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Oct 31, 2017 - Yields Homogeneous and Highly Potent. Antibody-Drug Conjugates. Ying Xu 1, Shijie Jin 2, Wenbin Zhao 1 ID , Wenhui Liu 1, Ding Ding 3, Jie ...
International Journal of

Molecular Sciences Article

A Versatile Chemo-Enzymatic Conjugation Approach Yields Homogeneous and Highly Potent Antibody-Drug Conjugates Ying Xu 1 , Shijie Jin 2 , Wenbin Zhao 1 Shuqing Chen 1, * 1

2 3

*

ID

, Wenhui Liu 1 , Ding Ding 3 , Jie Zhou 1, *

ID

and

Zhejiang Provincial Key Laboratory of Anti-Cancer Drug Research, Institute of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; [email protected] (Y.X.); [email protected] (W.Z.); [email protected] (W.L.) College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China; [email protected] Formulation and Analysis Laboratory, HisunPharma (Hangzhou) Co. Ltd., Xialian Village, Xukou Town, Fuyang, Hangzhou 311404, China; [email protected] Correspondence: [email protected] (J.Z.); [email protected] (S.C.); Tel.: +86-0571-8820-8410 (J.Z.); +86-0571-8820-8411 (S.C.)

Received: 7 October 2017; Accepted: 27 October 2017; Published: 31 October 2017

Abstract: The therapeutic efficacy of antibodies can be successfully improved through targeted delivery of potent cytotoxic drugs in the form of antibody-drug conjugates. However, conventional conjugation strategies lead to heterogeneous conjugates with undefined stoichiometry and sites, even with considerable batch-to-batch variability. In this study, we have developed a chemo-enzymatic strategy by equipping the C-terminus of anti-CD20 ofatumumab with a click handle using Sortase A, followed by ligation of the payload based on a strain-promoted azide-alkyne cycloaddition to produce homogeneous conjugates. The resulting antibody-drug conjugates fully retained their antigen binding capability and proved to be internalized and trafficked to the lysosome, which released the payload with a favorable efficacy in vitro and in vivo. Thus, this reported method is a versatile tool with maximum flexibility for development of antibody-drug conjugates and protein modification. Keywords: antibody-drug conjugate; site-specific; chemo-enzymatic conjugation; click chemistry

1. Introduction Antibody-drug conjugates (ADCs) selectively deliver potent toxic payloads to antigen-positive tumor cells via specific antibody binding to reduce off-target toxicity and widen the therapeutic window in comparison to chemotherapeutics or combination therapy. Recent successes in the development of ADCs for targeted cancer therapy, such as Brentuximab vedotin (Adcetris® , SGN-35, Seattle Genetics, Inc., Bothell, WA, USA) and Trastuzumab emtansine (Kadcyla® , T-DM1, Genentech, Inc., San Francisco, CA, USA, have proven that ADCs can be potent weapons used in the battle against cancer [1–4]. All approved ADCs are produced by chemical conjugation that covalently attaches the toxic payload to the antibody through surface-exposed lysine or cysteine residues generated by reducing intrachain disulfide bonds of the antibody, which leads to heterogeneous mixtures in terms of position and stoichiometry of the payload coupled to the antibody (defined as “drug to antibody ratio”, DAR). Furthermore, different DAR of ADCs will have different efficacy, pharmacokinetic properties, stability and safety profiles [5], which result in a huge challenge in quality control. Consequently, diverse site-specific conjugation technologies have been further developed to produce homogeneous ADCs. One option is engineering free cysteines into antibodies which can react with maleimide-functionalized

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homogeneous ADCs. One option is engineering free cysteines into antibodies which can react with maleimide-functionalized toxins, thereby yielding homogeneous ADCs with an improved therapeutic window [6].homogeneous Nonetheless, ADCs this approach requires a therapeutic reduction-reoxidation that toxins, thereby yielding with an improved window [6].procedure Nonetheless, can approach potentially lead toa the formation of faultyprocedure intrachainthat or interchain disulfide Besides, the this requires reduction-reoxidation can potentially lead bonds. to the formation of cysteine-maleimide linkages disulfide are instable in Besides, a circulatory system, becauselinkages the maleimide-linker faulty intrachain or interchain bonds. the cysteine-maleimide are instable in cansystem, be exchanged active thiol group of cysteine in humanby serum, resulting in the areaction circulatory because by thethe maleimide-linker reaction can be exchanged the active thiol group premature of toxins before internalization [7]. Thus, several conjugation of cysteine inrelease human serum, resulting in the premature release of toxins beforesite-specific internalization [7]. Thus, approaches have been reportedapproaches that utilizehave incorporated non-natural amino acids [8], or enzymatic several site-specific conjugation been reported that utilize incorporated non-natural modification, such as transglutaminase [9],such formylglycine generating[9], enzyme [10], andgenerating Sortase A amino acids [8], or enzymatic modification, as transglutaminase formylglycine (SrtA) [11]. enzyme [10], and Sortase A (SrtA) [11]. SrtA could could recognize substrates leucine; P, P, proline; T, SrtA substrates containing containingaahighly highlyconserved conservedLPXTG LPXTG(L,(L, leucine; proline; the amide amide bond bond T,threonine; threonine;G,G,glycine; glycine;XXcan canbe beany any amino amino acid)-sorting acid)-sorting sequence sequence motif, cleave the betweenthe the threonine (T) the andglycine the glycine (G),followed then, followed by the nucleophilic attack of between threonine (T) and (G), then, by the nucleophilic attack of oligoglycine oligoglycine substrates, produce covalently linked products (Figure 1).high Owing to its high substrates, produce covalently linked products (Figure 1). Owing to its specificity andspecificity the wide and the wide range substrates, of oligoglycine SrtA has been extensively exploited for protein range of oligoglycine SrtA substrates, has been extensively exploited for protein engineering and engineeringconjugation and site-specific a huge excessoligoglycine-modified of nucleophilic oligoglycinesite-specific [12]. conjugation However, a [12]. hugeHowever, excess of nucleophilic toxins modifiedmolar toxinsexcess) (100-fold excess) and enzyme are necessary to driveto the equilibrium to form (100-fold andmolar enzyme are necessary to drive the equilibrium form ADCs since the ADCs since the reaction is reversible [13]. Moreover, the toxins are usually expensive and cause the reaction is reversible [13]. Moreover, the toxins are usually expensive and cause the pollution with pollution with waste in theprocess, manufacturing its appeal in industrial largepoisonous wastepoisonous in the manufacturing losing itsprocess, appeal losing in industrial large-scale production. scale production. In addition, the larger oligoglycine-functionalized toxinsthan are less reactive than the In addition, the larger oligoglycine-functionalized toxins are less reactive the small molecular small molecular weight isotype due control substrate due to[14]. steric [14]. aAs an alternative, a weight isotype control substrate to steric hindrance Ashindrance an alternative, chemo-enzymatic chemo-enzymatic approach shown potential value for the site-specific conjugation. approach has shown potentialhas value for the site-specific conjugation.

Figure The reaction reaction mechanism mechanismofofSortase SortaseA.A.Sortase SortaseAAcould could recognize LPXTG sequence Figure 1.1. The recognize anan LPXTG sequence (L, (L, leucine; P, proline; T, threonine; G, glycine; X can be any amino acid), cleave the amide bond leucine; P, proline; T, threonine; G, glycine; X can be any amino acid), cleave the amide bond between between the threonine the (G) glycine (G)active-site via an active-site form a covalent the threonine (T) and (T) the and glycine via an Cys-184 Cys-184 and formand a covalent thioester thioester intermediate, then followed by the attack of oligoglycine substrates to produce covalently intermediate, then followed by the attack of oligoglycine substrates to produce covalently linked linked products. products.

Click especially the strain-promoted azide-alkyne cycloaddition (SPAAC),(SPAAC), is an emerging Clickchemistry, chemistry, especially the strain-promoted azide-alkyne cycloaddition is an tool for protein modification. The reaction azide with strained is highly is specific, emerging tool for protein modification. Theofreaction of azide with cyclooctyne strained cyclooctyne highly efficient, proceeds mild conditions (Figure 2).(Figure Herein,2). weHerein, presented chemo-enzymatic specific, and efficient, and under proceeds under mild conditions we apresented a chemoapproach sortase-mediated ligation and Cu(I)-free reaction (Figure 3A). In the3A). firstIn step, enzymaticusing approach using sortase-mediated ligation and click Cu(I)-free click reaction (Figure the we introduced an easy-to-synthesize bifunctional oligoglycine-modified small molecule (GGG-PEG-N first step, we introduced an easy-to-synthesize bifunctional oligoglycine-modified small molecule 3, GPN, Chart 1,3,1)GPN, comprising a bio-orthogonal functional group at the C-terminus through (GGG-PEG-N Chart 1, 1) comprising aazide bio-orthogonal azide functional group at the C-

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terminus through enzymatic catalysis to accomplish site-specific covalent attachment molecules of functional enzymatic catalysis to accomplish site-specific covalent attachment of functional to terminus to through enzymatic of catalysis tohumanized accomplish anti-CD20 site-specificantibody, covalent attachment of functional molecules theofC-terminus a fullyanti-CD20 ofatumumab (OFA), and the C-terminus a fully humanized antibody, ofatumumab (OFA), and subsequently molecules to the C-terminus of a fully humanized anti-CD20 antibody, ofatumumab (OFA), and subsequently a suitable auristatin monomethyl auristatin E (MMAE) derivative (DBCO-PEG-vcreacted with areacted suitablewith monomethyl E (MMAE) derivative (DBCO-PEG-vc-PAB-MMAE, subsequently reacted with a suitable monomethyl auristatin E (MMAE) derivative (DBCO-PEG-vcPAB-MMAE, 1, 2) via strain-promoted azide-alkynetocycloaddition generate theSince desired Chart 1, 2) viaChart strain-promoted azide-alkyne cycloaddition generate the to desired ADC. the PAB-MMAE, Chart 1, 2) via strain-promoted azide-alkyne cycloaddition to generate the desired ADC. Since the oligoglycine-modified molecule3(GGG-PEG-N ) and sortase enzymeavailable are readily oligoglycine-modified small moleculesmall (GGG-PEG-N ) and sortase 3enzyme are readily in ADC. Since the oligoglycine-modified small molecule (GGG-PEG-N3) and sortase enzyme are readily available in larger quantities from chemical synthesis and E. coli production respectively, it is larger quantities from quantities chemical synthesis and E.synthesis coli production it is conceivable available in larger from chemical and E.respectively, coli production respectively,that it isthis conceivable that this design is suitable for scale-up production. Compared with the direct enzymatic design is suitable Compared with theCompared direct enzymatic of toxic conceivable that for thisscale-up design isproduction. suitable for scale-up production. with theattachment direct enzymatic attachment of toxic payloads to the antibody (Figure 3B), the chemo-enzymatic approach only payloads to theofantibody (Figure to 3B), theantibody chemo-enzymatic approach only required aapproach 2.0 molaronly excess attachment toxic payloads the (Figure 3B), the chemo-enzymatic required a 2.0 molar excess ofoftoxin per to yield yieldhomogeneous ADCs with abeDAR of toxin per group to yield homogeneous ADCs with ahomogeneous DAR of 3.3, ADCs which could efficiently required aazide 2.0 molar excess toxin per azide azide group group to with a DAR of of 3.3,3.3, which could be efficiently internalized by CD20-positive tumor cells even though the noninternalized by CD20-positive tumor cells even though the non-internalization of CD20 antigen was which could be efficiently internalized by CD20-positive tumor cells even though the noninternalization of CD20 antigen was reported [15] and were highly efficacious in vitro and in vivo. reported [15] andofwere highly efficacious in vitro vivo. internalization CD20 antigen was reported [15]and andin were highly efficacious in vitro and in vivo.

Figure 2. A reactionscheme scheme ofstrain-promoted strain-promoted azide-alkyne cycloaddition. The azide was reacted Figure Figure 2. 2. A A reaction reaction scheme of of strain-promoted azide-alkyne azide-alkyne cycloaddition. cycloaddition. The The azide azide was was reacted reacted with the dibenzylcyclooctyne(DBCO) (DBCO) in in aa Cu(I)-free Cu(I)-free [2 ++ 3] cycloaddition under mild conditions to with the dibenzylcyclooctyne [2 3] cycloaddition under mild conditions with the dibenzylcyclooctyne (DBCO) in a Cu(I)-free [2 + 3] cycloaddition under mild conditions to to produce 1,4-substituted triazoles. produce produce 1,4-substituted 1,4-substituted triazoles. triazoles.

Figure 3. Schematic illustration of the two different strategies to generate homogeneous antibodydrug conjugates (ADCs). (A) For the chemo-enzymatic approach, a bifunctional oligoglycineFigure 3. 3. Schematic Schematicillustration illustrationofofthe the two different strategies to generate homogeneous antibodyFigure two different to generate homogeneous antibody-drug modified small molecule is enzymatically attachedstrategies to the LPETG-tagged antibody in a first reaction. drug conjugates (ADCs). (A) For the chemo-enzymatic approach, a bifunctional oligoglycineIn a second step, the which is modified with a suitable functional group, is conjugated to the conjugates (ADCs). (A)toxin, For the chemo-enzymatic approach, a bifunctional oligoglycine-modified small modified small molecule is attached enzymatically to the LPETG-tagged antibody in a first antibody strain-promoted azide-alkyne cycloaddition (SPAAC); The direct enzymatic molecule is via enzymatically to theattached LPETG-tagged antibody in a(B) first reaction. In areaction. second In approach a second step, thedirect toxin, whichwith is modified with a suitable functional group, istoconjugated to via the includes conjugation of an oligoglycine-functionalized toxin to the LPETG-tagged step, the toxin, which is modified a suitable functional group, is conjugated the antibody antibody strain-promoted azide-alkyne cycloaddition (SPAAC); (B) amino Theapproach direct antibody.via LPXTG: L, leucine; P, proline; T, threonine; G, (B) glycine; X can be any acid. enzymatic strain-promoted azide-alkyne cycloaddition (SPAAC); The direct enzymatic includes approach includes direct conjugation of an oligoglycine-functionalized toxin to the LPETG-tagged direct conjugation of an oligoglycine-functionalized toxin to the LPETG-tagged antibody. LPXTG: antibody. L, leucine; P, proline; T, threonine; glycine; can be any amino acid. L, leucine;LPXTG: P, proline; T, threonine; G, glycine; X can beG, any aminoXacid.

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Chart 1. Chemical substrates for for the the enzymatic enzymatic and and chemo-enzymatic chemo-enzymatic Chart 1. Chemical structures structures of of the the different different substrates approach. (1) GGG-PEG-N33 (GPN) (GPN) is is aa bifunctional bifunctional small small molecule molecule comprising an oligoglycine at the N-terminus and a bio-orthogonal azide functional group at the C-terminus through an oligoethylene glycol ligation; (2) DBCO-PEG-vc-PAB-MMAE was monomethyl auristatin E (MMAE) derivative containing aadibenzylcyclooctyne (DBCO), an oligoethylene glycol (PEG),(PEG), a protease-sensitive valinedibenzylcyclooctyne (DBCO), an oligoethylene glycol a protease-sensitive citrulline (vc) dipeptide and a self-immolative spacer, p-aminobenzylcarbamate (PAB); (3) GGG-vcvaline-citrulline (vc) dipeptide and a self-immolative spacer, p-aminobenzylcarbamate (PAB); PAB-MMAE was modified bymodified the addition of an oligoglycine to the vc-PAB-MMAE. (3) GGG-vc-PAB-MMAE was by the addition of an oligoglycine to the vc-PAB-MMAE.

2. 2. Results Results and and Discussion Discussion 2.1. Preparation of Optimized OFA OFA We previously thethe direct enzymatic conjugation usingusing a 100-fold excess excess GGGpreviously have haveshown shownthat that direct enzymatic conjugation a 100-fold vc-PAB-MMAE (Chart (Chart 1, 3) yielded 70% of modified chain for OFA-vcMMAE GGG-vc-PAB-MMAE 1, 3)approximately yielded approximately 70% heavy of modified heavy chain for (DAR = 1.4) [13].(DAR However, conjunction inefficient forvery the light chain,for which may be not OFA-vcMMAE = 1.4)the [13]. However,was the very conjunction was inefficient the light chain, accessible chain for the light enzyme and due the to steric hindrance. flexible linker which maytobethe notlight accessible to the chain forthe thesubstrate enzyme and substrate due to A steric hindrance. (Gflexible 4S)n, often used(Gto4 S) link the antibody formats [16], was inserted before the LPETG recognition motif A linker , often used to link the antibody formats [16], was inserted before the LPETG n (L, leucine; P, proline; E, glycine; T, threonine; G, glycine) at the C-terminus of light and heavy chains recognition motif (L, leucine; P, proline; E, glycine; T, threonine; G, glycine) at the C-terminus of light in order to chains improve conjugation Thus, six anti-CD20 OFA were engineered and heavy in the order to improveefficiency. the conjugation efficiency. Thus, sixvariants anti-CD20 OFA variants with aengineered C-terminalwith (GGGGS) nLPETG(GHHHHHH) m sequence (where n = 0, 1 or 2, and m = were a C-terminal (GGGGS)n LPETG(GHHHHHH) sequence (where n = 0,0 1oror1), 2, m and successfully expressed in HEK293F cells, stillcells, remained binding affinity to CD20affinity of cell m = 0 or 1), and successfully expressed in which HEK293F whichthe still remained the binding surface. order to seekInthe optimized antibody, six OFA variants werevariants tested for thetested relative to CD20 In of cell surface. order to seek the optimized antibody, six OFA were for conjugation efficiency using chemo-enzymatic approach, and MMAEand covalently to the light the relative conjugation efficiency using chemo-enzymatic approach, MMAEcoupled covalently coupled and heavy was visualized 3,3’-diaminobenzidine (DAB) color development after western to the lightchains and heavy chains waswith visualized with 3,3’-diaminobenzidine (DAB) color development blot of the conjugated antibody (Figure In accordance with previous studies [17,18], the after western blot of the conjugated antibody4).(Figure 4). In accordance with previous studies [17,18], conjugation efficiency to the light chain and heavy chain was progressively improved by increasing the conjugation efficiency to the light chain and heavy chain was progressively improved by increasing short peptide spacer (G44S)nn length length after after the the attachment attachment of of His-tag His-tag which will be lost in the enzymatic reaction. Therefore, we chose the (G (G44S)22LPETGGHHHHHH LPETGGHHHHHH tagged tagged OFA OFA to conjugate toxins using chemo-enzymatic method to generate ADCs in this study. study.

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Figure Figure 4. Conjugation Conjugation efficiency efficiency of of six six ofatumumab ofatumumab (OFA) (OFA) variants. Western Western blot blot analysis analysis was was performed performed for for determination determination of of monomethyl monomethyl auristatin auristatinEE(MMAE) (MMAE)levels levelsof oflight lightand andheavy heavychains. chains. Lane 1, 1,H/L-LPETG; H/L-LPETG;Lane Lane2,2, H/L-G Lane 3, H/L-(G 4, H/L-LPETGGH 4SLPETG; Lane 3, H/L-(G 4S)2LPETG; LaneLane 4, H/L-LPETGGH 6; Lane Lane H/L-G 4 SLPETG; 4 S)2 LPETG; 6; Lane 5, H/L-G ; Lane 6, H/L-(G heavy L, light chain. 4SLPETGGH 6; Lane66, H/L-(G 4S)2LPETGGH 6. H, heavy chain; L,chain; light chain. 5, H/L-G 4 SLPETGGH 4 S)2 LPETGGH 6 . H,

2.2. 2.2. Chemo-Enzymatic Chemo-Enzymatic Approach Approach to to Produce Produce ADC and Its Characterization We We developed a chemo-enzymatic chemo-enzymatic approach approach by by SrtA-mediated SrtA-mediated ligation ligation and and click click chemistry chemistry to to produce ADC (Table 1). In the first place, a small molecule substrate (GGG-PEG-N 3 , 2) suitable for produce ADC 3 , 2) suitable for the the SrtA-catalyzed SrtA-catalyzed transpeptidation transpeptidation was was used used to to transfer transfer an an additional additional azide azide functionality functionality onto onto the the antibody of 100:1 100:1 in in order orderto topromote promoteenzymatic enzymaticreaction, reaction,while while the antibody formats (scFv antibody at a rate of the antibody formats (scFv or or Fab) required less oligoglycine-modified substrate for their easy access [11]. In the second step, the Fab) required less oligoglycine-modified substrate for their easy access [11]. In the second step, the toxin toxin (DBCO-PEG-vc-PAB-MMAE, 3),was which was equipped with an alkynyl group reactive the (DBCO-PEG-vc-PAB-MMAE, 3), which equipped with an alkynyl group reactive to the azide,to could azide, thenwith be coupled with by thethe antibody by the strain-promoted azide-alkyne(SPAAC) cycloaddition then becould coupled the antibody strain-promoted azide-alkyne cycloaddition under (SPAAC) undertomild yieldThe theSPAAC desiredreaction ADC. The SPAAC waswith verya fast and mild condition yieldcondition the desiredtoADC. was very fast reaction and efficient minimal efficient with of a minimal molarhomogeneous excess of toxin to yield homogeneous (2 molar equivalent per molar excess toxin to yield ADCs (2 molar equivalentADCs per azide group), while direct azide group), while direct SrtA-based attachment of MMAE derivative required a 100 molar excess SrtA-based attachment of MMAE derivative required a 100 molar excess of drug since toxins are of drug since are expensive may causethe hazardous waste during the [13]. manufacturing expensive andtoxins may cause hazardousand waste during manufacturing procedure Moreover, procedure [13]. Moreover, the heterocyclic linkage generated by SPAAC has previously the heterocyclic triazole linkage generated triazole by SPAAC has been previously shown to been be highly stable shown to be highly stable both in vitro and in vivo [19]. Recent studies that successfully employed both in vitro and in vivo [19]. Recent studies that successfully employed enzyme-mediated ligation enzyme-mediated ligation and click chemistry to prepare PEGylated capsules [20] and streptavidinand click chemistry to prepare PEGylated capsules [20] and streptavidin-hydrogel [21], combined hydrogel combined our results, demonstrate broad utility this technology. with our [21], results, clearly with demonstrate the clearly broad utility of this the technology. Thisofchemo-enzymatic This chemo-enzymatic approach with flexibility versatility be readily adapted toand a variety approach with flexibility and versatility could be and readily adaptedcould to a variety of antibodies toxins, of antibodies and research toxins, and correlational research is under investigation. and correlational is under investigation. Table Table 1. The The comparison comparison of of the the different synthesis methods.

Characterizations Enzymatic ADC Chemo-Enzymatic ADC Characterizations Enzymatic ADC Chemo-Enzymatic ADC Derivative payloads 100-fold molar excess 2 molar equivalent per azide group 2 molar equivalent per azide DAR payloads 1.4 molar excess 3.3 Derivative 100-fold group Internalization (%) 39.44 49.31 DAR 1.4 3.3 IC50 on Ramos (nM)(%) 5.336 39.44 2.893 Internalization 49.31 on Ramos (nM) 2.893 IC50 IC on50Daudi (nM) 30.51 5.336 3.857 IC50 on 30.51 3.857 1 Effect in Daudi vivo (nM) *** p < 0.001 ** p < 0.01 1 Effect in vivo *** p < 0.001 1 ** p < 0.01 1 Safety in vitro and in vivo lowerlower higher Safety in vitro and in vivo higher 1 *** ***

p < 0.001 and ** p < 0.01 compared with untreated control group. ADC: antibody-drug conjugate; p < 0.001 and ** p < 0.01 compared with untreated control group. ADC: antibody-drug conjugate; DAR: drug to DAR: drug to antibody ratio. antibody ratio. 1

To successful conjugation chemo-enzymatic approach, To confirm confirm successful conjugation of of MMAE MMAE via via our our chemo-enzymatic approach, the the reduced reduced molecular weights of the light chain and heavy chain for OFA and OFA-GPN-vcMMAE molecular weights of the light chain and heavy chain for OFA and OFA-GPN-vcMMAE were were analyzed MSMS (Waters, Worcester, MA,MA, USA)USA) (Figure 5A). The mass of the light analyzed by byQ-TOF Q-TOF (Waters, Worcester, (Figure 5A). Thedifferences mass differences of chain and heavy corresponded to the loss of the thecleaved enzymatic reaction the light chain chain and heavy chain corresponded to peptide the losscleaved of theduring peptide during the (955 g/mol)reaction plus the(955 molecular GGG-PEG-N 3 (345 g/mol, 1) and DBCO-PEG-vc-PABenzymatic g/mol)weights plus theofmolecular weights of GGG-PEG-N3 (345 g/mol, 1) and MMAE (1657 g/mol, 2), approximately with the expected mass shift of 1049 DBCO-PEG-vc-PAB-MMAE (1657 g/mol, consistent 2), approximately consistent with the expected massDa. shiftAs of expected, we also confirmed the conjugation site on LPETG motif (Figure S1). Futhermore, the DAR 1049 Da. As expected, we also confirmed the conjugation site on LPETG motif (Figure S1). Futhermore, was determined by reversed phase high performance liquid chromatography (RP-HPLC) (Figure 5B) and hydrophobic interaction chromatography (HIC) (Figure 5C). The chemo-enzymatic approach

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the DAR was determined by reversed phase high performance liquid chromatography (RP-HPLC) Int. J. Mol. Sci. 2017, 18, 2284 6 of 17 (Figure 5B) and hydrophobic interaction chromatography (HIC) (Figure 5C). The chemo-enzymatic approach harvested a high ADCs withofa 3.3, DAR of 3.3, about 75% conjugation efficiency at the harvested a high yield ofyield ADCsofwith a DAR about 75% conjugation efficiency at the heavy heavy chain, and >90% conjugation efficiency at the light chain, achieving site-specific conjugation chain, and >90% conjugation efficiency at the light chain, achieving site-specific conjugation of drug of drug antibody, wherein both modification sites DARs could be more precisely controlled. totoantibody, wherein both thethe modification sites andand DARs could be more precisely controlled. Although obtained ADCcontaining containing aa small small amount species, its its homogeneity is is Although thethe obtained ADC amountofoflower lowerDAR DAR species, homogeneity higher than general ADCs constructedby bytraditional traditional lysine [1–4]. higher than general ADCs constructed lysineor orcysteine cysteineligation ligation [1–4].

Figure 5. The characterizationofofchemo-enzymatic chemo-enzymatic ADC. Q-TOF mass spectrum of of Figure 5. The characterization ADC.(A) (A)Deconvoluted Deconvoluted Q-TOF mass spectrum ofatumumab (OFA) and OFA-GPN-vcMMAE. The mass chain (25,446.4 Da)Da) andand heavy ofatumumab (OFA) and OFA-GPN-vcMMAE. masspeaks peaksofoflight light chain (25,446.4 heavy chain (53,005.8 OFA increaseby by1049 1049 Da Da after conjugation in accordance chain (53,005.8 Da)Da) forfor OFA increase afterthe thechemo-enzymatic chemo-enzymatic conjugation in accordance the expected mass (B) Reversed phase performance liquid chromatography (RPwithwith the expected mass shift;shift; (B) Reversed phase highhigh performance liquid chromatography (RP-HPLC) HPLC) evaluation of conjugation efficiency. Peak L0 and H0 indicate the light chain and heavy chain, evaluation of conjugation efficiency. Peak L0 and H0 indicate the light chain and heavy chain, and L1 and L1 and H1 represent the light chain and heavy chain conjugated with MMAE. Our chemoand H1 represent the light chain and heavy chain conjugated with MMAE. Our chemo-enzymatic ADC enzymatic ADC was difficult to be completely reduced even in the presence of 100 mM dithiothreitol was difficult to be completely reduced even in the presence of 100 mM dithiothreitol (DTT) due to (DTT) due to stable structure, which is one possible explanation of the unreduced peak; (C) stable structure, which is one possible explanation of the unreduced peak; (C) Hydrophobic interaction Hydrophobic interaction chromatography (HIC) analysis of the DAR under native condition. E0 (1, 2, chromatography (HIC) analysis of the DAR under native condition. E0 (1, 2, 3 or 4) means no (one, two, 3 or 4) means no (one, two, three or four) MMAE molecule was conjugated to the intact antibody. three or four) MMAE molecule was conjugated to the intact antibody.

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2.3. 2.3. ADC ADC Binding Binding to to CD20-Positive CD20-Positive Cells Cells To the effects effectsofofthe theconjugation conjugationprocesses processes toxins targeted antigen-binding, To investigate investigate the of of toxins on on targeted antigen-binding, the the qualitative cell-binding experiments were performed with CD20-positive Daudi cells using flow qualitative cell-binding experiments were performed with CD20-positive Daudi cells using flow cytometry. the antigen binding capability, which demonstrated thatthat the cytometry.The TheADCs ADCswere werefound foundtotoretain retain the antigen binding capability, which demonstrated modification of MMAE had minimal effect on the binding affinity to cell surface CD20 when compared the modification of MMAE had minimal effect on the binding affinity to cell surface CD20 when with the unconjugated antibody (Figure 6). This result is surprising the attachment sitethe of compared with the unconjugated antibody (Figure 6).not This result isbecause not surprising because drug is distant the is antigen-binding site. attachment sitefrom of drug distant from the antigen-binding site.

Figure 6. 6. Determining ADCs to to CD20-positive CD20-positive Daudi Daudi cells cells by by flow flow Figure Determining the the binding binding of of antibody antibody and and ADCs cytometry. PBS: phosphate-buffered saline; OFA-vcMMAE: the direct enzymatic ADC. cytometry. PBS: phosphate-buffered saline; OFA-vcMMAE: the direct enzymatic ADC.

2.4. Internalization of ADC and Subcellular Localization 2.4. Internalization of ADC and Subcellular Localization Next, we examined the internalization of OFA and the corresponding ADCs, which is a Next, we examined the internalization of OFA and the corresponding ADCs, which is predominant factor in the function of ADC after antigen binding. Daudi cells were incubated for 30 a predominant factor in the function of ADC after antigen binding. Daudi cells were incubated min at 4 °C with serial dilutions of either the unconjugated OFA or the corresponding ADCs, stained for 30 min at 4 ◦ C with serial dilutions of either the unconjugated OFA or the corresponding ADCs, with FITC-labeled secondary antibody, and examined by flow cytometry. Figure S2 showed that the stained with FITC-labeled secondary antibody, and examined by flow cytometry. Figure S2 showed relationship between the mean fluorescence intensity (MFI) and the concentration of antibody or that the relationship between the mean fluorescence intensity (MFI) and the concentration of antibody ADC is linear within an applicable scope, suggesting that the internalization of the antibody and or ADC is linear within an applicable scope, suggesting that the internalization of the antibody and ADCs by the target cells could be evaluated by the mean fluorescent intensity of cell surface. Change ADCs by the target cells could be evaluated by the mean fluorescent intensity of cell surface. Change in the surface levels of antibody or ADCs on Daudi cells was determined by flow cytometry after in the surface levels of antibody or ADCs on Daudi cells was determined by flow cytometry after incubation at a concentration of 2 μg/mL for 2 h at 37 °C (Figure 7). In accordance with previous incubation at a concentration of 2 µg/mL for 2 h at 37 ◦ C (Figure 7). In accordance with previous conclusions [22], cell surface loading with anti-CD20 antibody remained relatively constant after 2 h conclusions [22], cell surface loading with anti-CD20 antibody remained relatively constant after 2 h at at 37 °C. Unconjugated anti-CD20 antibody was hard to be internalized into CD20-positive cells since 37 ◦ C. Unconjugated anti-CD20 antibody was hard to be internalized into CD20-positive cells since CD20 is a noninternalizing antigen [23]. In contrast, fluorescent intensity of OFA-GPN-vcMMAE and CD20 is a noninternalizing antigen [23]. In contrast, fluorescent intensity of OFA-GPN-vcMMAE OFA-vcMMAE declined significantly. Within 2 h, >40% and >30% of OFA-GPN-vcMMAE and OFAand OFA-vcMMAE declined significantly. Within 2 h, >40% and >30% of OFA-GPN-vcMMAE and vcMMAE, respectively, were internalized into the Daudi cells, which was significantly enhanced OFA-vcMMAE, respectively, were internalized into the Daudi cells, which was significantly enhanced after conjugation with vcMMAE, suggesting that the conjugation of vcMMAE could promote the after conjugation with vcMMAE, suggesting that the conjugation of vcMMAE could promote the internalization of OFA in Daudi cells. Previous studies showed that internalization of type I antiinternalization of OFA in Daudi cells. Previous studies showed that internalization of type I anti-CD20 CD20 antibody was greatly augmented by their engagement with FcγRIIb on the cell surface via antibody was greatly augmented by their engagement with FcγRIIb on the cell surface via antibody antibody bipolar bridging and the rate of internalization positively correlated with cell surface bipolar bridging and the rate of internalization positively correlated with cell surface expression of expression of FcγRIIb [24]. Hence, we speculated that vcMMAE could engage with FcγRIIb on Daudi FcγRIIb [24]. Hence, we speculated that vcMMAE could engage with FcγRIIb on Daudi cells, followed cells, followed by the internalization of CD20-ADC-FcγRIIb complex. However, the biological by the internalization of CD20-ADC-FcγRIIb complex. However, the biological mechanisms need to mechanisms need to be further studied. be further studied.

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◦C The percentages internalization OFA, OFA-vcMMAE and OFA-GPN-vcMMAE atat37 Figure percentages of internalization OFA, OFA-vcMMAE and OFA-GPN-vcMMAE at Figure 7. 7. The percentages ofof internalization ofofof OFA, OFA-vcMMAE OFA-GPN-vcMMAE 3737 for 22h.h. p-value isisindicated as *** pp90% apoptotic cells and approximately 7% dead cells, suggesting the apoptosis-inducing ability ADCs mainly depends highly toxic MMAE, which has been reported apoptosis-inducing ability ofof ADCs mainly depends onon highly toxic MMAE, which has been reported apoptosis-inducing ability of ADCs mainly depends on highly toxic MMAE, which has been reported induce great growth arrest and apoptosis [26]. toto induce great growth arrest and apoptosis [26]. to induce great growth arrest and apoptosis [26].

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Figure 9. Effect antibodyand andADCs ADCson oninduction induction of Ramos cells were Figure 9. Effect of of antibody of apoptosis apoptosisininRamos Ramoscells. cells. Ramos cells were exposed to antibody, ADCs μg/mL for for 72 72 h. was detected using Annexin exposed to antibody, or or ADCs atat 5 5µg/mL h. Induction Inductionofofapoptosis apoptosis was detected using Annexin V-FITC PI-Staining flow cytometry.Late Lateapoptotic apoptotic and and necrotic V-FITC andand PI-Staining byby flow cytometry. necroticcells: cells:AnnexinV-FITC+/PI+ AnnexinV-FITC+/PI+ (upper-right). Early apoptotic cells: Annexin V-FITC+/PI− (lower right). (upper-right). Early apoptotic cells: Annexin V-FITC+/PI− (lower right).

Next, we further evaluated the cytotoxicity in vitro and selectivity of the ADC, two CD20Next, we further evaluated the cytotoxicity in vitro and selectivity of the ADC, two CD20-positive positive B-lineage lymphoma cell lines (Daudi, Ramos) and one CD20-negative cell line (K562) were B-lineage lymphoma cellshowed lines (Daudi, Ramos) and OFA one CD20-negative (K562) included. included. The results that unconjugated possessed very cell lowline toxicity on were two CD20Thepositive results showed that unconjugated OFA possessed very low toxicity on two CD20-positive cells (IC50 > 6.667 × 103 nM) (Figure 10). In contrast, OFA-GPN-vcMMAE (DAR = 3.3)cells 3 nM) (Figure 10). In contrast, OFA-GPN-vcMMAE (DAR = 3.3) effectively (IC50 > 6.667 killed × 10Ramos effectively and Daudi cells with an IC50 of 2.893 nM and 3.857 nM, respectively, while killed Ramos and Daudi cells with an IC50 of (DAR 2.893 nM andwas 3.857 nM, respectively, the direct enzymatic control OFA-vcMMAE = 1.4) slightly less cytotoxicwhile with the direct enzymatic control OFA-vcMMAE (DAR = 1.4) was slightly less cytotoxic with corresponding corresponding IC50 of 5.336 nM and 30.51 nM. The potency of these ADCs correlated well with the value.nM These 50 values comparable with published aboutwell anti-CD20-based ADCs IC50DAR of 5.336 andIC30.51 nM.are The potency of these ADCsvalues correlated with the DAR value. generated by reduced interchain disulfides [22,23]. values Significantly, anti-CD20-MMAE conjugates These IC50 values are comparable with published about two anti-CD20-based ADCs generated showed an approximately 1000-fold lowerSignificantly, IC50 than unconjugated OFA against two CD20-positive by reduced interchain disulfides [22,23]. two anti-CD20-MMAE conjugates showed cells, clearly demonstrating that the enhanced potency of the ADCs in comparison to the unmodified an approximately 1000-fold lower IC50 than unconjugated OFA against two CD20-positive cells, clearly antibody. Moreover, OFA-vcMMAE showed less potency on demonstrating that the both enhanced potency ofand theOFA-GPN-vcMMAE ADCs in comparison to thefar unmodified antibody. CD20-negative K562 cells compared to their effects on the CD20-positive cells, indicating the Moreover, both OFA-vcMMAE and OFA-GPN-vcMMAE showed far less potency on CD20-negative cytotoxicity is specific for CD20-positive cells.

K562 cells compared to their effects on the CD20-positive cells, indicating the cytotoxicity is specific Int. J. Mol. Sci. 2017, 18, 2284 10 of 17 for CD20-positive cells. Daudi(CD20+)

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Figure 10. Figure In vitro effects of of OFA-GPN-vcMMAE, OFA-vcMMAE and unconjugated OFA 10. Intoxic vitro toxic effects OFA-GPN-vcMMAE, OFA-vcMMAE and unconjugated OFA on CD20-positive (Daudi, Ramos) Ramos) andand negative (K562) cell lines. CD20+: CD20-positive; CD20-:CD20-positive; CD20on CD20-positive (Daudi, negative (K562) cell lines. CD20+: negative. CD20-: CD20-negative. 2.6. In vivo Antitumor Activity The in vivo efficacy of chemo-enzymatic ADC was explored using a Ramos B-lymphoma xenograft model in nude mice. Saline solution was used as negative control, and direct enzymatic ADC was included as a comparator. When mean tumor size in each group reached approximately 450 mm3 (3–9 fold of the regular starting tumor volume of 50–150 mm3), therapy was initiated once

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2.6. In vivo Antitumor Activity The in vivo efficacy of chemo-enzymatic ADC was explored using a Ramos B-lymphoma xenograft model in nude mice. Saline solution was used as negative control, and direct enzymatic ADC was included as a comparator. When mean tumor size in each group reached approximately 450 mm3 (3–9 fold of the regular starting tumor volume of 50–150 mm3 ), therapy was initiated once every four days for four times (q4d × 4), and the tumor growth was monitored (Figure 11A). Tumors treated with the saline control grew rapidly and reached an average tumor volume of more than 2000 mm3 . In contrast, OFA-GPN-vcMMAE and OFA-vcMMAE were able to significantly delay tumor growth (*** p < 0.001 and ** p < 0.01 compared with untreated control group). During the first week, OFA-GPN-vcMMAE and OFA-vcMMAE groups showed efficient tumor shrinkage. After one week, tumor sizes gradually increased for mice treated with OFA-vcMMAE but not for OFA-GPN-vcMMAE group, indicating OFA-vcMMAE showed lower potency in vivo compared with OFA-GPN-vcMMAE. Remarkably, the tumor disappeared completely in three of five mice in OFA-GPN-vcMMAE treated group, and no tumor recurrence was observed among them for the whole length of the study, leaving two slowly progressing. At the same time, mice were weighed to evaluate the in vivo toxicity of ADC (Figure 11B). Slight weight loss was observed in ADC-treated groups compared with untreated group. However, the mice in OFA-GPN-vcMMAE group gradually returned to their normal weights after treatment, indicating the absence of severe or nonrecoverable toxicity. In addition, the significant reduction in body weights was continuing in OFA-vcMMAE group, indicating that the chemo-enzymatic ADC exerted improved therapeutic effect and decreased toxicity compared to direct enzymatic ADC. For acute toxicity evaluation, mice were sacrificed 24 h after the last administration, and histological sections of the major organs (heart, liver and kidney) of the mice were examined after H&E staining. No obvious histomorphologic alterations were observed in any sections of organs except that hepatic steatosis was found in OFA-vcMMAE group (Figure 11C). To further evaluate the maximum tolerated dose (MTD) of our chemo-enzymatic ADC, we randomly selected a tumor-bearing nude mouse to treat intravenously high-dosage OFA-GPN-vcMMAE (20 mg/kg). No overt toxicity was found in the animal treated with 20 mg/kg OFA-GPN-vcMMAE (Figure 11C). Taken together, these suggested that a considerable therapeutic window may exist for OFA-GPN-vcMMAE. However, toxicology, pharmacokinetics and pharmacodynamics of ADC need to be further studied.

except that hepatic steatosis was found in OFA-vcMMAE group (Figure 11C). To further evaluate the maximum tolerated dose (MTD) of our chemo-enzymatic ADC, we randomly selected a tumorbearing nude mouse to treat intravenously high-dosage OFA-GPN-vcMMAE (20 mg/kg). No overt toxicity was found in the animal treated with 20 mg/kg OFA-GPN-vcMMAE (Figure 11C). Taken together, these that a considerable therapeutic window may exist for OFA-GPN-vcMMAE. Int. J. Mol. Sci. 2017,suggested 18, 2284 11 of 17 However, toxicology, pharmacokinetics and pharmacodynamics of ADC need to be further studied.

Figure 11. 11. In In vivo vivo efficacy efficacy and and toxicity toxicity of of OFA-vcMMAE OFA-vcMMAE and and OFA-GPN-vcMMAE OFA-GPN-vcMMAE in in human human tumor tumor Figure xenograft mouse mouse models. models. (A) (A) In In vivo and OFA-GPN-vcMMAE xenograft vivo antitumor antitumor activities activities of of OFA-vcMMAE OFA-vcMMAE and OFA-GPN-vcMMAE conjugates. The The p-value p-value is is indicated indicated as as follows: follows: ** ** pp