des-acetyl 7a and S-acetyl-dPEG12-NHS ester (Quanta Biodesign) proceeded in a similar fashion. Analytical data for SATA-7a and SATA-PEG-7a are available as Supporting Information. Antibody conjugation



Optimized conjugation reactions for photocrosslinking peptides were performed in Vbottom, clear, polystyrene 96-well plates (ThermoFisher, product # 2605) with a final reaction volume of 50 µL. Unused wells were filled with 150 µL of deionized water. Optimized reactions were performed in 20 mM histidine acetate buffer, pH=5.5, with a final concentration of 48 µM (7.2 mg/mL) antibody, 480 µM photo-crosslinking peptide, 480 µM 5-hydroxyindole (5-HT, Sigma-Aldrich), with 10% (v/v) DMSO. Photocrosslinking was initiated upon UV irradiation at a wavelength of 365 nm and energy of 100 J/cm2 in a UVP-crosslinker chamber (AnalytikJena, CL-1000L) for 4 hours with plates on an ice-pack frozen at -20 C. The DAR was assessed by LC-MS analysis of Fc/2 or heavy chain fragments generated by IdeS or DTT treatments of Trastuzumab, respectively (see Supporting Information). To prepare MMAE-linked ADCs, Trastuzumab was conjugated to SATA-7a and SATAPEG-7a using the optimized photocrosslinking reaction conditions described above. The resulting conjugates were treated with 50 mM hydroxylamine for 30 min at room temperature to effect removal of the acetyl groups and liberation of the free thiols on the conjugated peptides, as indicated by LC-MS. The deprotected Trastuzumab/SATA-7a or Trastuzumab/SATA-PEG-7a conjugates were purified with strong cation exchange spin columns (Pierce). Cation exchange columns were equilibrated with 20 mM histidine acetate, pH 5.5. The conjugated antibody samples, diluted first into equilibration buffer (histidine-acetate, pH 5.5), were bound to the column, washed with equilibration buffer and eluted with 20 mM histidine acetate, pH 5.5, 300 mM NaCl. Conjugation of mc-vc-PAB-MMAE to thiol-deprotected Trastuzumab/SATA-PEG-7a was carried out with 4 molar equivalents (relative to antibody) of mc-vc-PAB-MMAE in 50 mM Tris, pH 7.5 buffer with 10% (v/v) DMF overnight at room temperature. The resulting MMAE conjugates were purified by S maxi cation exchange columns and were characterized by LC-MS and SEC to determine DAR, aggregation and final ADC concentration. SPR binding experiments Kinetics of peptide binding to Trastuzumab were measured by surface plasmon resonance (SPR) on a Biacore 3000 instrument (GE Healthcare) using a previously established method.38 An amine coupling kit (GE Healthcare) was used to immobilize Trastuzumab to the surface of a CM5 Sensor Chip (GE Healthcare). All injections were double-referenced with real-time reference channel subtraction and buffer blank injections. Data were analyzed using the BiaEvaluation software (version 4.1, GE Healthcare). To determine the inhibition of FcRn binding to human IgG1 by Fc-III peptide, surface plasmon resonance (SPR) measurement with a BIAcore™ 8K instrument was used. Briefly, purified recombinant human IgG1 was captured on a series S protein A sensor chip. Serial dilutions of Fc-III peptide with 1 M FcRn in assay buffer (10 mM MES pH 6.0, 150 mM NaCl, 0.05% Tween-20) were injected on the sensor chip at a flow rate of 30 L/minute for 6 minutes, which allowed the system to be at steady-state for all


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concentrations. The SPR response was then measured, plotted against the concentration of peptide and an IC50 nonlinear fit was performed, restraining the top of the curve to the response of FcRn alone, using GraphPad Prism version 7.0c for Mac OS X (GraphPad Software, La Jolla California USA, www.graphpad.com). X-ray crystallography Human Fc for crystallization studies was prepared from limited digestion with lysine C (Wako) of Trastuzumab into Fab and Fc domains, the latter of which was purified by cation exchange chromatography on an Akta purification system (GE Healthcare). The purified Fc domain was concentrated to 20 mg/mL using a 10k Amicon centrifugal concentrator (EMD Millipore). Conjugation of the IgG1-Fc sample to 7a was performed using standard reaction conditions (see above) and the conjugate was purified by sizeexclusion chromatography (SEC). Monomeric 7a/Fc conjugate was pooled and concentrated to a final concentration of 6 mg/mL using a 10k Amicon centrifugal concentrator. The quality of the final conjugate was assessed by SDS-PAGE, SEC and LC-MS to ensure high purity and DAR (DAR = 1.9, 96.6% monomeric). Crystals of the photoconjugate were grown at 18 oC by mixing 2 µL of 100mM sodium acetate pH=5.6, 12%(w/v) PEG 1000 with 1 µL of 6 mg/mL 7a/Fc conjugate by hangingdrop vapor diffusion with 1 mL reservoirs. Crystals grew as thin plates after 1 week and were cryo-stabilized in 30% (v/v) ethylene glycol and flash frozen in liquid nitrogen. Data were collected to Bragg diffraction limit of 2.3Å at the ALS 5.0.2 and processed with XDS in space group P21 and unit cell of a=66.11 b=60.85 c=68.17 90.00, 103.13, 90.00.66 Molecular replacement was performed with a previous structure of Fc-III bound to the human Fc domain (PDB code: 1DN2) as the search model and using Phaser from the CCP4 suite.67 Refinement was performed using Phenix with rounds of manual fitting using Coot.68-69 The resolution of the final refined model was 2.58 Å with a Rcryst and Rfree of 0.226 and 0.261, respectively (Table S4). Measuring oxidation of Met-252 and impacts on photoconjugation Trastuzumab in storage buffer (5 mM L-histidine, 60 mM trehalose, 0.01% polysorbate, pH=6) was treated with 5% (w/v) of 2,2’-azobis(2-methylpropionamidine) (AAPH, Sigma-Aldrich) at 37 oC in a covered reaction vessel.47 Addition of AAPH increased the pH of the solution, so 1 M sodium acetate, pH=5, was added to a final concentration of 100 mM to bring the pH of both the AAPH oxidized and unoxidized control samples to ~5. Aliquots were extracted for each time point (0, 1, 4.5, 24, 123 hours) and buffer exchanged to remove excess AAPH using S maxi cation exchange columns (Thermofisher), eluting with phosphate buffered saline (PBS). Samples were then subjected to photocrosslinking with peptide 7a using standard reaction conditions. Methionine oxidation on AAPH from 0 to 24 hours was determined by LC-MS/MS on the digested protein. 20 ug of the 20 mg/mL Trastuzumab AAPH timepoint samples were diluted with 50 mM ammonium bicarbonate pH 8 (Burdick and Jackson, Muskegon, MI) then digested with modified trypsin (Promega, Madison, WI) at a 1:50 enzyme:substrate



ratio for 3 hours at 37°C. Digestions were quenched with 4ul of 2% trifluoroacetic acid and then subjected to c18 stage-tip clean up. Samples were injected via an autosampler onto a 75 µm × 100 mm column (BEH, 1.7 micron, Waters Corp) at a flow rate of 1 µL/min using a NanoAcquity UPLC (Waters Corp). A gradient from 98% solvent A (water +0.1% formic acid) to 80% solvent B (acetonitrile +0.08% formic acid) was applied over 40 min. Samples were analyzed on-line via nanospray ionization into QExactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Data was collected in data dependent mode with the top 15 most abundant ions selected for fragmentation to generate HCD spectra. Tandem mass spectrometric data were analyzed using Byonic™(Protein Metrics Inc, San Carlos, CA) software and interrogated with Byologic™ (Protein Metrics Inc., San Carlos, CA). The percentage of oxidized methionine at position 252 was determined by comparing the area under the curve (AUC) for the oxidized and unoxidized tryptic peptide, DTLMISR. Plasma Stability Analysis To evaluate stability, photoconjugates were spiked into plasma or buffer (1X PBS [pH7.4], 0.5% BSA, 15PPM Proclin) to a final concentration of 100ug/mL. After mixing, 100 μL aliquots were incubated for different time points (0, 48 and 96 hour) at 37oC in an incubator with shaking (~700rpm). After 48 and 96hrs, samples were stored in a 80oC freezer until AC LC-MS was performed as described previously.70 Briefly, washed streptavidin-coated (SA) magnetic beads (Thermo Fisher Scientific, Waltham, MA) were mixed with either biotinylated extracellular domain of target (e.g. human erb2) or antiidiotypic antibody for specific capture using a KingFisher Flex (Thermo Fisher Scientific, Waltham, MA) and incubated for 2hrs at room temperature with gentle agitation. After washing twice with HBS-EP buffer (GE Healthcare, Sunnyvale, CA), beads were added to stability samples diluted 16-fold and incubated for 2hrs at room temperature with gentle agitation. After ADC affinity capture, beads were washed twice with HBS-EP buffer and deglycosylated overnight with PNGase F (New England BioLabs, Ipswich, MA). Following two more washes with HBS-EP buffer, two washes with water and a final wash with 10% acetonitrile, the ADCs were eluted from the beads with 30% acetonitrile/0.1% formic acid for 30mins at room temperature with gentle agitation. The eluted samples were then analyzed by LC-MS (Synapt-G2S, Waters, Milford, MA) using a PepSwift reversed phase monolithic column (500 µm × 5 cm) (Thermo Fisher Scientific, Waltham, MA) maintained at 65oC using a Waters Acquity UPLC system at a flow rate of 20 µL/min with the following gradient: 20% B (95100% acetonitrile + 0.1% formic acid) at 0-2 min; 35% B at 2.5 min; 65% B at 5 min; 95% B at 5.5 min; 5% B at 6 min. The column was directly coupled for online detection with Waters Synapt G2-S Q-ToF mass spectrometry operated in positive ESI mode with an acquisition range from m/z 500 to 5000. Stability analysis was performed using Waters BiopharmaLynx 1.3.3 software and a custom Vortex script (Dotmatics, Bishops Stortford, United Kingdom). The relative ratios of ADC with different DARs were calculated by dividing the intensity of the specific ADC species with the intensity from the total ADC species and % DAR calculated as previously described.70


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A

B

Fc-III 1a,b,c 2a,b,c 3a,b,c 4a,b,c 5a,b,c 6a,b,c 7a,b,c 8a,b,c 9a,b,c 10

Ac-DCAWHLGELVWCT-NH2 Ac-XCAWHLGELVWCT-NH2 Ac-DCXWHLGELVWCT-NH2 Ac-DCAWXLGELVWCT-NH2 Ac-DCAWHXGELVWCT-NH2 Ac-DCAWHLGXLVWCT-NH2 Ac-DCAWHLGEXVWCT-NH2 Ac-DCAWHLGELXWCT-NH2 Ac-DCAWHLGELVXCT-NH2 Ac-DCAWHLGELVWCX-NH2 Ac-CDCAWHLGELXWCTC-NH2 O

N N

CF 3

N N

X= N H

peptid e s:

O

Bpa 1a-9a, 10

N H

O

N H

photo-Leu 1b-9b

O

Tdf 1c-9c

Figure 1. Design of Bpa site mutants of Fc-binding peptide Fc-III. (A) Previously reported crystal structure (PDB ID: 1DN2) of Fc-III peptide (magenta) bound to human Fc domain (surface). Positions of each amino acid on Fc-III are labeled numerically. (B) Peptides evaluated in these studies showing sequences and structures of photoconjugation residues (For “a” peptides, X = Bpa; “b” peptides, X = Photo-Leu; “c” peptides, X = Tdf). For all peptides except 10, cysteines were crosslinked as an intramolecular disulfide. Peptide 10 was subjected to the same crosslinking conditions as other peptides and, based on its mass (see Supporting Information), is assumed to have the disulfide connection pattern of the related peptide without a Bpa residue (Cys1-Cys15, Cys3-Cys13).38



Figure 2. Optimization of photoconjugation of peptide 7a to TMab. Conjugated antibody samples were treated with IdeS to create an Fc/2 fragment (left column) and a Fab’2 fragment (right column). Since efficient conjugation to Fc/2 and minimal conjugation and negative impacts of UV irradiation on the Fab’2 were desired, DAR and Fab’2 peak width at half-height (normalized to that of non-irradiated TMab) were monitored throughout optimization. Top row shows Fc/2 and Fab’2 for non-irradiated TMab. Rows A-E show these fragments after photoconjugation to 7a to 48 M (7.2 mg/mL) TMab under various conditions, as follows: (A) 267 M 7a, PBS, room temperature, 4 hours; (B) 267 M 7a, PBS, on ice, 4 hours; (C) 267 M 7a, histidine-acetate, pH 5.5, on ice, 4 hours; (D) 267 M 7a, PBS, 267 M 5-hydroxyindole, on ice, 4 hours (E) 480 M 7a, histidine-acetate, pH 5.5, 267 M 5-hydroxyindole, 6 hours, on ice.


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A

B

Figure 3. Surface plasmon resonance analysis of Bpa peptide binding and conjugation to TMab. Full SPR sensorgrams shown for (A) Fc-III and (B) 7a peptides with raw data (black) and curves fit with a one-site binding model (red). Microscopic rate constants from curve-fitting of sensorgrams for all Bpa peptides 1a-9a and 10, including association (ka) and dissociation (kd) rates, equilibrium binding dissociation constant (KD), and DAR are indicated in Table 1.



A

B

Page 18 of 29

C

Figure 4. (A) Crystal structure at 2.6 Å resolution of 7a conjugated to the Fc region of human IgG1 (PDB ID: 6N9T). Polder Fo-Fc omit map (grey mesh) is contoured at 4.0 σ r.m.s. within 5 Å of Met-252 and the unnatural Bpa residue on chain A. (B) Overlay of the previously-reported structure of the Fc-bound Fc-III peptide (green, 1DN2) and 7a (cyan, 6N9T) shown in sticks. The binding pose of the peptide is well maintained despite the Val-10  Bpa substitution (RMSD < 0.3 Å). (C) Overlay of the 7a/Fc and Fc-III/Fc complexes highlighting the movement of Met-428 in the Fc necessary to accommodate the terminal aromatic ring of the Bpa residue (arrow).


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Figure 5. Generation of site-specific ADCs using photocrosslinking peptides (A) Synthesis scheme for generation of TMab conjugated to SATA-7a and SATA-PEG-7a crosslinkers with thiols protected by acetylation. (B) Mass spectra for the Fc/2 fragment (generated by IdeS) of the starting TMab antibody, Intermediate I, Intermediate II and the final TMab-SATA-PEG-7aMMAE ADC. Insets indicate efficient removal of the S-acetyl groups (-42 Da) from Intermediate I to give Intermediate II. (C) Size-exclusion chromatogram of Tmab/SATA-PEG-7a/MMAE conjugate with indicated percentage of monomer.



A

B

Figure 6. Cytotoxicity of TMab/SATA-PEG-7a/MMAE photoconjugate (red) and standard THIOMAB™ antibody-drug conjugate (TDC, blue) against two cell lines, Sk-BR-3 and KPL-4, expressing high levels of Her2. The IC50 values in Sk-BR-3 cells were 1.7 and 2.0 ng/mL for the photoconjugate and TDC, respectively. The IC50 values in KPL-4 cells were 2.0 and 2.3 ng/mL for the photoconjugate and TDC, respectively.


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Figure 7. Stability of TMab/SATA-PEG-7a/MMAE conjugate in plasma from various species indicated, as monitored by affinity-capture LC-MS (see Supplementary Information for details).



Figure 8. FcRn binding to Tmab is inhibited by the presence of increasing amount of Fc-III. Different peptide concentrations were mixed with 1M FcRn in a buffer at pH 6.0 and injected on a sensor chip with captured Tmab. For each experiment, the system reached steady-state within 6 minutes and the response (in resonance units (RU)) was measured. A dose-response curve was measured by nonlinear fit to calculate an IC50 of 757 nM (dotted line is extrapolation to 0 M Fc-III concentration).


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Table 1. SPR binding data for Fc-III and Bpa peptides 1a-9a and 10 including association (ka) and dissociation (kd) rates, and equilibrium binding dissociation constant (KD).a Photoconjugation DAR is also shown for reference. Peptide

ka (M-1s-1) x 10-3

kd (s-1) x 103

KD (M)

2

DAR

Fc-III

1900 ± 25

31 ± 0.17

0.017 ± 0.00024

1.4

0.0

1a

80 ± 1.9

120 ± 1.4

1.5 ± 0.040

0.41

0.1

2a

5.2 ± 0.20

210 ± 4.2

41 ± 1.8

0.47

0.0

3a

0.031 ± 0.00071

1.5 ± 0.072

47 ± 2.6

1.1

0.3

4a

0.68 ± 0.030

21 ± 0.39

30 ± 1.4

3.1

0.9

5a

11 ± 0.35

60 ± 0.79

5.7 ± 0.21

3.8

0.0

6a

0.038 ± 0.00032

0.97 ± 0.019

26 ± 0.55

1.1

0.0

7a

0.034 ± 0.00026

2.4 ± 0.022

70 ± 0.83

1.0

1.9

8a

32 ± 1.3

530 ± 11

17 ± 0.77

0.75

0.1

9a

90 ± 1.3

42 ± 0.32

0.47 ± 0.0079

0.81

0.0

10

0.54 ± 0.013

6.1 ± 0.071

11.4 ± 0.31

46

1.2

a Data

obtained from fitting SPR sensorgrams to a one-site binding model. ka = association rate, kd = dissociation rate, KD = equilibrium binding dissociation constant, DAR = drug-toantibody ratio generated under optimized conjugation conditions (see text).



Page 24 of 29

Table 2. Photoconjugation of peptide 7a to recombinant monoclonal human, rabbit and mouse antibodies. Sequence alignment around Met-252 (human IgG1 numbering) and associated DARs are shown. Antibody Species Type Sequence DAR 252

Rituximab A B C D

Human Human Human Rabbit Mouse

IgG1 IgG4 IgG4 IgG IgG1

D D D D D

T T T T V

L L L L L

M M Y M T

I I I I I

S S T S T

R R R R L

T T E T T

P P P P P

E E E E K

V V V V V

T T T T T

C C C C C

2.0 2.0 0.0 1.2 0.0

Acknowledgements We thank Tom Pillow and Greg Lazar for helpful comments.

Supporting Information Additional methods, analytical characterization data for all peptides, Figures S1-S4 and Tables S1-S4.

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Development, optimization and structural characterization of an

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