Substrate Design Enables Heterobifunctional, Dual âClickâ Antibody

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Substrate Design Enables Heterobifunctional, Dual “Click” Antibody Modification via Microbial Transglutaminase Joshua A Walker, John J Bohn, Francis Ledesma, Michelle R Sorkin, Sneha R Kabaria, Dana N Thornlow, and Christopher A. Alabi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00522 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Bioconjugate Chemistry

Substrate Design Enables Heterobifunctional, Dual “Click” Antibody Modification via Microbial Transglutaminase Joshua A. Walker[a], John J. Bohn[b],¶, Francis Ledesma[a],¶, Michelle R. Sorkin[a], Sneha R. Kabaria[a], Dana N. Thornlow[a], and Christopher A. Alabi[a],* [a] Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 113 Ho Plaza, Ithaca, NY, 14850, United States [b] Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Urbana, IL, 61801, United States Keywords: click chemistry, antibodies, bioconjugate, microbial transglutaminase, bioorthogonal chemistry ABSTRACT: Site-specific modification of native antibodies has proven advantageous, as it enhances the properties of antibody-based bioconjugates without the need to manipulate the genetic code. However, native antibody modification is typically limited to strategies that introduce a single functional handle. In this work, we addressed this limitation by designing heterobifunctional substrates for microbial transglutaminase (MTG) that contain both azide and methyltetrazine “click” handles. Structure-conjugation relationships for these substrates were evaluated using the Her2-targeted antibody trastuzumab. Förster resonance energy transfer (FRET) was used to demonstrate that these chemical handles are mutually orthogonal. This orthogonality was leveraged for the one-pot synthesis of a bifunctional antibody-drug conjugate (ADC). This ADC, containing a maytansine-derived payload and a hydrophobicity-masking polyethylene glycol (PEG) side chain, demonstrated potent in vitro activity in SKOV3 cells. These studies establish the dual “click” approach as a powerful technique in the toolbox for native antibody modification.

antigen specificity of antibodies with the chemotherapeutic potential of small molecule drugs. These efforts have led to the FDA approval of five ADCs with many more in the clinical pipeline.34 Despite their clinical success, ADCs are not without their limitations. As with traditional chemotherapy, ADCs are susceptible to resistance due to tumor heterogeneity and acquired resistance.3,35-39 Additionally, the inherent hydrophobicity of many chemotherapeutic drugs limits the therapeutic window of ADCs.7,40 Researchers have sought to leverage bifunctional antibody conjugates to ameliorate these shortcomings. With an eye towards drug resistance, synergistic ADCs containing two complimentary payloads have been shown to improve the efficacy of a drug resistant mouse DEL-BVR xenograft model.41 Further, bifunctional ADCs containing branched, hydrophilic PEG-based side chains have been shown to mitigate the aggregation-inducing effects of high degrees of drug loading.7,40 Native, bifunctional ADCs are a particularly useful class of antibody conjugates. These ADCs have been synthesized using pre-synthesized cross-linkers7,40 and two-step approaches41,42. An advantage of two-step approaches is they enable a modular, mix-and-match approach to the attachment of functional cargo. However, they suffer from the need to perform stepwise

Monoclonal antibodies, the prototypical affinity reagent, are the foundational building block of numerous bioconjugate-based molecular probes1-3 and in vivo imaging agents4,5 as well as carriers for both small molecule6-8 and macromolecular therapeutics.9,10 In recent years, site-specific modification has been identified as a powerful approach to enhance the properties of antibody-based bioconjugates for applications in both immunodetection11,12 and drug delivery13-16. Broadly, site-specific antibody modification can be categorized as utilizing either engineered antibodies (e.g. engineered cysteines17,18, non-canonical amino acids19,20, and chemoenzymatically-recognized peptide tags21,22) or native antibodies (interchain disulfide conjugation7,23, disulfide re-bridging24,25, glycan remodeling26,27, and microbial transglutaminase28-30). Modification of native antibodies is advantageous as it circumvents the need to manipulate the genetic code. This mitigates the risk of negative effects on protein folding and function31,32 as well as low titers due to the inefficient read-through of non-canonical amino acids.33 However, native antibody modifications have traditionally been limited to conjugates modified at a single site with a single functional handle. One prominent application of antibody conjugates is antibody-drug conjugates (ADCs). ADCs combine the

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purification to remove excess reagents. In this work, we propose a new approach in which orthogonal chemical handles enable modular antibody modification without the need for stepwise purification. We were inspired by two bioorthogonal “click” chemistries, the strainpromoted azide-alkyne clycloaddition (SPAAC) reaction and the inverse electron demand diels-alder (IEDDA) reaction. With judicious selection of strained alkyne and dienophile, these chemistries are mutually orthogonal.43 This property has been exploited for simultaneous, multicolor labeling of live cells.44 We reasoned that these complimentary chemistries would enable simultaneous, one-pot synthesis of bifunctional antibody conjugates. To site-specifically incorporate these chemistries, we turned to the bioconjugation enzyme microbial transglutaminase (MTG). MTG belongs to a family of enzymes that catalyze the formation of interprotein isopeptide bonds between glutamine and lysine residues.45,46 Serendipitously, MTG recognizes glutamine 295 (Q295) within the heavy chain of aglycosylated, human IgGs.47 Co-treatment with Peptide:N-glycosidase F (PNGase F) removes the N-linked glycan at asparagine 297 (N297) and facilitates efficient bioconjugation.30 By supplying non-natural acyl acceptor substrates, this natural function has been co-opted for site-specific, homofunctional antibody modification.48,49 To realize our vision, we sought to design a heterobifunctional, dual “click“ substrate for MTG.

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To demonstrate the potential for this methodology to yield therapeutically relevant bioconjugates, trastuzumab was selected as a model protein. Trastuzumab, a humanized monoclonal antibody against the Her2 receptor, is employed as the targeting component in a variety of bioconjugates including the FDA approved antibody-drug conjugate Kadcyla.34 Trastuzumab was purified from the conditioned media of a stably expressing HEK293F suspension cell line using established protocols.50

Figure 2. A) Structures of linker 1 – 5. B) Analytical RP-HPLC of purified linkers. C) Analysis of linker 1 – 3 conjugation efficiency via hydrophobic shift analyzed by HIC. D) Analysis of linker 1 and 2 conjugation efficiency via hydrophobic shift analyzed by HIC.

To synthesize the heterobifunctional substrates, first, either mono-BOC-protected bis(amine) or azidefunctionalized, amine-bearing starting material was alkylated with the corresponding alkyl halide. The resulting secondary amine intermediate was reacted with an NHS ester-activated methyltetrazine (mTz) to yield the acylated, tertiary amide product. Liberation of the terminal primary amine conjugation site was achieved by removal of the BOC protecting group via acid treatment (Figures S1 – S6). The final substrates (Figure 2A), henceforth referred to as linkers 1 – 5, were purified via reverse-phase high-performance liquid chromatography (RP-HPLC). All linkers were isolated in high purity and displayed different degrees of hydrophobicity as measured by analytical RP-HPLC (Figure 2B).

Figure 1. Dual “click” modification of native antibodies. A) Overview of site-specific conjugation scheme B) Design of heterobifunctional substrates containing either short, alkyl spacers or long, ethylene oxide-based spacers.We report the design, synthesis, and characterization of five heterobifunctional substrates for MTG. These substrates contain two bioorthogonal chemical handles, azide and methyltetrazine (Figure 1). To the best of our knowledge, this represents the first report of heterobifunctional substrates for MTG.

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Hydrophobic interaction chromatography (HIC) was used to elucidate structure-conjugation relationships for these substrates. Linker-modified trastuzumab, henceforth referred to as conjugates T1 – T5, was analyzed via HIC to observe changes in conjugate hydrophobicity due to linker incorporation. Conjugates T1 – T3 showed a heterogeneous mixture of products (Figure 2C). Each conjugate T1 – T3 showed a peak which eluted at the same retention time as the control antibody as well as two other peaks. We interpreted peaks that eluted at approximately 46 and 48 minutes to correspond to singly and doubly conjugated antibodies, respectively. Conjugates T4 and T5 showed conversion to a single, homogeneous product (Figure 2D). This result points to the importance of a flexible ethylene oxide spacer alpha to the primary amine, which is present in both linker 4 and linker 5. The ability of conjugates T1 – T5 to facilitate the attachment of macromolecular cargo was assessed via SDS-PAGE analysis. In short, unpurified conjugates T1 – T5, were analyzed for a molecular weight shift following incubation with a dibenzocyclooctyne (DBCO)or transcyclooctene (TCO)-functionalized 5,000 g/mol polyethylene glycol (PEG5K) chain (Figures S7 – S8). DBCO and TCO functional handles are mutually orthogonal and compatible with the SPAAC and IEDDA “click” reactions, respectively.44 In agreement with heavy chain-directed modification, each conjugate displayed a molecular weight increase in the 50 kDa band upon addition of DBCO- and TCO-PEG5K (Figure S29). These results were quantified by relative band intensity (Figure S30). As expected, conjugates T1 – T3 showed incomplete conjugation. In agreement with HIC analysis, conjugates T4 and T5 showed improved conjugation efficiency. However, neither T4 (52% SPAAC and 68% IEDDA) or T5 (51% SPAAC and 63% IEDDA) showed complete conjugation. This result may speak to the limited solvent accessibility of the azide and mTz handles when conjugated to Q295 or the limited solvent accessibility of the DBCO and TCO functional groups when attached to a 5 kDa PEG chain. To validate the 2-to-1 conjugation, a multi-milligram scale synthesis of conjugate T5 was performed. Conjugate T5 was isolated via HIC and recovered in high purity at an overall yield of 1.1 mg (48%) (Figure S31). Successful conjugation of 2 substrates per antibody was confirmed via matrix-assisted laser desorption/ioniztion mass spectrometry (MALDI-MS). Treatment of trastutuzmab with PNGase F to remove the N-linked glycan at N297 produced a decrease of approximately 1630 Da in the molecular weight of the heavy chain fragment. Simultaneous treatment with PNGase F, MTG, and linker 5 resulted in a 500 Da increase in the molecular weight of the aglycosylated control, consistent with incorporation of one linker per heavy chain (Figure S32).

Figure 3. Fluorescence SDS-PAGE images of conjugate T5 upon treatment with DBCO-modified carboxyrhodamine 101 and/or TCO-modified sulfo-Cy5. Denatured and reduced 4 – 20% SDS-PAGE gel. Coomassie blue protein stain. Carboxyrhodamine 101 excitation: 488 nm, emission: 500 – 540 nm. Sulfo-Cy5 excitation: 633 nm, emission 655 – 685 nm. FRET excitation: 488 nm, emission: 655 – 685 nm

Dual “click” modification of conjugate T5 was demonstrated via fluorescent SDS-PAGE analysis (Figure 3). Briefly, conjugate 5 was reacted with a Förster resonance energy transfer (FRET) pair of fluorophores, DBCO-modified carboxyrhodamine 101 and TCO-modified sulfo-Cy5 (Figure S33). Upon excitation with 488 nm light, carboxyrhodamine 101 displays a characteristic fluorescence emission at 523 nm. Upon excitation with 633 nm light, sulfo-Cy5 displays a characteristic fluorescence emission at 655 nm. Dual modification leads to intramolecular energy transfer and subsequent emission at 655 nm (sulfo-Cy5) upon excitation at 488 nm (carboxyrhodamine 101). This data validates that these chemistries (SPAAC and IEDDA) are mutually orthogonal, and therefore enable the one-pot synthesis of bifunctional antibody conjugates.

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heterobifunctional PEG (Figures S10 and S28). Conjugate T5 was reacted with DBCO-PEG, TCO-DM1, or both (Figures S34) and analyzed via HIC to assess conjugation efficiency (Figure 4A). Addition of DBCOPEG fully consumed the parent conjugate to yield a homogeneous antibody conjugate with a degree of labeling (DOL) of 2. The molecular weight of this conjugate was confirmed via MALDI-MS (Figure S35). Addition of TCO-DM1 completely consumed the parent conjugate and yielded a mixed population of products. The peaks eluting at approximately 36 and 41 minutes were interpreted to correspond to a DOL of 1 and 2 respectively. Area under the curve (AUC) analysis estimated the DOL to be approximately 1.8. In agreement with this interpretation, MALDI-MS showed a mixture of conjugates corresponding to a DOL of 1 and 2 (Figure S35). Reaction with both cargoes also yielded a mixture of conjugates. Given the complete conversion observed after the addition of DBCO-PEG, incomplete labeling was attributed to the reaction with TCO-DM1. Under this assumption, AUC analysis indicated the PEG and DM1 DOL to be approximately 2 and 1.7 respectively. MALDI-MS analysis was consistent with this interpretation (Figure S35). However, the similar molecular weight of the DBCO-PEG and TCO-DM1 modifications makes it difficult to definitively identify the source of incomplete labeling by MALDI-MS. The potency of these ADCs was demonstrated using an in vitro cell viability assay of a model, Her2 positive cell line, SKOV3 cells (Figure 4B).52 Treatment of SKOV3 cells with the PEG-modified conjugate did not show appreciable toxicity at any of the test conditions. This is to be expected as PEG is inert and biocompatible, and thus should not be toxic. A positive control, DM1-S-Me, showed toxicity at all tested conditions (Figure S36A). Analysis of the DM1-loaded conjugate demonstrated an approximate IC50 value of 8.1 nM, in line with previously reported values for DM1.53 Dual modification of trastuzumab with DM1 and PEG did not negatively impact potency as the dual labeled conjugate yielded an approximate IC50 value of 6.6 nM. MCF7 cells, a Her2 negative cell line were used to confirm the Her2-specific nature of conjugate-induced toxicity (Figure 4C). Additional characterization, which is well beyond the scope of this manuscript, would be needed to determine the pharmacokinetics of these conjugates. These data demonstrate the potential for dual “click” conjugation to produce complex, bioactive antibody conjugates using native antibodies. In conclusion, we have synthesized, characterized, and tested the conjugation efficiency of five heterobifunctional substrates for MTG. Through this approach, we identified spacer flexibility alpha to the primary amine as the critical structural component for efficient conjugation. A heterobifunctional, dual “click” conjugate was synthesized and characterized at multimilligram scale. This conjugate was used to demonstrate the mutually orthogonal nature of the SPAAC and IEDDA reactions. This powerful feature was leveraged for the one-pot synthesis of a bifunctional ADC containing a

Figure 4. Synthesis of bifunctional antibody-drug conjugates. A) HIC analysis of both single and dual “click” modification efficiency. Evaluation of in vitro potency of DM1-loaded antibody-drug conjugates using B) SKOV3 cells, a model Her2 positive cell line, and C) MCF7cells, a Her2 negative cell line. Each point represents the average of three biological replicates measured in triplicate.

To further demonstrate the utility of our dual “click” approach, we sought to carry out a one-pot synthesis of a bifunctional ADC. This ADC contained DM1, a maytansine-derived cytotoxic payload, which inhibits microtubule assembly51, and a hydrophobicity-masking PEG side chain. A DBCO-modified discrete PEG was synthesized via NHS ester chemistry (Figure S9). A TCO-PEG modified, disulfide-linked version of DM1 was synthesized via the one-pot reaction of a discrete,

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maytansine-derived cytotoxic payload and hydrophobicity-masking PEG side chain. This bifunctional antibody conjugate was shown to induce Her2-specific toxicity in an in vitro cell viability assay. Taken together, these data demonstrate the power of substrate design in developing new approaches to sitespecific antibody modification. Further, the conjugates described here may find utility in the field of pre-targeted, bioorthogonal labeling for the delivery of therapeutic cargo and imaging agents54-56. Further, an iterative methodology for linker synthesis, could be used to elaborate on the principles outlined in this work. This could enable the synthesis of antibody conjugates that contain multiple functionalities, including combinations of imaging agents, drug payloads, and stabilizing PEG chains. Ultimately, work in this area could yield multifunctional ADCs with high drug loading, diagnostic capabilities, and optimized pharmacokinetics.

ASSOCIATED CONTENT The supporting information is available free of charge on the ACS Publications website at DOI: . Materials, methods, synthesis schemes, NMR spectra, and MS spectra; supplementary figures and tables (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions J.A.W. and C.A.A. conceived the research. J.A.W., J.J.B., F.L., and M.R.S. designed the experiments. J.A.W., J.J.B., F.L., M.R.S., S.R.K., and D.N.T performed experiments and analyzed results. J.A.W. and C.A.A. wrote the manuscript. All authors edited and commented on the manuscript. All authors have given approval of the final version of the manuscript. ¶

J.J.B. and F.L. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by funds from Cornell University and the Nancy and Peter Meinig Investigator Fellowship. MRS acknowledges the Fleming fellowship for financial support. JAW acknowledges the NSF GRFP (DGE-1650441) for financial support. JJB was funded by the "Keeping Ezra's Promise" program through the RF Smith School of Chemical and Biomolecular Engineering. Equipment used to perform this research was funded in part by the NSF MRI (CHE-1531632). The authors thank Professor Joelle Pelletier (Université de Montréal, Montreal, Canada) for kindly providing plasmid pDJ1-3 for the expression of microbial transglutaminase.

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Figure 1. Dual “click” modification of native antibodies. A) Overview of site-specific conjugation scheme B) Design of heterobifunctional substrates containing either short, alkyl spacers or long, ethylene oxide-based spacers.



Figure 2. A) Structures of linker 1 – 5. B) Analytical RP-HPLC of purified linkers. C) Analysis of linker 1 – 3 conjugation efficiency via hydrophobic shift analyzed by HIC. D) Analysis of linker 1 and 2 conjugation efficiency via hydrophobic shift analyzed by HIC.


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Figure 3. Fluorescence SDS-PAGE images of conjugate T5 upon treatment with DBCO-modified carboxyrhodamine 101 and/or TCO-modified sulfo-Cy5. Denatured and reduced 4 – 20% SDS-PAGE gel. Coomassie blue protein stain. Carboxyrhodamine 101 excitation: 488 nm, emission: 500 – 540 nm. SulfoCy5 excitation: 633 nm, emission 655 – 685 nm. FRET excitation: 488 nm, emission: 655 – 685 nm



Figure 4. Synthesis of multifunctional antibody-drug conjugates. A) HIC analysis of both single and dual “click” modification efficiency. Evaluation of in vitro potency of DM1-loaded antibody-drug conjugates using B) SKOV3 cells, a model Her2 positive cell line, and C) MCF7cells, a Her2 negative cell line. Each point represents the average of three biological replicates measured in triplicate.


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Substrate Design Enables Heterobifunctional, Dual âClickâ Antibody

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Substrate Design Enables Heterobifunctional, Dual âClickâ Antibody