Genetically Encoded Azide Containing Amino Acid in Mammalian

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Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody−Drug Conjugates Using Click Cycloaddition Chemistry Michael P. VanBrunt,† Kurt Shanebeck,† Zachary Caldwell,† Jeffrey Johnson,† Pamela Thompson,‡ Thomas Martin,‡ Huifang Dong,‡ Gary Li,† Hengyu Xu,† Francois D’Hooge,§ Luke Masterson,§ Pauline Bariola,† Arnaud Tiberghien,§ Ebele Ezeadi,§ David G. Williams,§ John A. Hartley,∥,§ Philip W. Howard,§ Kenneth H. Grabstein,† Michael A. Bowen,‡ and Marcello Marelli*,†,‡ †

Allozyne, Inc., 1600 Fairview Avenue East, Seattle, Washington 98102, United States MedImmune, LLC, One MedImmune Way, Gaithersburg, Maryland 20878, United States § Spirogen MedImmune, The QMB Innovation Centre, 42 New Road, London E1 2AX, United Kingdom ∥ UCL Cancer Institute, 72 Huntley Street, London WC1E 6BT, United Kingdom

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S Supporting Information *

ABSTRACT: Antibody−drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-L-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide−alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.



INTRODUCTION Antibody−drug conjugates (ADCs) show great promise as therapies for the treatment of cancer by piggybacking potent cytotoxins onto targeting antibodies.1 These can be very effective for cytoplasmic delivery of a toxin to tumor cells, while avoiding cytotoxicity by the free drug to normal tissues.2,3 The construction of an ADC requires the chemical ligation of a small molecule toxin to the targeting antibody while preserving the functional characteristics of both elements. The specific conjugation chemistry for attachment of the drug and linker to the antibody plays a very important role in the activity and tolerability of the ADC. This chemistry determines the extent of drug conjugation and the position of the drugs on the antibody. Perhaps most importantly, it determines the stability of the bond in circulation, and thus the extent of undesirable drug release. The conjugation chemistry used by many ADCs currently in development targets existing lysine side-chain © XXXX American Chemical Society

amines or cysteine sulfhydryl groups. These methods have been used successfully for the generation of two commercially available ADCs, Kadcyla (Immunogen/Roche) and Adcetris (Seattle Genetics), as well as many others currently in development.3−5 However, these methods result in the generation of heterogeneous ADCs, in terms of number (drug to antibody ratio (DAR)) and position of the payload.6−8 The intrinsic heterogeneity that results from using naturally occurring amino acids for conjugation affects the stability and thus the pharmacokinetic properties of the products.9−11 Special Issue: Antibody-Drug Conjugates Received: June 30, 2015 Revised: September 1, 2015

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DOI: 10.1021/acs.bioconjchem.5b00359 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Site-specific PEGylation of azide containing antibodies. (A) 1 was introduced at different positions on the LC (L70 and L81) or HC (274, 359) of 4D5. The expressed AzAbs were conjugated to 20 kDa PEG-BCN and the products of the reaction resolved by SDS-PAGE and stained with Coomassie-Blue. Addition of PEG results in a shift in mobility of the PEGylated HC or LC (PEG-HC and PEG-LC) relative to the untreated antibody (4D5 AzAb). Antibodies containing four conjugation sites (H274/L70 and H274/L81) show mobility of both HC and LC bands. (B) Three antibodies were expressed containing 1 at position H274 and the purified antibodies were subjected to a PEGylation reaction with 20KDa PEG-BCN. The azide containing (4D5 H274, Her H274 and a-IL6 H274) and the control antibodies (4D5 and a-IL6) lacking 1 were PEGylated and resolved by SDS-PAGE under reducing conditions and stained with Coomassie Blue. Complete conversion of the heavy chain bands (HC) to PEGylated forms (PEG-HC) is observed. Light chains (LC) and control antibodies remain unmodified demonstrating the strict bio-orthogonality of the conjugation chemistry.

protein conjugates.31,32 A variety of nnAAs have been successfully introduced that enable a range of bio-orthogonal conjugation chemistries and provide increased control over the site and number of conjugation sites. Incorporation of nnAAs in mammalian cells has been achieved by orthogonal aminoacyl tRNA synthetases and their cognate tRNA in engineered cells,33−36 as well as in cell-free expression systems.37 In one system, the introduction of para-acetylphenylalanine enables an oxime conjugation that has been shown to be stable under physiological conditions and generated homogeneous ADCs.33,35 This method is specific and generates stable conjugates, but the kinetics of the conjugation reaction are slow and requires extended incubation in acidic buffers (pH4).38 We have utilized the pyrrolysyl-tRNA synthetase (pylRS) and its cognate tRNA (tRNA pyl), derived from Methanosarcina mazei, to engineer Chinese Hamster Ovary (CHO) cells to genetically encode nnAAs in response to an amber stop codon.39,40 This enzyme has a number of advantages over tRNA synthetase/tRNA pairs engineered to be orthogonal in their expression hosts. The native pylRS/ tRNA pair is orthogonal in a variety of host cells, including mammalian cells, and the tRNA that is naturally directed to amber codons.41−52 The pylRS is also naturally promiscuous and able to recognize a variety lysine analogues, including azides, alkynes, and alkenes, without mutation.40,52,53 Here we have incorporated N6-((2-azidoethoxy)carbonyl)-Llysine40 (1) (Supporting Information Figure S1A), that enables azide−alkyne cycloaddition, or click chemistry (Supporting Information Figure S1B).54−59 This conjugation chemistry is specific, efficient, and rapid and results in the formation of a heterocyclic triazole that is irreversible in vivo.55,60,61 Antibody conjugates with the toxins auristatin F (AF) and a pyrrolobenzodiazepine (PBD) dimer show specific in vitro and in vivo potency to target cells. This system provides a means for the generation of ADCs with unparalleled stability and with precise control over both number and site of conjugation.

The stability of the conjugate is a key determinant in the performance of the drug. Stable ADCs have shown improved potency in vitro and lower toxicities in vivo than less stable conjugates.12 This is especially important in ADCs generated using very toxic compounds like auristatins, maytansinoids, and calicheamicins that are not well tolerated as free drugs. Most recently, Adcetris, an anti CD30 mAb conjugated to monomethyl auristatin E, and Kadcyla, Herceptin (trastuzumab) conjugated to maytansinoid DM1 toxin, showed enhanced survival of patients and were approved for clinical use. The success of these ADCs demonstrates the potential of this class of drugs for the treatment of cancer. However, the instability of the linkers used to in these products results in the early release of the toxin payload and lower intratumoral drug exposures.6,13−15 Increasing the stability of these ADCs will greatly improve their potency and tolerability to patients. Site-specific conjugation technologies have been developed that increase the stability of ADCs and limit premature release of toxin. Engineered unpaired cysteines allow for better control over the drug load and site of conjugation and improve their pharmacokinetic properties.16 However, few stable sites have been identified.15,17 Thus, there exists a need for the development of conjugation strategies which are not subject to the limitations of thiol-based conjugation. Several approaches have been examined to develop more stable conjugates. Recently, self-hydrolyzing maleimides were developed that catalyze the maleimide ring after conjugation.18,19 The hydrolyzed thiosuccinimide is not susceptible to elimination reactions and improved the stability of the conjugate in vivo. Alternate strategies make use of enzymes to modify canonical amino acids within a target sequence, or glyco-engineering to generate conjugation compatible sites.20−30 The process is specific and can be used to produce very stable ADCs, but requires modification of the IgG sequence. Technologies that enable the site-specific introduction of non-natural amino acids (nnAAs) into a target protein provide a significant advantage for the generation of functionalized B

DOI: 10.1021/acs.bioconjchem.5b00359 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 2. Azide−alkyne cycloaddition generates potent and specific ADCs. (A) 4D5 toxin conjugates were generated by SPAAC (with AF-BCN) or CuAAC (with AF-Alkyne or a PBD-Alkyne dimer) to produce 4D5-AF and 4D5-PBD conjugates. Untreated antibody (4D5) and the product of the conjugation reactions were reduced, resolved by SDS-PAGE and stained with Coomassie Blue. The conjugation of toxin to 4D5 results in a mobility shift relative to the untreated antibody. Both CuAAC and SPAAC reactions are efficient and specific to the HC. (B) 4D5-AF generated by CuAAC or SPAAC resulted in equivalent potency against a tumor cell line expressing high levels of the receptor Her2/neu (BT474). A gain in potency over the unconjugated drug (AF) was observed. Both ADCs showed no cytotoxic activity in PC3 cells, which show low levels of Her2/neu receptor expression, while the unconjugated toxin retained cytotoxicity. (C) The functions of 4D5 conjugates generated by CuAAC to AF or PBD dimer were compared to an ADC generated using thiol-maleimide conjugation chemistry (Her2-malPBD) in vitro. All conjugates were cytotoxic to BT474 cells, but not PC3 cells. 4D5-PBD retained cytotoxicity, but showed decreased potency relative to 4D5-AF and Her2-malPBD. (D) 4D5-PBD conjugate, a control antibody conjugate (a-IL6-PBD), or 4D5-AF (CuAAC) was administered to mice bearing BT474 tumors. For each group 10 mice were treated once per week, for 3 weeks, starting on day 1 with 1 mg/kg of conjugate. Tumor volume was monitored over 90 days. For each treatment the mean tumor volume and standard deviation are shown. 4D5-PBD and Her2-malPBD conjugates led to sustained regressions. Lack of treatment (vehicle) or treatment with a-hIL-6-PBD or 4D5-AF at the same dosage resulted in continuous growth of the tumors.



RESULTS Incorporation of 1 into an anti-Her2/neu Antibody (4D5) Enables Site-Specific Modification of Antibodies. The site of conjugation is an important consideration in the construction of ADCs in order to generate conjugates capable of efficient antigen binding without impacting functional domains that control pharmacokinetics and stability. Using a crystal structure for IgG1,62 we selected four conjugation sites that were distal to the antigen binding sites and avoided the hinge region, and also residues known to be critical for Fc receptor binding and complement activation. In addition, we selected sites predicted to be solvent exposed and outwardly oriented to enable efficient conjugation. Two IgG heavy chain (HC) positions H274, H359 (Kabat numbering), and two light chain (LC) positions, L70 and L81, satisfied the criteria stated above (Supporting Information, Figure S2). Both HC positions are found in the constant domains, CH2 and CH3, respectively, and generally applicable to all IgG1 Abs. The selected LC sites occupy positions between the CDR2 and CDR3 in the variable domains of the LC. These positions were of interest because

they occupy sites on exposed loops and may be transferrable to scFv formats. An antibody directed against Her2/neu (4D5) was utilized in this study as it provides a well characterized target in oncology, and for which in vitro assays are readily available to assess ADC function. Thus, 1 was incorporated at each site using an orthogonal pylRS/tRNA pair derived from M. mazei by transient transfections in HEK29334 or in stably transfected CHO cells. Expression titers of antibody containing 1 (AzAb) of 30−80 mg/L in transient transfections, and 1.7 g/ L in stable pools, have been reached. These yields compare favorably with those previously reported using the Eucode system for the incorporation of para-acetylphenylalanine.33,36 To assess the suitability of these sites for conjugation, the expressed AzAbs were purified by affinity chromatography, and subjected to a conjugation reaction with a 20 kDa polyethylene glycol (PEG) containing a strained alkyne (PEG-BCN; Supporting Information Figure S1C). The reaction used a 10fold molar excess of PEG-BCN to ensure the complete consumption of the available azides. A comparison of the untreated and PEGylated antibodies by reducing SDS-PAGE shows a specific molecular weight shift of the desired proteins C

DOI: 10.1021/acs.bioconjchem.5b00359 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. In Vitro Cytotoxicity of SPAAC and CuAAC ADC Conjugates EC50 (nM)a sample

PC3

SKOV3

SKBR3

HCC1954

BT474

4D5-AF (CuAAC) 4D5-AFc (SPAAC) AF

ndb ndb 131.9 ± 34.325

0.048 ± 0.183 0.028 ± 0.002 20.1 ± 3.34

0.013 ± 0.004 0.011 ± 0.006 24.5 ± 8.7

0.02 ± 0.003 0.016 ± 0.007 11.064 ± 1.836

0.101 ± 0.032 0.082 ± 0.02 50.230 ± 8.58

a

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Cells were exposed to indicated toxins and viability assessed 72 h post treatment using Alamar Blue dye conversion as a measure of viability. The average half maximal effective concentration (EC50) and standard deviation for each sample was calculated from two independent assays. bEC50 could not be determined due to lack of efficacy. cAF-BCN used as a payload.

cycloaddition (SPAAC) using AF-BCN, and the products were analyzed by SDS-PAGE (Figure 2A). In each case, the 4D5-AF conjugates showed an increase in molecular weight over the untreated antibody (4D5 AzAb) due to the addition of the toxin, with no detected unreacted material. SPAAC is notable for its use of strained cyclooctyne functional groups and offers a copper free method of generating stable conjugates.59 The reaction has been also been shown to be effective in the generation of active ADCs.37 Several different reactive cyclooctynes are commercially available and vary in structure and hydrophobicity. To assess the utility of an alternate cyclooctyne moiety, MMAF was functionalized with DBCO and used in a coupling reaction with the 4D5 H274 AzAb. The specificity and efficiency of adduct formation was confirmed by mass spectrometric analyses (Supporting Information Figure S4A). To do this the intact masses of both the HC and LC for untreated AzAb and 4D5MMAF were examined. The HC of the 4D5-MMAF show a 1266.84 mass increase over the unconjugated HC that corresponds to the addition of a single MMAF-DBCO molecule with an efficiency of 97.8% and the generation of a DAR 1.96 ADC. No mass change was observed in the light chain. These data confirm that adduct formation occurs at a single site, with high efficiency and specificity. Next, we examined the efficacy of conjugation and function of ADCs generated by CuAAC. 4D5 H274 AzAb was subjected to a conjugation reaction with AF armed with a linear alkyne (Supporting Information Figure S1). SDS-PAGE analyses of CuAAC reactions showed efficient conjugate formation resulting in complete consumption of the HC to a higher molecular weight species (Figure 2A). The efficiency of conjugation was confirmed by analytical HPLC using hydrophobic interaction chromatography (HIC) (Supporting Information Figure S4C). This method distinguishes unconjugated AzAb (DAR0) from AF conjugates based on their hydrophobicity. A discrete increase in retention time of 4D5AF over that of the unmodified antibody was observed. To assess the difference in hydrophobicity profiles between CuAAC and SPAAC ADCs, HIC analyses were performed on ADCs generated with AF-Alkyne or AF-BCN. Then AFBCN payload was selected because it represents the least hydrophobic SPAAC compatible moiety. In each case the ADCs resulted in a monomeric peak confirming the efficiency of conjugation and homogeneity of the products. The AF-BCN derived conjugate was more hydrophobic than the copper catalyzed conjugate; this is likely due to the presence of the large hydrophobic cyclooctyne ring. The function of the ADC requires efficient binding of the conjugate to target cells and delivery of the toxic cargo to the cytoplasm. An ELISA assay was developed that measures binding to the Her2 extracellular domain and was used to determine whether 4D5-AF retained the ability to bind its

(PEG-HC or PEG-LC) relative to the untreated antibody (Figure 1A). The near-complete conversion of the targeted proteins indicates that the selected sites are accessible to the conjugation chemistry and enable very efficient adduct formation. Furthermore, LC bands in H274 and H359, or HC bands in L81 and L70, were not affected by the conjugation reaction, illustrating the specificity of the azide−alkyne cycloaddition chemistry. It should be noted that the additional bands in the PEGylation reactions for L70 and L81 are copurifying contaminating proteins that were present in the untreated samples and do not represent off-site PEGylations of the Ab or aggregate formation as a result of the reaction (see Supporting Information Figure S3). Antibodies with four attachment sites were generated by pairing expression cassettes with amber codons in both the heavy and light chains in transient transfection. The observed yields from these transient transfections were similar to those obtained by antibodies targeting a single site (24 mg/L). Efficient PEGylation was observed as shown by the mobility shift for both HC and LC in these samples (Figure 1A; H274/L70 and H274/L81) demonstrating that this technology is amenable to the expression of antibodies with multiple conjugation sites. Position H274 was selected for further characterization. To demonstrate that the incorporation of 1 at H274 is generally applicable we expressed three antibodies; two directed against Her2/neu (4D5 and trastuzumab (Her)), as well as an antibody directed against human IL-6 (a-IL663). An amber codon was encoded at position H274 in each antibody in order to produce the corresponding AzAb by transient transfection. The expressed and purified antibodies were subjected to a PEGylation reaction using 20KDa PEG-BCN. SDS-PAGE analyses of these reactions under reducing conditions showed complete and specific conversion of the HC to a PEGylated form (Figure 1B; PEG-HC). No change in LC mobility was observed. To further examine specificity of the conjugation reaction, antibodies lacking 1 (Figure 1B; 4D5 and a-IL6) were subjected to the same conjugation reaction. The products of these reactions showed no change in mobility illustrating the bio-orthogonality of the conjugation chemistry. SPAAC and CuAAC Conjugations Generate Fully Functional Conjugates. Antibody conjugates with AF were constructed using click chemistry to assess the use of this technology for the construction of ADCs. AF was selected due to its potent cytotoxic function, the ability to incorporate a terminal cycloalkyne in its structure, and its demonstrated cytotoxicity as part of several ADCs.37,64−66 Two AF derivatives including either a terminal alkyne (AF-Alkyne) or cycloalkyne (AF-BCN) (Supporting Information Figure S1C) were prepared in which a short noncleavable PEG linkage was placed at the carboxyl terminus of the peptide based cytotoxin. 4D5-AF conjugates were generated by copper catalyzed cycloaddition (CuAAC) or by strain promoter azide−alkyne D

DOI: 10.1021/acs.bioconjchem.5b00359 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 2. In Vitro Cell Cytotoxicity of PBD ADC Conjugates EC50 (nM)a sample

PC3

4D5-PBDc Her2-malPBDb a-IL6-PBDc 4D5-AFc AF PBD

ndd ndd ndd ndd 104 ± 10.5 4.5 ± 1.5

SKBR3 0.061 0.022 >100 0.008 30.3 2.1

SKOV3

± 0.02 ± 0.007

0.504 0.17 >100 0.025 75.0 33.17

± 0.003 ± 20.4 ± 1.2

± 0.281 ± 0.086 ± 0.023 ± 8.5 ± 4.3

HCC1954 0.665 0.292 >100 0.016 20.5 12.4

BT474

± 0.255 ± 0.077 ± 0.002 ± 3.25 ± 1.9

0.464 0.189 >100 0.048 45.4 57.2

± 0.248 ± 0.139 ± 0.052 ± 5.34 ± 9.2

Cytotoxicity assays were conducted as described in Table 1. bThiol-maleimide conjugate with a DAR = 3. cCuAAC conjugate. dEC50 could not be calculated due to lack of efficacy.

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a

Figure 3. Azide−alkyne cycloaddition forms a stable conjugate. (A) 4D5-FITC conjugate (SPAAC) was incubated in human serum for up to 4 weeks at 37°C. Samples were collected at the start of the incubation (T0), and after one, two, and four weeks. An ELISA was developed to detect the intact conjugate and used to quantify the conjugate levels at each time point. 4D5-FITC levels were determined in a titration of each sample and the concentrations plotted. No loss of conjugate was observed over the span of the experiment. (B) Male CD rats were injected intravenously with 1 mg/kg 4D5-FITC and levels of the intact 4D5-FITC as well as total human IgG were quantified by ELISA at various time points over 280 h. The circulating concentrations of the 4D5-FITC and human IgG overlap indicating no deconjugation of the FITC cargo. (C) The pharmacokinetics of 4D5-FITC was compared with those of unconjugated antibody (trastuzumab) in the same rat sera as described above in (B). The circulating levels of 4D5-FITC largely match those of trastuzumab. (D) The potency of 4D5-AF, prepared 6 months prior to the experiment and stored at 4 °C, was compared to that of freshly prepared conjugates in an in vitro cell cytotoxicity assay against the Her2/neu expressing cell line BT474. The potency between these samples is comparable and indicates that these conjugates can be stored at 4 °C for long periods without loss of function.

that Cu mediated conjugation does not measurably reduce the activity of the ADC. Her2-PBD Conjugates Are Efficacious in Vivo. The utility of this conjugation method, and its general applicability to different payloads, was assessed through the generation of conjugates with a PBD dimer. 4D5-PBD (Figure 2A), a-IL6PBD, a control antibody directed against human cytokine IL6 (a-IL6-PBD), and 4D5-AF conjugates were generated using CuAAC at position H274. RP-HPLC analyses showed a drug load of 1.8 DAR for both PBD conjugates and a DAR of 1.9 for the AF constructs (not shown) with no increase in high molecular weight species over the unconjugated Ab (Supporting Information Figure S6). The conjugates were first assessed in vitro in tumor cell cytotoxicity assays using a panel of Her2 positive cell lines. The data show that the 4D5-PBD conjugates retained subnanomolar potency with all the cell lines tested, but appeared to be less effective than the AF conjugates, illustrating the high potency of AF in these in vitro cytotoxicity assays (Figure 2C; Table 2). In contrast, a-IL-6-PBD was not effective in these assays. Interestingly, the CuAAC conjugated 4D5-PBD was less potent on some cell lines than trastuzumab-PBD (Her2-malPBD) generated using conventional maleimide chemistry.68 The enhanced potency of the latter construct may be due in part to the higher drug load of this conjugate (DAR3). The efficacy of these conjugates was also examined in vivo against human Her2-positive BT474 tumor xenographs. 4D5AF, 4D5-PBD, a-IL6-PBD, and Her2-malPBD conjugates were administered intravenously into CB.17 severe combined immunodeficient (SCID) mice implanted with BT474 tumors. The data show that 4D5-PBD and conventional Her-malPDB

target epitope. The unmodified 4D5 AzAb and 4D5-AF showed similar levels of binding to Her2 indicating that the conjugate retained the full binding activity of the antibody (Supporting Information Figure S5A). Furthermore, SPAAC and CuAAC conjugates showed similar binding efficiencies suggesting that the linkers and payloads, at this position, do not interfere with the target binding of the antibody. The tumor cell killing function of the ADC was initially tested with an in vitro cytotoxicity assay using tumor cell lines known to express Her2/neu. Cells expressing either high levels (SKOV3, SKBR3, HCC1954, and BT474) or low levels (PC3) of Her2/neu were incubated with 4D5-AF or AF alone and viability was assessed after 3 days. AF is a potent toxin that is cytotoxic against all cell lines tested (Table 1). The 4D5-AF conjugate was 1000-fold more potent than the unconjugated drug in cells expressing high Her2/neu levels. The potency observed here is consistent with previous observations of ADCAF conjugates.37 The greater potency of the ADC is attributed to receptor mediated internalization of the conjugate,67 while the unconjugated toxin penetrates cells through passive diffusion. The cytotoxicity of the ADCs was not due solely to antibody binding, as trastuzumab was poorly cytotoxic (not shown). 4D5-AF toxicity appears specific for cells expressing higher levels of Her2, demonstrated by the lack of cytotoxicity against PC3 tumor cell lines (Figure 2B and Table 1). In addition, SPAAC and CuAAC ADCs were equally potent against each of the Her2/neu expressing tumor cell lines tested (Table 1; Figure 2B). These data show that the increased hydrophobicity of the SPAAC conjugate does not affect the function of this ADC in vitro. Moreover, the data also show E

DOI: 10.1021/acs.bioconjchem.5b00359 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 3. Activity of Positional 4D5-AF Variants EC50 (nM)a

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a

sample

A549

274-AFb 70-AFb 81-AFb 359-AFb 274/70-AFb 274/81-AFb AF trastuzumab

>100 >100 >100 >100 >100 >100 104.97 ± 7.42 >100

SKBR3 0.007 0.005 0.019 0.012 0.013 0.008 40.01 >100

± ± ± ± ± ± ±

SKOV3

0.002 0.001 0.011 0.004 0.009 0.003 23.26

0.110 0.051 0.121 0.083 0.065 0.031 43.39 >100

± ± ± ± ± ± ±

0.077 0.009 0.079 0.077 0.042 0.021 21.47

HCC1954 0.048 0.020 0.054 0.047 0.018 0.015 14.02 >100

± ± ± ± ± ± ±

BT474

0.043 0.002 0.04 0.023 0.012 0.007 4.79

0.165 0.314 0.693 ntc 0.052 ntc 45.42 >100

± 0.125 ± 0.015 ± 0.077 ± 0.01 ± 3.77

Cytotoxicity assays were conducted as described in Table 1. bAF-BCN used as a payload. cNot tested.

at both HC and LC locations (H274/L70 and H274/L81). The specificity of conjugation and the distribution of the drug in each sample were analyzed by reverse phase HPLC PLRP under reducing conditions. This method allows for the separation of the antibody HC and LC and the determination of drug load at each chain relative to the unmodified controls (AzAb control). These data show that the conjugations occurred within the targeted proteins (Figure S4B). HIC analyses of these products showed near complete conjugations to each site (generating ADCs with DAR > 1.9 and DAR > 3.8 for antibodies containing four conjugation sites) (Supporting Information Figure S7A). The relative hydrophobicity of these ADCs varied by position (unconjugated < H274 < L81 < H274-L81 < H359) and is likely related to the accessibility of the hydrophobic cargo to the HIC resin. As expected, the addition of four AF conjugates (H274/L81) showed an additive increase in hydrophobicity over their respective H274 and L81 counterparts, each containing two AF molecules. Interestingly, H359 led to an ADC with increased hydrophobicity over the DAR4 variants (Supporting Information Figure S8). The elevated hydrophobicity of H359 ADCs may adversely affect the function of this ADC. Indeed, we have observed that AF conjugates generated at position H359 retain potency in in vitro cytotoxicity assays (Supporting Information Figure S7C; Table 3) but are prone to aggregation upon longterm storage. The effect of position and drug load on ADC function was assessed using in vitro cell cytotoxicity assays. Here trastuzumab, DAR2 ADCs (H274, H359, L70, L81), or DAR4 ADCs (H274/L70 and H274/LC81) generated using AF-BCN (SPAAC) were incubated with tumor cell lines expressing Her2/neu and viability assessed. The half maximal effective concentration (EC50) was calculated for each treatment and each cell line tested (Table 3). We observed that all ADCs retained high potency against the Her2 expressing cell lines (Supporting Information Figure S7C). H274/L70 showed a slight increase in potency over other cytotoxin conjugates that is most apparent in the BT474 cells, which provide the least sensitive and most discriminatory model. This gain in potency is likely due to the higher doses of toxin delivered by this conjugate pair. The 274/81 conjugate retains potency but is not improved over the H274-AF conjugate alone, indicating that drug load alone is not sufficient to increase ADC activity. Interestingly, a slight decrease in potency was observed for L81AF conjugates with respect to H274-AF in BT474. These results suggest that the placement of the AF toxin at this site may be attenuating antibody function. Overall, the data shows that the position of conjugation is an important consideration in the construction of an ADC that impacts the overall

conjugates were similarly effective in producing sustained regression of tumors (Figure 2D). In contrast, mice treated with the control ADC (a-IL6-PBD) or vehicle alone showed continual growth of the tumors. 4D5-AF did not show curative effects at this dose level despite the high potency observed in vitro. This is consistent with previous in vivo studies comparing PBD dimer and tubulin inhibitor containing ADCs.68,69 In Vitro and in Vivo Stability of the Triazole Based ADC. One of the key advantages of site-specific conjugation technologies, and more specifically the use of click chemistry as a conjugation method, is the high stability of the linkage that is formed. The heterocyclic triazole generated by azide−alkyne cycloaddition has been previously shown to be extremely stable both in vitro and in vivo with PEGylated proteins60 and more recently with ADCs.37,70 To confirm these observations and assess the stability of conjugates at H274 a 4D5-FITC conjugate, was assembled using DBCO-FITC (SPAAC). The availability of antibodies directed against the FITC moiety allowed for the development of a quantitative ELISA capable of detecting loss of payload (Supporting Information Figure S5B). No decrease in 4D5-FITC conjugate levels were observed in human serum at 37 °C over 4 weeks (Figure 3A), nor over 11 days in rats (Figure 3B). To examine the effect of the payload (FITC) on the pharmacokinetics of this conjugate the circulating levels of trastuzumab were compared to those of the 4D5-FITC conjugate in vivo. The 4D5-FITC conjugate has a PK profile that is very similar to that of trastuzumab (Figure 3C). The observed divergence between the two antibodies is likely the result of a reduced bioavailability of the FITC conjugate, potentially due to the increased hydrophobicity of the 4D5-FITC linker-conjugate. Finally, we assessed the shelf life of the conjugates; the in vitro potency of a freshly prepared 4D5-AF was compared to a sample stored at 4 °C for 6 months. Comparable potency between the two samples indicates high in vitro stability without loss of the toxin payload (Figure 3D). Taken together, these data show that click-cycloaddition chemistry generated stable conjugates that exhibit an in vivo half-life that is similar to that of a normal antibody, and points to the utility of position H274 as a viable conjugation site. Alternate Positions for ADC Construction. The sitespecific nature of this expression technology enables the precise placement of conjugation sites at desired positions on a target protein. We have shown that the H274 position is accessible for conjugation and can produce active conjugates. Furthermore, we have shown that positions H359, L70, and L81 are also accessible to conjugation. To test the effect of position on the activity of an ADC, AzAbs at each of these sites were generated and conjugated to AF-BCN. In addition, AzAbs containing four toxins were produced by expressing AzAbs with amber codons F

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therapeutic index. Kadcyla/T-DM1, the maytansinoid conjugate of trastuzumab, was required to reduce the dose from 3.6 mg/kg to 2.4 mg/kg during the Phase 3 efficacy trial due to toxicity, compared to the 8.0 mg/kg dose approved for trastuzumab.71 The result is a product that cannot reach its maximum effective dose, which turns out to be greater than the maximum tolerated dose. This difference in dose essentially captures the unmet need that could be satisfied with a sitespecific and highly stable version of this ADC. In spite of these limitations, the existing ADCs are effective. The improvement of the technology with site-specific conjugation and stable linker chemistry will expand the therapeutic index and potentially increase the efficacy of ADCs beyond what is achieved using conventional conjugation methods. The data reported here describe the bioanalytical evidence that 4D5-AF and 4D5-PBD are very stable and very toxic specifically to antigen bearing tumor cells. The site of conjugation is a key element in the construction of an ADC and must be carefully selected to enable efficient conjugation. This is especially the case for conjugates generated using thiol-maleimide chemistry. Site-specific approaches using engineered cysteines have provided better stability to these conjugates, but few sites have been identified that are not susceptible to uncoupling.15,16 Emerging technologies using enzyme dependent modifications show promise but also variability in conjugation efficacy at different sites.29 We have identified four effective sites, and two others have been reported that allow efficient conjugation to a payload and point to click cycloaddition as having wider access to viable sites than other site-specific conjugation methods.37,70 The efficiency of conjugation is not the only parameter that needs to be considered. The conjugate can have meaningful effects on the stability of the protein. For example, position H359 is a viable site that allows for efficient conjugate formation (DAR > 1.9); however, conjugation of AF at this site resulted in a more hydrophobic ADC with a propensity for aggregation. It is likely that at this position, the cytotoxin is unfavorably exposed and capable of forming undesired interactions. The site of conjugation is also important to prevent premature release of toxins attached through cleavable linkers.29 Thus, conjugate position must be optimized for each payload and linker to ensure the stability and optimal function of the ADC. This sitespecific technology amenable to click conjugation grants access to a variety of sites that are not stable as maleimide linkages, and allows for positional flexibility that will enable the optimization of ADCs for function and stability, all aimed at increasing the therapeutic indices of these drugs. One of the greatest challenges for genetically encoding nnAA has been achieving high productivity. Cell lines containing an amber suppressing orthogonal RS/tRNA pair have been shown to achieve 1 g/L in stable expressions here and in previous studies.33,36 Early stable cell lines developed for this study have demonstrated yields of over 1.7 g/L in stable pools, and yields are expected to significantly improve with additional development.51 These titers are still below the 3−10 g/L yields that can be achieved with conventional antibodies. However, the productivity of these cell lines is currently commercially viable especially when one considers that the doses of ADCs will likely be much lower and less frequent than those necessary for therapeutic antibodies due to the added functionality of the cytotoxic payload. In addition, the homogeneity of the products will improve the processing and manufacturing yields over those of conventional methods. Most importantly, this system

hydrophobicity and in vitro potency of the conjugate. The combined characteristics of these variants will likely have important effects in vivo, but these remain to be tested.

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DISCUSSION We have developed a mammalian cell based expression method for the generation of antibodies capable of site-specific bioconjugation and the production of ADCs. The molecules presented here are distinguished from those currently in development, by virtue of its homogeneous site-specific conjugation and the chemistry used to conjugate the toxins. The site-specificity of this approach provides molecules on which a toxin is uniformly positioned at a precisely defined site (or sites) that does not impair the natural Ig functions of the ADC, including in particular Fc dependent half-life, and antigen binding for efficient internalization. Site specific conjugation also enables manufacture of a uniform, reproducible product, at high yield. This homogeneous product is easier to purify and characterize than ADCs that have multiple possible conjugation sites distributed on the antibody. The heterogeneity of ADCs produced by alternative methods may suffer from difficulties in purification and analytical characterization and loss in purification yields. Moreover, the final product will contain ADC related contaminants that have lost function and stability. The most common bioconjugation method targets cysteines that are normally engaged in protein stabilizing disulfide bonds and requires their reduction, conjugation, and reoxidation. This method has been successfully applied and the first product (Adcetris) has been approved. However, there is little doubt that site-specific conjugation will greatly enhance the manufacturability, stability, and potency of future ADCs. The chemistry for conjugation described here is enabled by the introduction of an azide containing amino acid. The azide moiety permits the “click” cycloaddition conjugation when paired with an alkyne group to form a triazole linkage. The reaction can be conducted with a terminal alkyne in a copper catalyzed reaction (CuAAC),54,55 or with a strained alkyne moiety (SPAAC) in the absence of copper.59 Both ligations occurred rapidly, in aqueous buffers at room temperature, and with high efficiency, to produce conjugates with comparable potency and functional properties. The development of CuAAC for bioconjugation is an important advance that enables the generation of payloads that are not able to arm with the more reactive, and more hydrophobic, cycloalkyne moieties. Thus, CuAAC expands our reach to a wider range of payloads that result in conjugates with improved hydrophobicity profiles than those generated by SPAAC. The stability of the triazole linker has been verified both in preclinical studies, including the one shown here, and in clinical studies using the same linker to attach a PEG to IFN beta.37,60,70 In the case of ADCs generated using this chemistry, the stability of the linkage ensures that the drug remains attached to the antibody until the ADC is internalized and the toxin is released into the cytoplasm of the cell. This is in contrast to other products in development including Adcetris. The published data indicate that Adcetris loses its toxic payload in circulation at a high rate with as much as 50% lost over 6 days in vivo.6 The premature loss of a payload decreases the efficacy of ADCs by exposing normal tissues to the free toxin not only resulting in off-site and nonspecific toxicity, but also resulting in the presence of drug-free antibody that competes with the ADC for binding sites on the tumor cells. These negative attributes result in reduced potency and a narrow G

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human IgG1 frameworks encoded in pFUSE-CHIg-hG1 (Invivogen) and pFUSE-CHLIg-hK (Invivogen) to generate a mouse−human hybrid. Amber codons were introduced into the heavy chain (Gamma) or light chains (Kappa) at selected positions by site directed mutagenesis. Surface exposed positions on an IgG1 molecule were selected as conjugation sites by analyses of the IgG structure.62 Four sites were initially selected for modification: heavy chain positions K274, T359, and light chain positions D70 and E81 (numbering is based on the Kabat numbering system for IgG residues; see also Supporting Information Figure S2). pGFPY40, pTracer (Invivogen), mutated to contain an amber codon corresponding to amino acid 40 of the GFP reading frame. Stable Expression of Ab Containing N6-((2azidoethoxy)carbonyl)-L-Lysine. A cell line stably expressing pylRS and tRNA-pyl was generated by transfection with pMOAV2 into CHO DG44 and subsequently selected with the antibiotic hygromycin (0.5 mg/mL). Cells showing pylRS and tRNA-pyl activity were isolated by fluorescence activated cell sorting after transient transfection with a reporter construct pGFPY40. Cells demonstrating high GFP levels upon exposure of 1 were collected and expanded. Selected cells were subsequently transfected with additional copies of the tRNA expression vector pSZ-9x-tRNA and pSB-9xtRNA and the functional selection repeated. The resulting pool (CHO−DG44 223) was used for the generation of stable cells for the expression of target genes. Stable cell lines for the expression of 4D5 H274 were constructed in CHO−DG44 223 cells. Genes encoding the HC and LC of 4D5 H274 were cloned into pOptivec (Life Technologies). The construct was transfected into CHO−DG44 223 and cells selected in CD CHO medium (life Technologies) containing 1 μM MTX. Clones showing expression of the IgG were identified by ELISA and adapted for serum free suspension growth in Excell CHO DHFR (Sigma) supplemented with 6 mM glutamine. For expression of azide containing antibody (AzAb), cells were grown to a cell density of (1−2) × 106 cells/mL, and 2 mM 1 added to the medium and incubated with shaking for 7−14 days. Expressed AzAb was purified by affinity chromatography using IgSelect resin (GE Healthcare) at 1−5 mL/min flow rate using an AKTA chromatography system. Peak protein fractions were dialyzed to PBS at 4 °C for 16 h, and concentrated to 5−20 mg/mL using Amicon centrifugation concentrators (EMD Millipore, Darmstadt, Germany). Site-Specific Conjugation to Expressed AzAb. Purified AzAbs were subjected to cycloaddition reactions using SPAAC or CuAAC. The preparation of AF and PEG conjugation precursors modified to contain a terminal alkyne moiety or a BCN group (Figure S1C) is available in the Supporting Information. A PBD N10-linked payload as previously described with a Val-Ala cleavable site was modified to contain a terminal alkyne.75 For SPAAC conjugations, 10-fold molar excess of the payload was used. Briefly, a solution of the AzAb at a concentration of 2 mg/mL (25uL) was mixed with a solution of conjugate fitted with a SPAAC moiety (PEG-BCN, DBCO-FITC, AF-BCN, or AF-DBCO) (60 mg/mL, 33 μL) and 50 mM sodium phosphate buffer (pH = 7.4, 12 μL) and incubated for 4−16 h at room temperature. Conjugates were purified from unconjugated payload by HIC chromatography as described below. For the assembly of 4D5-AF or the 4D5-PBD conjugates using CuAAC, phosphate buffer (13.6 μL, 150 mM Pi, pH = 7.4), a solution of the azide containing antibody (14.3 μL, 20

can produce ADCs with superior characteristics; more efficacious antitumor drugs requiring lower and less frequent dosing. We have shown that the system enables the generation of conjugates with two classes of toxin payloads, AF and PBD. PBD dimers have been shown to cross-link DNA and exert potent antitumor activity.72,73 Their method of action sets them apart from the microtubule binders (AF and maytansinoids) commonly used for ADCs. These highly potent agents have been shown to be effective in the treatment of hematological tumors and are ideal payloads to eliminate tumor stem cells.74,75 The increased stability and positional flexibility of this conjugation method may enable the identification of a new generation of PBD-ADCs with improved therapeutic indices to address a wide variety of tumor types.

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EXPERIMENTAL PROCEDURES Materials. AF and MMAF-DBCO were purchased from Concortis Biosystems, Inc. (Sand Diego, CA). DBCO-FITC was purchased from Click Chemistry Tools (Scottsdale, AZ). THPTA was generously provided by Professor M.G. Finn (Georgia Institute of Technology). Cell Culture Conditions. All mammalian cell cultures were maintained in humidified incubators at 37 °C and with 7.5% CO2 content and grown in complete Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies), containing 2 mM Glutamax, 1 mM sodium pyruvate, 1× nonessential amino acids, 10% fetal bovine serum, and HT supplement unless otherwise noted. Transfections were conducted using standard lab procedures and methodologies. The transfected cells were incubated for 1−3 h and 1 was added to the growth medium to a final concentration of 0.5−2 mM. After 5−7 days incubation, medium was collected for purification. Stable CHO cell lines were maintained in serum free Ex-Cell CHO DHFR-medium (SAFC Biosciences) in the presence of 20 μg/mL blasticidin, 0.2 mg/mL Zeocin, 0.5 mg/mL puromycin, and 0.25 μg/mL hygromycin B. Molecular Biology and Construction of Expression Vectors. General molecular biological techniques were conducted as previously described.76 The following plasmids were used in this work: pCEP4-pylRS, the gene encoding for pylRS (reference sequence WP_011033391) was generated by gene synthesis77 and subcloned into pCEP4 (Life Technologies) under control of a CMV promoter. The gene was codon optimized for expression in CHO cells and mutated to contain a Y384F mutation, shown to increase aminoacylation rate of the synthetase.48 pOriP-9x-tRNA, nine tandem copies of a tRNA expression cassette consisting of the U6 snRNP promoter, and the wild type tRNA-pyl sequence from M. mazei were introduced into a pOriP containing vector.34 pSZ-9xtRNA and pSB-9x tRNA were constructed by restriction enzyme digestion of pOriP-9x-tRNA and insertion into sites in pSelectBlast and pSelect Zeo vectors (Invivogen). For construction of a cell line expressing pylRS and tRNA-pyl an integrating construct was generated in pJTI-FAST-DEST using Gateway technology (Life Technologies). To do this, the CMV-pylRS expression cassette and 9xtRNA cassettes were combined into pJTI-FAST DEST using LR clonase to generate a vector containing pCMV-pylRS and 18 copies of the tRNA expression cassettes (pMOAV2). An antibody directed to the extracellular domain of Her2/neu was generated based on the mouse antibody 4D5.78 The variable regions of 4D5 were generated by gene synthesis using overlapping oligomers and cloned into the H

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Bioconjugate Chemistry mg/mL) and the cytotoxic alkyne (3.8 μL, 5 mM) were combined. In a separate tube a solution containing copper sulfate (35.5 μL, 15 mM), THPTA ligand (6.7 μL, 160 mM), amino guanidine (26.6 μL, 100 mM), and sodium ascorbate (20 μL, 400 mM) was made. The THPTA-CuSO4 complex was capped, vortexed, and allowed to stand for 10 min. A portion of this complex (6.3 μL) was added to the antibody−alkyne mixture. The final mixture was capped, vortexed, and allowed to incubate for 1−2 h at room temperature. Conjugation of MMAF-DBCO to an azide-containing antibody by SPAAC. To a solution of AzAb (532 μL, 9.4 mg/mL) in 1× PBS pH 7.2 (468 μL) containing 8% v/v DMSO (80 μL) was added 6 mol equiv (1:3 azide:DBCO) of MMAF-DBCO (20 μL, 10 mM in DMSO). The reaction was allowed to rotate gently for 16 h at room temperature. The crude reaction mixture was purified by buffer exchange using a PD-MidiTrap column into 1× PBS, pH 7.2. Prior to in vitro cytotoxicity assays, the excess, unconjugated toxins were removed using HIC chromatography. The conjugation reaction mix was diluted greater than 5-fold in water and incubated with HIC resin in batch for 1 h. The bound material was washed in 10 mM phosphate [pH 7.4], and bound Ab-conjugates were eluted with 500 mM phosphate [pH 7.4] containing 0.1% Tween and the samples dialyzed into PBS. Analytical Methods. All conjugation products were analyzed by SDS-PAGE and Coomassie staining and quantified by densitometry analysis (GE-Molecular Dynamics Personal Densitometer SI, ImageQuant TL software) to determine the conversion rate of the conjugated protein. Analytical HIC, SEC, and PLRP HPLC methods used in this study are available as Supporting Information. Stability and PK Assays for of 4D5-Conjugate. A 4D5FITC conjugate was incubated in human serum (at a 1:20 dilution) at 37 °C for over 4 days. Stability was measured using a Her2 Extracellular Domain (ECD) sandwich ELISA. The Her2/neu extracellular domain (ECD) was bound to 96 well plates and samples applied at room temperature for 1 h. Antibodies captured by the ECD were detected using an HRP conjugated antibody directed against the FITC fluorophore. Only intact antibody conjugate is detected using this assay since it requires antigen binding and the presence of the dye for detection to occur. The in vivo PK of the conjugate was measured in male CD rats (3/group) injected with 4D5-FITC or trastuzumab at 1 mg/kg IV. Serum samples were collected at various times after injection (0.5−264 h) and assayed for human IgG by ELISA to determine antibody levels. The concentration of the conjugate was determined in the same serum samples using the Her2 Extracellular Domain (ECD) sandwich ELISA. In Vitro Cytotoxicity Assays. Tumor cell cytotoxicity assays were conducted with tumor cell lines including PC3 (CRL-1435), SKBR3 (HTB-30), SKOV3 (HTB-77), BT747 (HTB-20), and HCC1954 (CRL-2338), all obtained from ATCC. Tumor cell lines were plated in 96 well plates at 1000 cells/well in RPMI medium (Life Technologies, Carlsbad CA) supplemented with 10% heat inactivated FCS, pyruvate, nonessential amino acids, and gentamicin. For each assay, growth inhibition was assessed in duplicate samples by incubating conjugates with the tumor cell lines for 72 h followed by treatment with Alamar blue (Life Technologies) for 1−4 h. The fluorescence emission at 545 nm (excitation 590 nm) of reduced resazurin was quantified using a Molecular Diagnostics plate reader. Each assay was conducted in

duplicate, the data log transformed, and average values and standard deviations plotted on a semilog graph. The halfmaximal effective concentration (EC50) for each treatment was calculated using a nonlinear least-squares fit to a four parameter logistic equation using Prism v 6 software (GraphPad Software Inc., San Diego CA, USA). The average and standard deviation of EC50s in two or three experimental replicates are reported for each treatment in the in vitro cytotoxicity tables. In vivo tumor xenograft model. In vivo studies were conducted at Charles River Laboratories. Briefly, 8−12-weekold female CB.17 SCID mice were implanted subcutaneously with 1 mm3 BT474 tumor fragments. When tumors reached an average size of 100−150 mm3, animals were divided randomly into groups of ten mice each. Mice were treated with 1 mg/kg ADC generated through CuAAC bioconjugation (a-4D5-PBD, a-4D5-AF) or an ADC conjugated through cysteine based linkage (Her2-malPBD) once a week for 3 weeks or with a placebo treatment. A control arm was conducted with mice receiving an ADC (CuAAC) treatment using an antibody directed against the human cytokine Interleukin-6 (IL-6) that does not recognize mouse antigens (a-hIl6-PBD). The weight of the mice and the size of the tumor were monitored regularly throughout the treatment. Animals were sacrificed when the tumor load reached 1000 μm3 or 90 days whichever occurred first.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00359. Analytical methods and conjugate precursor preparation, supporting figures referenced in the text, structures of compounds, antibody sequence, and analytical data (PDF)



AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]. T elephone: 301.398.2556. Fax: 301.398.0000. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors would like to thank Professor M.G. Finn for his generosity in making ligands available to us and for his suggestions for optimizing the CuAAC conjugation conditions, William Brady for helpful suggestions in identifying suitable conjugation sites for IgG1.



ABBREVIATIONS aaRS, aminoacyl tRNA synthetase; AF, auristatin F; AzAb, Azide containing antibody; ADC, antibody drug conjugate; CuAAC, copper mediated azide−alkyne cycloaddition; DAR, drug antibody ratio; DMEM, Dulbecco’s Modified Eagle’s Medium; ELISA, enzyme-linked immunosorbent assay; HIC, hydrophobic interaction chromatography; HPLC, high pressure liquid chromatography; nnAA, non-natural amino acid; PBD, pyrrolobenzodiazepine dimer; PBS, phosphate buffered saline; PLRP, polymeric reverse phase; pylRS, pyrrolysine tRNA synthetase; SEC, size exclusion chromatography; SPAAC, strain I

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promoted azide−alkyne cycloaddition; tRNA, transfer ribonucleic acid

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