Hydrolytically Stable Site-Specific Conjugation at the N-Terminus of an

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Hydrolytically stable site-specific conjugation at the N-terminus of an engineered antibody. Nazzareno Dimasi, Binyam Bezabeh, Ryan Fleming, Shenlan Mao, Patrick Strout, Cui Chen, Song Cho, Haihong Zhong, Herren Wu, Changshou Gao, Monica Pruitt, and Pamela Thompson Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00355 • Publication Date (Web): 04 Sep 2015 Downloaded from http://pubs.acs.org on September 8, 2015

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Bioconjugate Chemistry

Hydrolytically stable site-specific conjugation at the N-terminus of an engineered antibody

Pamela Thompson1, Binyam Bezabeh1, Ryan Fleming1, Monica Pruitt1, Shenlan Mao2, Patrick Strout2, Cui Chen2, Song Cho2, Haihong Zhong2, Herren Wu1, Changshou Gao1,* and Nazzareno Dimasi1,*

1

Antibody Discovery and Protein Engineering, 2Oncology Research, MedImmune, Gaithersburg,

Maryland, United States of America

*

Corresponding authors’ emails: [email protected], [email protected]

ABSTRACT Antibody drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combines the target specificity of antibodies with the killing activity of

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anti-cancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogenous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the Nterminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition. KEYWORDS antibody-drug conjugate; N-terminal serine; site-specific conjugation; site-selective oxidation of antibodies; oxime ligation; in vivo efficacy

INTRODUCTION Antibody drug conjugates (ADCs) represent a growing class of therapeutics that combine the target specificity of a monoclonal antibody with the killing ability of a cytotoxic drug conjugated to the antibody. To date, the primary application of ADCs has been in oncology, with two approved drugs, Kadcyla® and Adcetris®. Kadcyla® is composed of the anti-Her2 antibody conjugated with mertansine at lysine residues, while Adcetris® is an anti-CD30 antibody conjugated with monomethyl auristatine E (MMAE) at cysteine residues.1,2 Several other ADCs armed with a variety of chemotherapeutics with distinct mechanisms of action are currently in clinical development for hematological cancers and solid tumors. The two approved ADCs,


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along with a majority of the ADCs in clinical development, use bioconjugation methods that produce heterogeneous mixtures of conjugates, which complicate analytical characterization. In addition to product heterogeneity, problems with ADC stability in serum have been identified for ADCs prepared by the reaction of native cysteine thiols with maleimides.3 The realization of these unfavorable characteristics of ADCs prepared using traditional biorthogonal methods has contributed to the design and development of a new class of ADCs which use site-specific conjugation, wherein the site of conjugation and the drug load can be precisely controlled.4-6 With the exception of glycoconjugation7-10, in which protein engineering is not required, site-specific preparation of ADCs requires the use of antibody modifications to introduce the preferred conjugation site. The most advanced and validated method for site-specific conjugation has been to take advantage of the unique reactivity of cysteines engineered at exposed surfaces of the antibody.3, 11-12 The cysteine-based conjugation approach allows for a fast and specific conjugation with maleimide-functionalized drugs, minimizes structural perturbation, and results in the preparation of homogeneous ADCs with drug-to-antibody ratios (DAR) of two. Moreover, stabilization of the conjugate can be achieved using a linker which favors hydrolysis of the succinimide ring.13,14 One disadvantage of this method is that efficient conjugation at engineered cysteines requires reduction of the antibody and subsequent reoxidation to reform the interchain disulfides. This process can potentially generate structural heterogeneity, leaving free thiols at native cysteines in the antibody which are available for conjugation to the drug, and may represent a development liability. Another site-specific conjugation modality relies on genetically encoding unnatural amino acids.15,16 An antibody engineered with an amber stop codon can be expressed in mammalian cells that have been engineered with an orthogonal tRNA/aminoacyl-tRNA synthetase pair,



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which will install the unnatural amino acid. This technique offers minimal structural perturbation of the antibody, no off-site conjugation, and the ability to use a variety of biorthogonal chemistries for conjugation.17 From the examples described above, it is evident that site-specific chemistry will help produce ADCs with optimal properties, such as defined drug load and improved in vivo properties. However, site-specific ADCs are still in their nascent stage of development and further improvements such as additional site-specific conjugation modalities may contribute to the development of next wave of ADCs. Inspired by the work of Georghegan and Stroh18 in which site-directed conjugation of a peptide to a protein was obtained via periodate oxidation of the 2-amino alcohol of a N-terminal serine or threonine residue, we envisaged the use of an anti-EphA2 antibody containing an engineered N-terminal serine residue for the site-specific attachment of an anti-cancer drug. The two step procedure described herein for the preparation of the oxime-linked ADCs is mild and robust. We demonstrate that the chemical modification of engineered serine residue positioned outside the complementarity determining region (CDR) of the antibody does not disrupt antigen binding. Furthermore, we show that these oxime-based ADCs are homogeneous, stable in serum, and have potent in vitro cytotoxicity and in vivo tumor growth inhibition. RESULTS Engineering the N-terminal serine in the antibody. The site-specific conjugation strategy described herein involves the modification of a 2-amino alcohol engineered at the N-terminus of an antibody. To this end, we engineered a serine residue at the N-terminus of the light chain domain of an anti-EphA2 antibody (1C1) and a negative control antibody (R347). Figure 1


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shows a schematic representation of an antibody showing the structure of the engineered serine at the N-terminus of the light chain. The serine engineered antibodies, 1C1-Ser and R347-Ser, were transiently expressed in 293-F cells, with expression levels of 150 mg/L, which are similar to the parental 1C1 and R347 antibodies.

Figure 1. (A) Top-view of a representative Fv domain of a human antibody (PDB: 1F8T; CH1 and VL structural information not shown). The VL and VH domains and their respective Ntermini are schematically labeled. The antigen binding domain is shown in the dotted line box.



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(B) Cartoon representation of an antibody with the serine residue engineered at the N-terminus of the light chain. The CH1 and CL structural information of the antibody are not shown.

N-terminal site-specific conjugation. The conjugation process described herein consists of two steps (Figure 2). First, the 2-amino alcohol of the terminal serine residue was oxidized to an aldehyde using 4 molar equivalents of NaIO4 in quantitative yields. Second, quantitative formation of the glyoxylic oxime proceeded overnight by the addition of the alkoxyaminebearing monomethyl auristatine E payload at pH 6 in the presence of p-anisidine19,20 as a catalyst, as detected by reduced reverse phase liquid chromatography mass spectrometry (rLCMS).


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Figure 2. Site-specific oxime-based conjugation process. An antibody with an engineered Nterminal serine residue on the light chain (A) is selectively oxidized using NaIO4 to form an



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aldehyde. The resulting antibody (B), with two glyoxylates, is reacted (C) with alkoxyaminefunctionalized monomethyl auristatine E payload in the presence of p-anisidine to form a sitespecific antibody-drug conjugate with a DAR of 2.


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Figure 3. Quantitative oxidation of (A) 1C1 and (B) R347 antibodies by NaIO4 was determined by rLCMS. (C) Equilibrium of an aldehyde and geminal diol under aqueous conditions. Expected masses (Da): 1C1-Ser, 23541; 1C1-Aldehyde, 23511; 1C1-geminal diol, 23539; R347Ser, 22832; R347-Aldehyde, 22801; R347-geminal diol, 22819. Analytical characterization of the ADCs. Quantitative oxidation of the N-terminal serine residue in both 1C1-Ser and R347-Ser was confirmed using rLCMS (Figures 3A & 3B). The periodate oxidation of an N-terminal serine residue results in the loss of 31 Da; under aqueous conditions, the resulting aldehyde is present in equilibrium as a geminal diol (Figure 3C). Ions corresponding to both the aldehyde and the geminal diol were identified in the reduced mass spectra for oxidized 1C1-Ser and R347-Ser. Oxidation and subsequent conjugation of the Nterminus did not generate in process aggregate, as shown by analytical size-exclusion chromatography (SEC-HPLC, Figure 4A). The oxime formation was highly efficient, which was demonstrated by analytical hydrophobic interaction chromatography (HIC-HPLC, Figure 4B), with 95% of ADC having a drug-to-antibody ratio (DAR) of 2. Moreover, reduced reverse phase chromatography (rRP-HPLC) confirmed that the conjugation was specific to the light chain (Figure 4C). Additionally, a DAR of 2 was determined by rLCMS (Figure 5).



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Figure 4. Analytical characterization of the antibody-drug conjugates. (A) Size-exclusion chromatography, (B) hydrophobic interaction chromatography, and (C) reduced reverse phase chromatography of the oxidized (black) and conjugated (red) 1C1 antibody.

Figure 5. Conjugation of the alkoxyamine-PEG4-MMAE to the N-terminal aldehyde determined by rLCMS. Expected masses (Da): 1C1-Ser-MMAE, 22474; R347-Ser-MMAE, 23764. Binding of serine engineered antibodies and ADCs to human EphA2. Binding of 1C1, 1C1Ser, and 1C1-Ser-MMAE to human EphA2 expressed on PC-3 cells and recombinant EphA2 was demonstrated by flow cytometry and ELISA, respectively (Figures 6A and B). PC-3 cells, which express EphA2 on cell surface,21 were incubated with antibodies and ADCs and the



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binding was detected using a fluorescently labeled secondary antibody. As shown in Figure 6A, the parental anti-EphA2 antibody 1C1, 1C1-Ser, and 1C1-Ser-MMAE bind to PC-3 with equivalent maximal binding, with EC50 values ranging from 0.22 to 0.47 µg/mL. The controls antibodies have no measurable binding to PC-3 cells. In addition, specific binding to EphA2 was further confirmed using antigen negative SKBR3 cells (data not shown). Binding of 1C1, 1C1Ser, and 1C1-Ser-MMAE to recombinant human EphA2 was verified using ELISA, as shown in Figure 6B. All 1C1 antibodies were found to have similar EC50 values ranging from 5.8 to 6.4 µg/mL for human EphA2, while all negative controls R347 did not bind to EphA2 (Figure 6B). Additionally, no binding was observed by all constructs when human serum albumin was used as a negative control in the ELISA assay (data not shown).


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Figure 6. Binding (n = 3) of wild-type antibodies, N-terminal serine engineered antibodies, and ADCs to (A) cell surface and (B) recombinant human EphA2 by flow cytometry and ELISA, respectively. Hydrolytic stability of the oxime-linked ADC. To assess the hydrolytic stability of the oximelinked ADC, we incubated 1C1-Ser-MMAE formulated at pH 7.2, for 6 days at 4oC and 40oC and at pH 4.5 for 4 days at 37oC. As shown in Figure 7, the antibody-drug conjugate was found to be stable with negligible drug loss in all tested conditions.



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Figure 7. Hydrolytic stability of 1C1-Ser-MMAE. 1C1-Ser-MMAE was incubated in (A) 1X PBS, pH 7.2 at 4°C and (B) 40°C for 6 days and (C) 5 mM sodium acetate, pH 4.5 at 37°C for 4 days and then analyzed by rLCMS. In serum stability of the ADC. One important attribute of an ADC is its stability in serum. 1C1Ser-MMAE was incubated at 200 µg/mL in rat serum at 37°C for 4 days. After four days, the ADC was affinity purified from the rat serum using an anti-human Fc specific antibody and analyzed using rLCMS. Over the course of four days, we observed no detectable loss of the oxime-linked MMAE (Figure 8).

Figure 8. 1C1-Ser-MMAE demonstrates ex vivo serum stability. The ADC was incubated in rat serum for 4 days at 37°C, affinity purified using an anti-human Fc-specific antibody, and analyzed using rLCMS.


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In vitro cytotoxicity of the ADC. To test the cytotoxicity of 1C1-Ser-MMAE towards cells expressing EphA2, we incubated the 1C1, 1C1-Ser, 1C1-Ser-MMAE, R347, R347-Ser, and R347-Ser-MMAE with PC-3 cells for six days at 37°C and measured cell viability. As shown in Figure 9, 1C1-Ser-MMAE was able to specifically kill PC-3 cells in a dose dependent manner with an IC50 of 12.98 ng/mL, whereas the unconjugated 1C1 and negative control antibodies were inactive.

Figure 9. 1C1-Ser-MMAE is highly cytotoxic against EphA2 positive PC-3 cells. In vivo anti-tumor activity. Mouse xenograft studies were conducted with PC-3 cells using nude mice. Tumor-bearing mice were treated once weekly with an intravenous injection of 3 mg/kg of 1C1-Ser, 1C1-Ser-MMAE, R347-Ser, and R347-Ser-MMAE for a total of three weeks. Significant tumor growth inhibition was observed for the 1C1-Ser-MMAE (Figure 10), while 1C1-Ser, R347-Ser, and R347-Ser-MMAE had no anti-tumor activity (Figure 10). Moreover, mouse body weights were unaffected in all treatment groups (data not shown).



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Figure 10. 1C1-Ser-MMAE has potent and specific anti-tumor activity in a mouse xenograft model of human prostate cancer. DISCUSSION In recent years intense effort has been devoted to improving the therapeutic index (TI, defined as maximum tolerated dose/minimum effective dose) of ADCs.22 Some advancements in TI improvement have come from the development of site-specific ADCs which offer precise drug load and optimal in serum stability.23 Herein we report a bioorthogonal site-specific conjugation strategy for preparing ADCs based on the engineering of a N-terminal serine residue into an antibody. This approach is broadly applicable to any IgG since it involves introducing the serine residue at the N-terminus of the light chain, which is positioned outside the antigen binding site and far from the antibody domains that are critical for binding to FcγRs and FcRn (Figure 1). Similarly, engineering a serine residue at the N-terminus of the heavy chain and its subsequent use for conjugation is also


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possible. Moreover, introducing serine residues at both N-termini of the variable domains is possible if a DAR of 4 is desired. It has been reported that the use of excess NaIO4 can oxidize the vicinal diols of both fucose and sialic acid on an antibody.7, 24 To ensure oxidation of only the engineered serine residue, the light chain of the antibody was selected for the introduction of the N-terminal serine, which allows for simultaneous monitoring of both light and heavy chain during the treatment with NaIO4. The stoichiometry of the NaIO4 was optimized to control the reaction, leaving the reactive glycans and amino acids, such as lysine, histidine, and cysteine25 intact, as determined by rLCMS in Figure 3. Subsequent conjugation of the aldehyde with an alkoxyamine-bearing monomethyl auristatine E in the presence of p-anisidine as a catalyst allowed for a complete reaction after 16 hours at pH 6. Biophysical analysis of the N-terminally conjugated ADCs showed the conjugate to be homogeneous (Figures 4B & 4C), with negligible in process aggregate formation (Figure 4A), which are both indications of manufacturability. Having demonstrated the robust formation of the ADCs at near neutral pH, we next confirmed the hydrolytic stability of the oxime-linked conjugates under both neutral and acidic pH. While the instability of aldoxime bioconjugates has been reported26, we observed no significant hydrolysis of the oxime-linked drug at both pH 7.2 and 4.5 over the course of six days (Figure 7). To address the in serum stability of the novel oxime-linked ADC, the 1C1 conjugate was incubated in rat serum for four days. An aldehyde-tagged protein conjugated via oxime chemistry with Alexa Fluor 488 showed evidence of degradation in plasma after one day.27 We, however, have found that the conjugation of an alkoxyamine-bearing drug to the N-terminus of



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the light chain of an antibody results in a stable oxime-linked conjugate, with no appreciable drug loss after a four day incubation in serum (Figure 8). This enhanced stability may be attributed to the extended pi system of the oxime. Analysis by flow cytometry and ELISA did not detect any significant difference in either 1C1Ser or 1C1-Ser-MMAE’s binding to the EphA2 antigen when compared to the parental antibody (Figure 6). In addition, when conjugated to the anti-mitotic MMAE, specific and potent cytotoxic activity is observed in antigen positive PC-3 cells (Figure 9). The 1C1-Ser-MMAE has specific and potent anti-tumor activity in a xenograft prostate cancer model expressing EphA2 (Figure 10). The in vitro cytotoxicity and in vivo tumor growth inhibition indicate that the 1C1-Ser-MMAE is efficiently internalized into target positive cells. In conclusion, this is the first report of a stable ADC conjugated at the N-terminus of an antibody using oxime chemistry. We have demonstrated that for antibody conjugations, the oxime ligation at the N-terminus is a robust reaction that occurs at near neutral pH. Additionally, the resulting oxime-linked ADC demonstrates excellent hydrolytic stability in not only neutral and acidic environments, but also in rat serum. The two-step procedure described above promises to be a valuable method for the scalable preparation of site-specific antibody-drug conjugates. Furthermore, when used in combination with bioorthogonal conjugation modalities, like the engineered cysteine approach, this N-terminal conjugation strategy offers an opportunity to combine conjugation partners, including fluorophores and other anti-cancer therapeutics, like the DNA alkylating pyrrolobenzodiazepine dimers11,12. Current efforts in our laboratory are devoted to studying (i) the in vivo pharmacology in multiple murine models, (ii) the biodistribution, pharmacokinetics, and tolerability in animal models, (iii) preparing ADCs in which the serine


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residue is engineered at the N-terminus of the heavy chain, and (iv) preparing ADCs with a DAR of 4 where serine residues are introduced at both N-termini of the light and heavy chains. EXPERIMENTAL SECTION General Remarks. All reagents were purchased from Thermo Scientific or Sigma Aldrich and were used without further purification, unless otherwise stated. The payload used in this study, alkoxyamine-PEG4-MMAE, was purchased from Concortis (Sorrento Therapeutics) and was 95% pure as judged by analytical HPLC. An Agilent 1100 series HPLC system equipped with an autosampler, diode-array detector (DAD), and ChemStation software was used to carry out analytical characterization of the ADCs. All antibodies and ADCs were detected using a wavelength of 280 nm. Monoclonal antibodies. An anti-EphA2 IgG1 antibody21 (1C1, with a kappa light chain) and negative-control antibody (R347, with a lambda light chain) were used for the N-terminal serine engineering on the light chain (1C1-Ser and R347-Ser). Standard molecular biology methods were used to generate the N-terminal serine antibody mutants. The antibodies were produced and purified at MedImmune as described by Dimasi et al.28 Expression and secretion of the recombinant antibodies were under the control of the cytomegalovirus promoter and the native immunoglobulin light chain signal peptide (Supplementary Figure 1), respectively. Antibodies were formulated into 1X PBS, pH 7.2. The 3D-structural information of the Fv domain was retrieved from the Protein Data Bank using entry code 1F8T. Oxidation of the N-terminal serine engineered antibodies. 1C1-Ser and R347-Ser antibodies were treated with 4 molar equivalents of sodium periodate (NaIO4) for 20 minutes at room temperature. The excess NaIO4 was quenched by the addition of 70 molar equivalents of serine.29



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The antibodies were dialyzed using 10 KDa molecular weight cut off (MWCO) cassettes into 1X PBS, pH 7.2. Conjugation of alkoxyamine-PEG4-MMAE to aldehyde-functionalized monoclonal antibodies. To a solution of glyoxylate-functionalized antibodies was added 1 M sodium phosphate, pH 6, and 500 mM p-anisidine (in acetonitrile) to a final concentration of 100 mM and 10 mM, respectively. 15 molar equivalents of alkoxyamine-PEG4-MMAE were then added to the reaction mixture, which was gently rotated in the dark for 16 hours. Purification and formulation of antibodies and N-terminally conjugated ADCs. The crude ADC reaction mixtures were diluted four-fold with water and purified using type II ceramic hydroxyapatite column chromatography (Biorad). Each ADC was eluted with a linear gradient of 0 M to 2 M sodium chloride in 10 mM sodium phosphate pH 7 over 20 minutes. The purified ADCs and antibodies were concentrated to 2 mg/mL and formulated by buffer exchange into 1X PBS, pH 7.2. Analytical characterization of antibodies and ADCs. The purity of antibodies and ADCs was determined by SEC-HPLC, as described by Dimasi et al.,28 with the addition of 10% isopropanol to the running buffer. 100 µg (100 µL of volume) of antibodies and ADCs were used for the SEC-HPLC analysis. Hydrophobic interaction chromatography (HIC-HPLC) was used to determine the conjugation efficiency of the ADCs and drug load distribution. HIC-HPLC was carried out using a ButylNPR column (4.6 µm ID x 3.5 cm, 2.5 µm, Tosoh Bioscience) and 25 mM Tris-HCl, 1.5 M (NH4)2SO4, pH 7.0 (buffer A), and 25 mM Tris-HCl, 5% isopropanol, pH 7.0 (buffer B). 100 µg (100 µL of volume) of antibody or ADC were loaded and eluted at a flow rate of 0.8 mL/min


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with a gradient of 5% B to 100% B over 13 minutes. Efficiency of conjugation and drug-toantibody ratio (DAR) were determined by integration of the observed peaks at 280 nm. Reduced reverse-phase chromatography (rRP-HPLC) was used to confirm conjugation to the Nterminus of the engineered light chain. Antibodies and ADCs were reduced at 37°C for 20 minutes using 42 mM dithiothreitol (DTT) in PBS pH 7.2. 10 µg (20 µL of volume) of reduced antibodies or ADCs were loaded onto a PLRP-S, 1000Å column (2.1 x 50 mm, Agilent) and eluted at 80°C at a flow rate of 1 mL/min with a gradient of 5% B to 100% B over 25 minutes (solvent A: 0.1% Trifluoroacetic acid in water and solvent B: 0.1% Trifluoroacetic acid in acetonitrile). Conjugation efficiency and DAR were determined using reduced reverse phase mass spectrometry (rLCMS) on an Agilent 1200 series HPLC coupled to an Agilent 6520 AccurateMass Q-TOF LC/MS with an electrospray ionization source. 2 µg (4 µL of volume) of reduced glycosylated antibodies or ADCs were loaded onto a Poroshell 300SB-C3 column (2.1 x 75 mm, Agilent) and eluted at a flow rate of 0.4 mL/min using a step gradient of 60% B after 6 minutes (solvent A: 0.1% Formic acid in water and solvent B: 0.1% Formic acid in acetonitrile). Agilent MassHunter was used for data acquisition and chromatogram processing. Binding of antibodies and ADCs to human EphA2. Binding of antibodies and ADCs to soluble and membrane bound human EphA2 was carried out using ELISA and flow cytometry (FACS), respectively. For the flow cytometry binding, PC-3 (ATCC CRL-1435) and SK-BR-3 (ATCC HTB-30) cells were dissociated using TrypLE (Life Technologies) and suspended at a concentration of 0.56 cells/mL in FACS buffer (1X PBS + 2% FBS). 100 µL of this cell suspension (50K cells) was pipetted into each well of a 96 well U-bottomed plate (Thermo



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Scientific), washed with 150 µL of FACS buffer, and centrifuged at 1500 rpm for 5 minutes. The resulting cell pellets were suspended in 1:4 serial dilutions of antibodies in FACS buffer in concentrations ranging from 20 µg/mL to 1.22 ng/mL. Cells were then incubated with the antibody for 2 hours on wet ice. After 2 hours, cells were spun down at 1200 rpm for 5 minutes and washed twice with 250 µL of FACS buffer. After the second wash step, the resulting cell pellets were suspended with 150 µL of 1:250 dilution of goat anti-human AlexaFluor® 647 secondary antibody (Life Technologies). Cells were incubated with the secondary antibody in wet ice for 1 hour and washed twice with FACS buffer. After the last centrifugation, the FACS buffer was removed from the plate, and 150µL of 3 µM concentration of DAPI (Life Technologies) was added to the wells to determine cell viability. Stained cells were acquired using a BD LSRII flow cytometry system (BD Biosciences), and data from the LSRII were analyzed using the software FlowJo. For the ELISA binding assay 60 ng/well of human EphA2-FLAG (MedImmune) and Bovine Serum Albumin (Thermo Fisher) were coated on 96 well plate (Corning) overnight at 4oC in 1X PBS. The plates were washed three times with 1X PBS containing 0.1% Tween-20 (PBST) and blocked with 150 µl of 3% non-fat dry milk (Biorad) in PBST at room temperature. After one hour incubation, the plates were washed with PBST and a two-fold serial dilution of the antibodies (starting from 10 µg/mL) in blocking buffer were added and incubated for one hour at room temperature. The antibody solution was removed by washing with PBST followed by one hour incubation at room temperature with a 1:3000 dilution of an anti-human-Fc-HRP antibody (Thermo Scientific) prepared in PBST. Binding was visualized with the addition of 30 µl of SureBlue Reserve TMB substrate (KPL) for 5 minutes at room temperature and the reaction was


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stopped by adding 30 µl of TMB Stop Solution (KPL). The absorbance at 450 nm was measured using a microtiter plate reader. The data were analyzed using Prism 5 software (GraphPad). Hydrolytic stability studies of the ADCs. 1C1-Ser-MMAE, at 2 mg/mL in 1X PBS, pH 7.2, was subjected to incubation at 4°C and 40°C for 6 days. The ADC was reformulated into 5 mM sodium acetate, pH 4.5, by dialysis using 10 KDa MWCO cassettes and subjected to incubation at 37°C for 4 days. All samples were analyzed by rLCMS to detect changes in DAR. Rat serum stability analysis of the 1C1-Ser-ADC. Rat serum was purchased from Jackson ImmunoResearch Labs. The serum was filtered through a 0.2 µm syringe filter into sterile polypropylene tubes and kept on ice. 100 µL of the ADC solution at 2 mg/mL was added to 900 µL of rat serum to a final concentration of 200 µg/mL. An aliquot of 300 µL was taken from the sample at time zero, placed on dry ice, and stored at -80°C within one minute of the ADC addition to serum. Following incubation for 4 days at 37°C, the sample was stored at -80°C until analysis. Anti-human Fc-specific agarose-immobilized antibody (Sigma-Aldrich) was used to affinity capture the ADC from serum. For each time point, 25 µg of immobilized anti-human IgG was mixed with 300 µL 1X PBS and 100 µL serum sample for 30 minutes at room temperature under continuous and gentle rotation. The mixture was washed in triplicate with 1X PBS to remove any unbound serum proteins and the ADC was eluted using 100 µL IgG elution buffer (Thermo Scientific) and neutralized with 20 µL 1 M Tris-HCl pH 8. To prepare the ADC for rLCMS analysis, 55 µL of ADC was treated with 3 µL 0.5 M DTT at 37°C for 30 minutes. 25 µL of the reduced ADC sample was analyzed by rLCMS. In vitro cytotoxicity. 1C1, 1C1-Ser, 1C1-Ser-MMAE, and R347-Ser-MMAE were analyzed for in vitro cytotoxicity using the human EphA2-positive PC-3 prostate cancer cell lines. PC-3 cells



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

were obtained from ATCC, maintained in RPMI1640 media (Life Technologies), and supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS) (Life Technologies) at 37°C in 5% CO2. The cells in exponential growth phase were seeded in 96-well culture plates at 1500 cells/well and then treated on the following day with the antibodies and ADCs at 4-fold serial dilution at 9 concentrations in duplicate starting from 4000 ng/mL. The treated cells were cultured for 6 days and cell viability was determined using the CellTiter-Glo Luminescent Viability Assay (Promega) following the manufacturer’s protocol. Cell viability was calculated as a percentage of control untreated cells. IC50 for 1C1-Ser-MMAE was determined using logistic non-linear regression analysis with Prism software (GraphPad). In vivo efficacy. In vivo efficacy studies were performed using 4- to 6-week-old female athymic (nu/nu) mice (Harlan Sprague Dawley Inc.). A total of 5 × 106 PC-3 cells were implanted into the right flank of mice to establish a subcutaneous tumor disease model. When the mean tumor volume was 100 to 150 mm3, the tumor-bearing mice were randomly divided into groups (n = 8, each group), and treated once a week for a total of three weeks with intravenous dose of R347Ser, 1C1-Ser, R347-Ser-MMAE, and 1C1-Ser-MMAE at 3 mg/kg. Untreated mice were included as control. Mice were monitored daily and tumors were measured twice weekly using calipers. The tumor volumes were calculated using the formula ½ × L × W2 (L = length; W = width). Body weights were measured daily to assess tolerability of the treatments. The study was terminated 41 days from tumor implantation or when the tumor volumes reached ~2000 mm3. The tumor growth inhibition was plotted using Prism software. Tumor volumes are expressed as mean ± SEM. The efficacy studies were approved by the MedImmune Institutional Animal Care and Use Committee and were conducted in accordance with the Guide of the Care and Use of Laboratory Animals.30


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FINANCIAL SUPPORT: This work was supported by MedImmune. CONFLICT OF INTEREST: All authors are employees of MedImmune and may be stockholders of AstraZeneca. ACKNOWLEDGMENTS: We would like to thank our colleague Manuel Baca for scientific discussion. REFERENCES •

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SUPPLEMENTARY INFORMATION

Supplementary Figure 1. (A) Amino acid sequence of the leader peptide and the native light chain of the anti-EphA2 antibody 1C1. (B) Amino acid sequence of the leader peptide and the Nterminal serine engineered light chain of the anti-EphA2 antibody 1C1. Leader sequences are



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

underlined; variable domains are highlighted in grey; constant domains are in italic; the engineered N-terminal serine is in red and highlighted in yellow. Identical leader sequence was used for the native and N-terminal serine engineered control antibody (R347) used in this study. In silico signal peptide cleavage prediction for the native (C) and for the N-terminal serine engineered (D) light chain of the anti-EphA2 antibody 1C1. The prediction shows that cleavage of the signal peptide occurs before the aspartic acid and the serine for the native and for the Nterminal serine engineered antibodies, respectively. Signal peptide cleavage prediction was carried out using SignalP 4.1 Server (Petersen, N., Brunak, S., von Heijne, G., and Nielsen, E. (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8, 785-786). TABLE OF CONTENT GRAPHIC

Graphical abstract: Stable antibody-drug conjugates can be rapidly prepared by the mild and selective oxidation of an N-terminally engineered serine and subsequent oxime ligation.


Page 30 of 42

Page 31 of 42 Thompson P.

et al.


1 2 3 4 5 6 7 8 Sodium periodate 9 Aldehyde N-terminal 10 Mild and selective Serine 11 oxidation 12 13 14 15 N-terminal serine engineered antibody Antibody with N-terminal aldehyde 16 17 18 19 20 21 22 23 Oxime Toxin 24 p-Anisidine 25 26 27 28 29 Alkoxyamine-toxin 30 31 Antibody-drug conjugate 32 33 34 35 36 37 38 39 ACS Plus Environment 40 Graphical abstract: Stable antibody-drug conjugates can be Paragon rapidly prepared by the mild and selective oxidation of an N-terminally 41 engineered serine and subsequent oxime ligation. 42

Thompson P. et al. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58


Page 32 of 42

A

VH N-terminal VH

N-terminal VL

VL

B VH VL

VH VL N-terminal Serine

N-terminal Serine


1


et al.


A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 B 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 C 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 2 58

VH

VH

VL

VL N-terminal Serine

N-terminal Serine

Mild and selective oxidation

VH

4 Molar Equivalent of Sodium Periodate 1X PBS, pH 7.4

VH

VL

VL

Aldehyde

Aldehyde

Oxime ligation

VH VL

Alkoxyamine-funtionalized 10 mM p-Anisidine, anti-cancer drug 100 mM Sodium phosphate pH 6

VH VL

Alkoxyamine-PEG4-monomethyl-auristatine-E (MW = 995.31 Da)


Antibody Drug Conjugate

Thompson P. et al.

x10 6 1.2

A

Page 34 of 42

23541.8 ←LC-N-Terminal-Serine 51270.6 ←HC

1.0 0.8

Anti-EphA2 (1C1)

0.6 0.4 0.2 0

20000

25000

30000

35000 40000 45000 50000 Counts vs Deconvoluted Mass (amu)

x10 5 7 6 5 4 3 2 1 0

55000

23511.6 ←LC-N-Terminal-Aldehyde

20000

25000

30000

35000 40000 45000 50000 Counts vs Deconvoluted Mass (amu)

55000

←LC-N-Terminal-Serine ←HC

20000

←HC

←LC-N-Terminal-Geminal diol

C


3

60000

51271.2 ←HC

B

Isotype control (R347)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58


60000



A

300

Monomer (99%)

A b so rb a n c e (2 8 0 n m )

250 200 150 100

Aggregate (1%)

50 0 0

2

4

6

8

10

12

14

16

18

20

R e te n tio n tim e (m in )

B

Unconjugated


125

DAR 2, 95%

100

Conjugated 75

50

DAR 1, 5%

25

0 7

8

9

10

11

12

13

14

15


Unconjugated HC C 20


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58

et al.

15

Unconjugated LC Conjugated LC DAR 1, 100%

10

5

0

ACS1 0Paragon Plus Environment 12 14

4


16

18



Page 36 of 42

LC + 1 MMAE → 24474.41

Anti-EphA2(1C1)

DAR = 2

←LC + 1 MMAE

←HC

LC (23511) ↓

LC (23511) ↓

23764.02 ← LC + 1 MMAE

DAR = 2

Isotype control (R347)

←HC ←LC + 1 MMAE

LC (22819) ↓

LC (22819) ↓


5



A 1C 1 1 C 1 -S er

M e a n flo u r e s c e n c e in te n c ity

2000

1 C 1 -S e r-M M A E

1500

1000

500 R 347 R 3 4 7 -S e r 0 1 0 -3

R 3 4 7 -S e r-M M A E 10

-2

10

-1

10

0

10

1

A b [ g /m L ]

B 3 .0 1C 1 1 C 1 -S er 2 .5 A b s o r b a n c e (4 5 0 n m )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58

et al.


2 .0

1 .5

1 .0

0 .5 R 347 0 .0 1 0 -5

R 3 4 7 -S e r 10

-4

-3

10 10 A b [ g /m L ]

-2

10

-1


6

10

0

R 3 4 7 -S e r-M M A E



A

6 days, 4°C, pH 7.2 ←LC-oxime-PEG4-MMAE

LC (23511) ↓

B 6 days, 40°C, pH 7.2 ←LC-oxime-PEG4-MMAE

LC (23511) ↓

C 4 days, 37°C, pH 4.5 ←LC-oxime-PEG4-MMAE

LC (23511) ↓


7

Page 38 of 42

Page 39 of 42 Thompson P. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58

et al.


A 1C1-Ser-ADC, time zero day in rat serum at 37°C ←LC-oxime-PEG4-MMAE

LC (23511) ↓

B 1C1-Ser-ADC, time four days in rat serum at 37°C ←LC-oxime-PEG4-MMAE

LC (23511) ↓


8

Thompson P. et al.

Page 40 of 42

1C 1

100

1 C 1 -S er R 3 4 7 -S e r-M M A E 80 V ia b ility %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Figure 58


60

40


20 0 .0 1

0 .1

1

10

100

1000

A b [n g /m L ]


9

10000


et al.


3

T u m o r v o lu m e (m m )

1 2 3 4 5 6 7 8 9 1200 10 11 12 13 1000 14 15 16 U n tre a te d 17 800 18 R 3 4 7 -S e r 19 20 R 3 4 7 -S e r-M M A E 21 600 22 1 C 1 -S er 23 24 1 C 1 -S e r-M M A E 25 400 26 27 28 29 200 30 31 32 33 0 34 D ays 35 24 31 38 36 37 38 ( = 3 m g /k g in tr a v e n o u s w e e k ly f o r th re e w e e k s ) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 ACS Paragon Plus Environment 56 57 Figure 10 58

Bioconjugate at Chemistry Page 42 of 42 Hydrolytically stable site-specific conjugation the N-terminus of an engineered antibody 1Pamela Thompson, Binyam Bezabeh, Ryan Fleming, Monica Pruitt, Shenlan Mao, Patrick Strout, 2 Cui Chen, Song Cho, Haihong Zhong, Herren Wu, Changshou Gao,* and Nazzareno Dimasi* 3 4 5 MedImmune, Gaithersburg, Maryland, United States of America 6 7 *Corresponding authors’ emails: [email protected], [email protected] 8 9 10 (A) 11 MDMRVPAQLLGLLLLWLPGARCDIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLI 12 YKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIKRTVAAPSVFIFP 13 PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK 14 HKVYACEVTHQGLSSPVTKSFNRGEC 15 16 (B) 17 18 MDMRVPAQLLGLLLLWLPGARCSDIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPG 19 KAPKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIK 20 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS 21 TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 22 23 24 25 (C) (D) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Supplementary Figure 1. (A) Amino acid sequence of the leader peptide and the native light 43 44 chain of the anti-EphA2 antibody 1C1. (B) Amino acid sequence of the leader peptide and the N45 terminal serine engineered light chain of the anti-EphA2 antibody 1C1. Leader sequences are 46 underlined; variable domains are highlighted in grey; constant domains are in italic; the engineered 47 N-terminal serine is in red and highlighted in yellow. Identical leader sequence was used for the 48 49 native and N-terminal serine engineered control antibody (R347) used in this study. In silico signal 50 peptide cleavage prediction for the native (C) and for the N-terminal serine engineered (D) light 51 chain of the anti-EphA2 antibody 1C1. The prediction shows that cleavage of the signal peptide 52 occurs before the aspartic acid and the serine for the native and for the N-terminal serine 53 54 engineered antibodies, respectively. Signal peptide cleavage prediction was carried out using 55 SignalP 4.1 Server (Petersen, N.; S.; Environment von Heijne, G.; Nielsen, E. SignalP 4.0: ACS Brunak, Paragon Plus 56 discriminating signal peptides from transmembrane regions. Nature Methods, 2011, 8, 785-786). 57 58

Hydrolytically Stable Site-Specific Conjugation at the N-Terminus of an

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