Enzyme-Based Labeling Strategies for Antibody–Drug Conjugates and Antibody Mimetics

19 Pages·2017·2.95 MB·English

antibodies
Review
Enzyme-Based Labeling Strategies for
Antibody–Drug Conjugates and Antibody Mimetics
GeorgFalck ID andKristianM.Müller* ID
CellularandMolecularBiotechnology,FacultyofTechnology,BielefeldUniversity,Universitätsstr.25,
33615Bielefeld,Germany;[email protected]
* Correspondence:[email protected];Tel.:[phone]
Received:1November2017;Accepted:18December2017;Published:4January2018
Abstract: Strategiesforsite-specificmodificationofproteinshaveincreasedinnumber,complexity,
andspecificityoverthelastyears. Suchmodificationsholdthepromisetobroadentheuseofexisting
biopharmaceuticalsortotailornovelproteinsfortherapeuticordiagnosticapplications. Therecent
questfornext-generationantibody–drugconjugates(ADCs)sparkedresearchintotechniqueswith
site selectivity. While purely chemical approaches often impede control of dosage or locus of
derivatization,naturallyoccurringenzymesandproteinsbeartheabilityofco-orpost-translational
protein modifications at particular residues, thus enabling unique coupling reactions or protein
fusions.Thisreviewprovidesageneraloverviewandfocusesonchemo-enzymaticmethodsincluding
enzymessuchasformylglycine-generatingenzyme,sortase,andtransglutaminase. Applicationsfor
theconjugationofantibodiesandantibodymimeticsarereported.
Keywords: chemo-enzymaticlabeling;armedantibody;antibodycoupling;antibodyconjugation
1. Introduction
1.1. Antibody–DrugConjugates
Inthefightagainstcancer,antibody–drugconjugates(ADC)havegainedconsiderableattention,
especiallysincethemarketreleaseofKadcylaandAdcetris,whichhavedoubledinyearlysalessince
2013,closinginonabilliondollars[1,2]. AvastnumberofnewADCsareinthepipeline,withseven
oftheminpivotalclinicaltrials[3]. AfactorfuelingthesuccessofADCsisthepromisingexpansion
of the therapeutic window compared to “naked” therapeutic monoclonal antibodies (mABs) and
classicalchemotherapy. Furthermore,thisapproachcouldreviveantibodieswithsuitableaffinities
yetinsufficientcytotoxicity,andhighlypotentdrugsunfavorableforunspecificsystemicapplication.
Improvement of therapeutic efficacy has been proven for the trastuzumab–maytansine conjugate
T-DM1ontumormodelsrefractorytotrastuzumab[4]. However,saidpotentialtherapeuticwindowis
limitedbytheheterogeneity,stability,andpharmacokineticsofcurrentADCsandcouldberestored
bysite-specificconjugationstrategies[5,6]. Thedrugtoantibodyratio(DAR)emergedasakeyterm
in thediscussionof theideal ADC,which hasto behigh enoughto provide sufficienttherapeutic
potency as well as low enough to not generate heavily drug-modified conjugates with impaired
bindingproperties,stability,andcirculationhalf-life[7,8]. Dependingonthepropertiesofthedrug,
theidealDARformostADCsisbetween2and4[7,9]. CouplingstrategiesforfirstgenerationADCs
havebeenexclusivelychemicalwithrandomcouplingofthiolgroupsonreducedcysteines,ofside
chainaminegroupsonlysinesandaldehydegroupsonoxidizedglycostructures[10]. Theabundance
ofsuchmodificationtargetsleadstoabroaddistributionofDARs. Subsequentstrategiestherefore
focusedonalteringinterchaindisulfidebonds,changingrespectivecysteinestoserinesorintroducing
newfreecysteines[11],asutilizedfortheTHIOMABconjugationstrategy[12,13],ormodulatingthe
Antibodies 2018,7,4;doi:10.3390/antib7010004 www.mdpi.com/journal/antibodies
Antibodies 2018,7,4 2of19
Antibodies 2018, 7, 4 2 of 18
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1.2. Antibody Fragments and Mimetics
1.2. AntibodyFragmentsandMimetics
Antibody fragments and mimetics were created to generate economical and more convenient high-
Antibodyfragmentsandmimeticswerecreatedtogenerateeconomicalandmoreconvenient
affinity proteins with alternative properties, though in part similar to those of full antibodies. Intended
high-affinityproteinswithalternativeproperties,thoughinpartsimilartothoseoffullantibodies.
differences to full antibodies are their smaller size and the possibility to be produced in prokaryotic
Intended differences to full antibodies are their smaller size and the possibility to be produced in
cells. Some of these mimetics are directly derived from their full antibody counterparts, like antigen-
prokaryoticcells.Someofthesemimeticsaredirectlyderivedfromtheirfullantibodycounterparts,like
binding fragments (Fab), single-chain variable fragments (scFv) or domain antibodies, such as VHH
antigen-bindingfragments(Fab),single-chainvariablefragments(scFv)ordomainantibodies,suchas
domains (also termed Nanobodies). Nanobodies are derived from camelid heavy–chain–only
V Hdomains(alsotermedNanobodies). Nanobodiesarederivedfromcamelidheavy–chain–only
anHtibodies and have been used extensively for imaging and even therapeutic applications [15]. They
antibodiesandhavebeenusedextensivelyforimagingandeventherapeuticapplications[15]. They
inhibited antigen binding or were loaded with toxins and thereby delayed tumor growth [16,17]. ScFv
inhibitedantigenbindingorwereloadedwithtoxinsandtherebydelayedtumorgrowth[16,17]. ScFv
antibodies for therapy are human or humanized light and heavy chain variable fragments fused by a
antibodiesfortherapyarehumanorhumanizedlightandheavychainvariablefragmentsfusedby
flexible (glycine-serine) linker. They have shown increased distribution across tumors while retaining
aflexible(glycine-serine)linker. Theyhaveshownincreaseddistributionacrosstumorswhileretaining
antigen affinity. Their potential use for cancer therapy has been discussed [18–20] with Blincyto
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sduuceh taos tthhee liarcakb oilfi taynt oFcp penaertt,r athteeitru mmiosrssin,gb uatnatilbsoodthye-direrpeelantdivenelty csehllo-mrtehdailaft-elidfe ciyntobtlooxoidcitcyir (cAulDatCioCn) aanndd,
dcoumeptoletmheenlat-cdkepoefnadneFnct pcyartot,tothxeiciirtym i(sCsDinCg a[2n4t,i2b5o]d. yW-dheiplee nthdee nsthcoerltle-rm ceidrciautleadtiocny totitmoxe icoift ya(nAtiDboCdCy)
amnidmceotimcsp nleamrreonwt-sd tehpee tnhdeernaptecuyttioct owxiincidtoyw(C fDorC c[a2n4c,e2r5 ]t.hWerahpilye, tiht eiss hoothreterrwciisrec ubleanteiofincitailm foero dfiaangtniboostdicy
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penetration, due to smaller size and typically lower affinities, can be advantageous in the treatment of
approaches,asthepatient’sexposuretosubstancessuchasradiolabelsisshorter. Theenhancedtumor
solid tumors. Whereas whole antibodies usually accumulate between tumor interstitium and tumor
penetration,duetosmallersizeandtypicallyloweraffinities,canbeadvantageousinthetreatment
surface cells, antibody fragments and mimetics are able to reach the inner tissue faster [24,26]. Antibody
of solid tumors. Whereas whole antibodies usually accumulate between tumor interstitium and
mimetics can benefit from modification, like PEGylation and fusion to an albumin-binding domain for
tumorsurfacecells,antibodyfragmentsandmimeticsareabletoreachtheinnertissuefaster[24,26].
longer circulation and site-specific drug coupling for enhanced toxicity.
Antibodymimeticscanbenefitfrommodification,likePEGylationandfusiontoanalbumin-binding

domainforlongercirculationandsite-specificdrugcouplingforenhancedtoxicity.
Antibodies 2018,7,4 3of19
2. Site-SpecificProteinModificationStrategies
2.1. Formylglycine-GeneratingEnzymes
Firstfoundduringaninvestigationofthemultiplesulfatasedeficiency,formylglycine-generating
enzymes(FGE)havetheabilitytoconvertcysteinessite-specificallytoformylglycines, presenting
analdehyderesidueuniqueinproteinsandsuitableforbio-orthogonalcoupling(Figure2)[27,28].
Theirnaturalsubstratesaresulfatasesbearingahighlyconservedhexapeptide,inwhichthecysteineis
convertedtoformylglycineintheendoplasmicreticulum(ER)[29,30]. FGEscanbefoundinavariety
oforganisms,bothprokaryoticandeukaryotic,suggestingpossiblevariousinvivoaswellasinvitro
applications. Since its discovery in 2003, human FGE (hFGE) has been predominantly employed
informylglycineconversionintheeukaryoticcontext[31,32]. Whenoverexpressed,excesshFGEis
truncatedandsecretedfromtheER.Thissecretedformisstillcatalyticallyfunctionalandutilizable
forinvitroreactions[33]. Forprokaryoticsystems,differentFGEsareavailable,withMycobacterium
tuberculosis, Streptomyces coelicolor, and Thermomonospora curvata leading the way. Formylglycine,
asanovelposttranslationalmodification,hasdrawninterestinthefieldofsite-specificmodification.
Carricoetal. coinedtheterm“aldehydetag”in2007fortheshortuniversalFGErecognitionmotif
LCTPSR,latershortenedtoCXPXR[34,35].TheyusedthistagforN-orC-terminalproteinmodification
inE.colico-expressedwithM.tuberculosisFGE[36]. ThetagwasalsointroducedinaCHO(Chinese
HamsterOvary)cellsystemforIgG-Fcandwholeantibodymodification,showingthepreservationof
binding functionalities, as well as for labelling of cytosolic and cell surface proteins [37]. For the
generation of site-specific coupled antibodies and antibody fragments expression platforms for
aldehydetaggedproteinsweretestedinbothE.coliandCHOcellswhilestablycoexpressinghFGE[38].
TheeffectofpositioningofthedruginADCshasbeenaddressedandaldehydetagfunctionalityat
differentsitesofanIgG1antibodyhasbeenvalidated. Whileaggregationoccurredafterintroduction
atcertainpositions,especiallyintheCH2andCH3domains,forthemostparttheimplementedtags
wereapplicable. Theflexibilityofthetagsusewasthusextendedtoalmostallgenerallyaccessible
areas of the antibody [39]. Formylglycine conversion rates of 75% to over 90% could be achieved,
yettotalconversionstillremainsanissue. Inanattempttoenhanceconversion,differentmediaand
copper(II) supplementation were tested yielding higher conversion rates even with 5 µM copper
addition. Mediacompositionseemstoplayacrucialroleinconversionresultsaswell,butdetails
remainelusive[40]. CoppersupplementationandreconstitutionofFGEinvitrohavebeendiscussed
inthelightofnewfindingsregardingthemechanismoftheFGEcatalysis. HumanFGEconvertsthe
substratecysteineafterdisulfideformationwiththeCys-341inanoxygenase-typereactionrequiring
molecularoxygenandareductant[41,42]. Recentlycopper(I)wasproposedasacofactor[43]. Invitro
reconstitutionwithcoppercouldsuccessfullyincreasethecatalyticefficiencyofbacterialFGEs[44,45].
TheoverallsimilarityofthemycobacteriumtuberculosisandS.coelicolorFGEtohFGEincludingthe
two cysteines in the catalytic center suggests a common mechanism [46]. Setbacks of the method
includingprecipitationinvitrohavebeenreported,especiallyinhighionicstrengthreactionbuffers,
anddiolformationoftheformylglycine[44]. Atthesametime,thepossibilityofusingthisenzyme
strategyineukaryoticandprokaryoticenvironments, aswellasbothinvitroandincells, andthe
numberofaldehydegroup-basedcouplingchemistries,e.g.,hydrazino–iso–Pictet–Spengler,theWittig
reactionortrapped-Knoevenagel,showitsflexibility.
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the reaction is only depicted in one heavy chain. R represents a moiety with an aldehyde reactive
thereactionisonlydepictedinoneheavychain. Rrepresentsamoietywithanaldehydereactive
function capable of coupling via, e.g., hydrazino-iso-Pictet-Spengler Ligation, trapped Knoevenagel
functioncapableofcouplingvia,e.g.,hydrazino-iso-Pictet-SpenglerLigation,trappedKnoevenagel
Ligation, or Wittig reaction.
Ligation,orWittigreaction.
2.2. Sortases
2.2. Sortases
Gram positive bacteria produce sortases for the attachment of surface proteins on pentaglycine
Grampositivebacteriaproducesortasesfortheattachmentofsurfaceproteinsonpentaglycine
structures of the peptidoglycane cell wall [47]. Staphylococcus aureus sortase A (srtA) is the most
structures of the peptidoglycane cell wall [47]. Staphylococcus aureus sortase A (srtA) is the most
frequently used enzyme for site-specific protein labelling with this transpeptidase reaction (Figure 3).
frequentlyusedenzymeforsite-specificproteinlabellingwiththistranspeptidasereaction(Figure3).
Its natural substrate recognition motif is the pentapeptide LPXTG, where X is usually glutamic acid,
Its natural substrate recognition motif is the pentapeptide LPXTG, where X is usually glutamic
besides aspartic acid or lysine [48]. In a ping-pong hydrolytic shunt-like mechanism, srtA binds the
acid, besides aspartic acid or lysine [48]. In a ping-pong hydrolytic shunt-like mechanism, srtA
LPXTG substrate, hydrolyzes the backbone between threonine and glycine, and generates an acyl-
bindstheLPXTGsubstrate,hydrolyzesthebackbonebetweenthreonineandglycine,andgenerates
enzyme intermediate. The terminal amine of the oligoglycine then acts as a nucleophile, resulting in an
an acyl-enzyme intermediate. The terminal amine of the oligoglycine then acts as a nucleophile,
amide bond formation with the C-terminal threonine of the substrate [49,50]. The nucleophilic tag can
resulting in an amide bond formation with the C-terminal threonine of the substrate [49,50].
be as short as two or three glycines for the protein ligation to work, making it popular for protein–
The nucleophilic tag can be as short as two or three glycines for the protein ligation to work,
peptide ligation [51] with biologically as well as chemically synthetized compounds. This has been
makingitpopularforprotein–peptideligation[51]withbiologicallyaswellaschemicallysynthetized
successfully demonstrated by either using LPXTG or Gn at the N- or C-terminus with in vitro
compounds. This has been successfully demonstrated by either using LPXTG or G at the N- or
modification of purified recombinant proteins and on living cells [52–54]. Attempting to cnircumvent the
C-terminuswithinvitromodificationofpurifiedrecombinantproteinsandonlivingcells[52–54].
limitation to either terminus Antos et al. introduced a dual labeling strategy featuring Streptococcus
AttemptingtocircumventthelimitationtoeitherterminusAntosetal. introducedaduallabeling
pyogenes sortase A. This sortase A is capable of accepting alanines as nucleophiles. The newly ligated C-
strategyfeaturingStreptococcuspyogenessortaseA.ThissortaseAiscapableofacceptingalaninesas
terminus of the protein will therefore be no substrate for srtA in the second modification step. In
nucleophiles. ThenewlyligatedC-terminusoftheproteinwillthereforebenosubstrateforsrtAin
combination with srtA twofold modifications via distinct reactions at N- and C-terminus can be
thesecondmodificationstep. IncombinationwithsrtAtwofoldmodificationsviadistinctreactions
conducted [55]. Most of srtA’s use in antibody systems has revolved around diagnostic approaches with
atN-andC-terminuscanbeconducted[55]. MostofsrtA’suseinantibodysystemshasrevolved
single chain antibody fragments derivatized with fluorescent or radioactive markers [56–61].
arounddiagnosticapproacheswithsinglechainantibodyfragmentsderivatizedwithfluorescentor
Kornberger and Skerra fused a whole protein, the plant toxin gelonin, to the trastuzumab-Fab with an
radioactivemarkers[56–61]. KornbergerandSkerrafusedawholeprotein,theplanttoxingelonin,
srtA approach, yielding 50% conversion [62]. Nanobodies have likewise been effectively labeled with
to the trastuzumab-Fab with an srtA approach, yielding 50% conversion [62]. Nanobodies have
fluorophores and cytotoxic payloads [63,64]. Due to the limitation to modifications of either terminus
olifk tehwei staerbgeeet nperoffteecitniv, serltyAla hbaesl endowt bitehenfl uthoero pprheoferrerseadn cdhcoyictoe tfooxri cApDaCyl goeandesr[a6t3io,6n4.] C.Donuseidtoerthinegl itmheit saoti ofanr
ptoremfeordreifidc aDtiAonRs ooff efiothuerr, tBeremerilni uest oaflt.h ecotaurlgde tpprorodtueicne, sKrtaAdchyalsa-n oatnbde eAndthceetprirse-fseimrreildarc haonitciebofodrieAsD bCy
sgeepnaerraatteiloyn f.uCsionngs imdaeyritnagnstihnee-s oanfdar apurreisfteartriend-oDligAoRgloyfcifnoeu rt,oB teheer lliigehtta aln. dco hueladvpyr cohdauince oKf tardacsytulaz-uamnadb
Aanddc ebtrreisn-tsuimximilaarba [n6t5i]b. oTdhieeys baychsieepvaerda taerloyufnuds i8n0g%m taoyxitnan csoiunpe-lianngd, caourrriesstpaotinnd-oinligg otog lay cDinAeRt ooft hroeuligghhlyt
3a.n2d. Wheaagvnyerc ehta ainl. corfetartaesdtu bzisupmecaibfica nandtibbroedniteusx bimy laibga[t6i5n]g. tTwhoe yfualcl hanietvibeoddaierso uwnitdh 8a0 c%omtobxiinnedco sutrpaltienggy,
ocof rsroerstpaosen dreinacgtitoona aDndA Rcliocfk rcohuegmhilsytr3y.2 [.6W6].a gInn ethree tmael.acnrteimatee,d thbeis preepceifirtcoiarnet iobfo tdhiee smbeythloigda thinasg btweeon
efuxltlenandteidbo dbiye sdwivitehrsaifcyoinmgb itnheed tsatrga t[e6g7y] oafnsdo rtsaoslevrineagc tsioonluabnilditycl icisksuchese mainstdr ys[i6d6e] .rIenatchtieomnse a[n56ti,m68e],.
Fthuertrheepremrtoorier,e doefptshipeempteidtheos dhahvaes bbeeeenn inetxrtoednudceedd bays sduivbestrrsaitfeysin fogrt Nhe-tteargm[i6n7a]l panrodtesionl vminogdisfioclautbioinli ttyo
aisdsdureessas nrdevseirdseibrieliatcyt ioofn tsh[e5 r6e,6a8c]t.ioFnu [r6th9]e.r Tmhoorueg,dh ecposmippeaprtaibdleys hhiagvhe ebnezeynmine tcroondcuecnetdraatisosnusb asrter anteesedfoedr
aNn-dte trhmei nmaeltphroodte iisn limmoitdeidfi ctaot iionn vtiotroad pdrroetsesinr emveordsiifbicilaittyioonfs,t hite hraesa cbteioenn [e6x9t]e.nTshivoeulyg husceodm fpoarr saubclycehssigfuhl
benioz-ycomnejucgoantcioenn.t rationsareneededandthemethodislimitedtoinvitroproteinmodifications,ithas
beenextensivelyusedforsuccessfulbio-conjugation.
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2.3. Transglutaminases
2.3. Transglutaminases
Transglutaminases catalyze an acyl-transfer reaction to the side chain of glutamine residues of their
Transglutaminasescatalyzeanacyl-transferreactiontothesidechainofglutamineresiduesof
protein substrate (Figure 4) [70]. Depending on the acyl donor, this can result in an amide bond between
theirproteinsubstrate(Figure4)[70]. Dependingontheacyldonor,thiscanresultinanamidebond
the glutamine and a primary amine, crosslinking between two proteins via a side chain lysine of the
between the glutamine and a primary amine, crosslinking between two proteins via a side chain
donor protein or the deamidation of glutamine [71]. For protein labelling purposes, the acyl-transfer
lysine of the donor protein or the deamidation of glutamine [71]. For protein labelling purposes,
reaction is preferred. While transglutaminases are specific for glutamine on the target protein, the
the acyl-transfer reaction is preferred. While transglutaminases are specific for glutamine on the
flexibility in terms of the amine containing acyl-donor offers diverse possibilities for modification. In
targetprotein,theflexibilityintermsoftheaminecontainingacyl-donoroffersdiversepossibilities
contrast to other protein-ligation strategies, the probe containing reactant is not required to be a peptide
formodification. Incontrasttootherprotein-ligationstrategies,theprobecontainingreactantisnot
and can simply be an alkylamine or an oligoamine as long as it contains a primary amine [72].
required to be a peptide and can simply be an alkylamine or an oligoamine as long as it contains
Eukaryotic transglutaminases used for protein modification are derived from the guinea pig liver as
aprimaryamine[72]. Eukaryotictransglutaminasesusedforproteinmodificationarederivedfrom
well as the human transglutaminase 2. However, the bacterial transglutaminase from Streptomyces
theguineapigliveraswellasthehumantransglutaminase2. However,thebacterialtransglutaminase
mobaraensis (MTG) is the enzyme of choice due to its independency from calcium and its lower
fromStreptomycesmobaraensis(MTG)istheenzymeofchoiceduetoitsindependencyfromcalcium
deamidation activity [73]. Transglutaminases display a certain promiscuity when it comes to the
and its lower deamidation activity [73]. Transglutaminases display a certain promiscuity when it
glutamine containing recognition sequence, which has spawned a search for a universal minimal tag.
comestotheglutaminecontainingrecognitionsequence,whichhasspawnedasearchforauniversal
Besides phage display derived peptides, different heptapeptide tags with hydrophobic residues N-
minimaltag. Besidesphagedisplayderivedpeptides,differentheptapeptidetagswithhydrophobic
terminal to the central glutamine have been used [74–76]. Farias et al. shortened the tag to an LLQGA
residues N-terminal to the central glutamine have been used [74–76]. Farias et al. shortened the
motif [77]. A novel strategy was presented by Siegmund et al. by generating a disulfide bridge stabilized
tag to an LLQGA motif [77]. A novel strategy was presented by Siegmund et al. by generating
handle with an exposed glutamine modelled on a natural MTG substrate reaching ligation efficiencies
adisulfidebridgestabilizedhandlewithanexposedglutaminemodelledonanaturalMTGsubstrate
of 85% [78]. They successfully biotinylated the therapeutic antibody cetuximab, showing the
reaching ligation efficiencies of 85% [78]. They successfully biotinylated the therapeutic antibody
amenability of the tag for possible antibody–drug conjugation. The feasibility of the generation of
cetuximab,showingtheamenabilityofthetagforpossibleantibody–drugconjugation. Thefeasibility
antibody conjugates with the aid of MTG has been proven repeatedly at different surface accessible sites
ofthegenerationofantibodyconjugateswiththeaidofMTGhasbeenprovenrepeatedlyatdifferent
[6,79–81]. The product yields for the latter approaches vary between 80% and 90%. The promiscuity of
surface accessible sites [6,79–81]. The product yields for the latter approaches vary between 80%
the enzyme may generate side products, like deamidated glutamine or a transesteferification product,
and90%. Thepromiscuityoftheenzymemaygeneratesideproducts,likedeamidatedglutamineor
and the need for experimental verification of sites for tag introduction remains. MTG catalysis is mainly
atransesteferificationproduct,andtheneedforexperimentalverificationofsitesfortagintroduction
used in vitro, but has been done on cells as well [75]. By combining transglutaminase catalysis with
remains. MTGcatalysisismainlyusedinvitro,buthasbeendoneoncellsaswell[75]. Bycombining
engineered cysteine maleimide conjugation, Puthenveetil et al. created a dually labeled antibody
transglutaminasecatalysiswithengineeredcysteinemaleimideconjugation,Puthenveetiletal. created
reaching a DAR of 4 [82].
aduallylabeledantibodyreachingaDARof4[82].
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2.4. Inteins
2.4. Inteins
Inteins are protein sequences that can cleave themselves off to form a mature protein. Since they
Inteinsareproteinsequencesthatcancleavethemselvesofftoformamatureprotein. Sincethey
bear an internal endoprotease activity, their application is not an enzyme strategy in the classical sense.
bear an internal endoprotease activity, their application is not an enzyme strategy in the classical
For site-specific protein modification, inteins are deployed in variations of two different ligation
sense. Forsite-specificproteinmodification,inteinsaredeployedinvariationsoftwodifferentligation
strategies, expressed protein ligation (EPL) (Figure 5) and protein trans-splicing (PTS) (Figure 6). The
strategies, expressed protein ligation (EPL) (Figure 5) and protein trans-splicing (PTS) (Figure 6).
reaction mechanism involves the formation of a thioester intermediate. It is formed by the N-terminal
The reaction mechanism involves the formation of a thioester intermediate. It is formed by the
cysteine of the intein. Subsequently, the transfer of the thioester to an intramolecular cysteine
N-terminal cysteine of the intein. Subsequently, the transfer of the thioester to an intramolecular
downstream of the intein sequence and the release of the intein, through the generation of a peptide
cysteine downstream of the intein sequence and the release of the intein, through the generation
bond between the exteins, completes the reaction. Muir et al. described EPL in 1998, therein exploiting
of a peptide bond between the exteins, completes the reaction. Muir et al. described EPL in 1998,
an intein mutation capable of inhibiting the reaction downstream of the thioester intermediate and
thereinexploitinganinteinmutationcapableofinhibitingthereactiondownstreamofthethioester
intercepting it with a synthetic peptide containing an N-terminal cysteine [83,84]. The synthetic peptide
intermediateandinterceptingitwithasyntheticpeptidecontaininganN-terminalcysteine[83,84].
can in turn be a carrier for C-terminal site-specific protein modification. This has been successfully
ThesyntheticpeptidecaninturnbeacarrierforC-terminalsite-specificproteinmodification. This
applied for fluorescein coupling and PEGylation [85,86]. EPL has also been used to generate monoclonal
has been successfully applied for fluorescein coupling and PEGylation [85,86]. EPL has also been
antibody conjugates [87,88]. Unfavorably for ADC generation reducing conditions have to be
usedtogeneratemonoclonalantibodyconjugates[87,88]. UnfavorablyforADCgenerationreducing
maintained to prevent oxidation of the accepting cysteine. For antibodies, Möhlmann et al. could
conditions have to be maintained to prevent oxidation of the accepting cysteine. For antibodies,
maintain intact interchain disulfide bonds and retained binding properties [87], albeit with a product
Möhlmannetal. couldmaintainintactinterchaindisulfidebondsandretainedbindingproperties[87],
yield of 60%. PTS on the other hand is carried out by split inteins, first characterized in Synechocystis sp.
albeit with a product yield of 60%. PTS on the other hand is carried out by split inteins, first
PCC6803. This example consisted of separately expressed N- and C-inteins with high affinity towards
characterized in Synechocystis sp. PCC6803. This example consisted of separately expressed N-
each other fused to the N- and C-terminal half of the DnaE protein respectively. Once they assembled,
andC-inteinswithhighaffinitytowardseachotherfusedtotheN-andC-terminalhalfoftheDnaE
self-cleavage similar to inteins took place forming a DnaE fusion protein [89]. This strategy also has
proteinrespectively. Oncetheyassembled,self-cleavagesimilartointeinstookplaceformingaDnaE
been used in vitro and in the cellular environment with a protein transduction domain fused to the C-
fusionprotein[89]. Thisstrategyalsohasbeenusedinvitroandinthecellularenvironmentwith
intein to enter the cell for labeling of the intracellular protein equipped with the N-intein [90]. The
aproteintransductiondomainfusedtotheC-inteintoenterthecellforlabelingoftheintracellular
comparatively small size of the C-intein enables coupling of synthetic peptides similar to EPL. Product
proteinequippedwiththeN-intein[90]. ThecomparativelysmallsizeoftheC-inteinenablescoupling
yields of up to 80% could be achieved using PTS with antibodies. Discovery and engineering of shorter
ofsyntheticpeptidessimilartoEPL.Productyieldsofupto80%couldbeachievedusingPTSwith
C-intein (6 aa) and N-intein (12 aa) sequences has extended the applicability of the method to N-terminal
antibodies. DiscoveryandengineeringofshorterC-intein(6aa)andN-intein(12aa)sequenceshas
protein modification [91,92]. Faster acting split inteins and inteins less sequence dependent on the extein
extendedtheapplicabilityof themethodtoN-terminalproteinmodification[91,92]. Fasteracting
sequence have since emerged [93,94]. For comfortable labeling with thiol reactive probes the Cys-Tag
splitinteinsandinteinslesssequencedependentontheexteinsequencehavesinceemerged[93,94].
(EAGSCS) has been employed with split inteins [95]. Recently Bachman et al. modified nanobodies N-
ForcomfortablelabelingwiththiolreactiveprobestheCys-Tag(EAGSCS)hasbeenemployedwith
terminally with the help of split inteins and the Cys-Tag [96]. Han et al. generated bispecific IgG
splitinteins[95]. RecentlyBachmanetal. modifiednanobodiesN-terminallywiththehelpofsplit
antibodies by producing full dimeric antibodies with a C-Intein instead of a second Fab-fragment. The
inteinsandtheCys-Tag[96]. Hanetal. generatedbispecificIgGantibodiesbyproducingfulldimeric
Fab-fragment of second specificity was engineered with an N-intein and fused to the first antibody via
antibodieswithaC-InteininsteadofasecondFab-fragment. TheFab-fragmentofsecondspecificity
PTS [97]. Downsides of both EPL and PTS are the long terminal inteins of approximately 100–150 aa
wasengineeredwithanN-inteinandfusedtothefirstantibodyviaPTS[97]. DownsidesofbothEPL
that have to be appended to the protein of interest. While this might not be an issue for whole antibodies,
andPTSarethelongterminalinteinsofapproximately100–150aathathavetobeappendedtothe
opr rhoatse ibneeonf isnhtoewrens tt.oW beh mileatnhaigsemabigleh ftonr oVtHbHe,a fnori sssoumeef oarntwibhoodlye amnitmibeotdicise sth,iosr choausldb eleeands thoo dwifnfictoulbtiees
in production.
manageableforV H,forsomeantibodymimeticsthiscouldleadtodifficultiesinproduction.
H
Antibodies 2018,7,4 7of19
AAnnttiibbooddiieess 22001188,, 77,, 44 77 ooff 1188

FFFiiiggguuurrreee 555... SSSccchhheeemmmeee ooofff aaannn aaannntttiiibbbooodddyyy mmmooodddiiififfiicccaaatttiiiooonnn b bbyyye eexxxppprrereessssseseedddp pprorrotoettieeniinnli glliiaggtaaiottiinoo.nnM.. MMESEENSSNNAAAis iitssh ttehhaeeb aabbbrbebvrreeiavvtiiiaaottniioonn
fffooorrr 222---mmmeeerrrcccaaappptttoooeeettthhhaaannneeesssuuulllfffooonnnaaattteee...

FFFiigigguuurrreee6 66...S SSccchhheeemmmeeeo oofffa aannna aannnttitbiibbooodddyyym mmooodddifiiiffciiccaaatittoiioonnnb bbyyyt rttraraannnss-ss--spspplillciicicniinnggg...
222...555... TTTuuubbbuuullliiinnn TTTyyyrrrooosssiiinnneee LLLiiigggaaassseee
CCCaaatttaaalllyyyzzzeeeddd bbbyyy tttuuubbbuuulliilnnin ttytyyrrrooosssiinnineee lliilggigaaassseee ((TT(TTTTLLL)),,) ,ααα--tt-uutubbbuuulliilnnin iissi smmmooodddiiffiiifieedded pppoosostts--ttt-rrtaarnnasnsllsaalttaiiotoinnoaanllallyyll ybbyyb yCC--
ttCeerr-mmteriinnmaailln aaattlttaaacctthhammcheemnntte onofft aao fttyyarrtooyssriionnseei n[[99e88[]]9.. 8 BB].aannBeearrnjjeeeerej eeeett aaellt.. asslhh.ooswwhoeewdd ettdhhaattth TTatTTTLLT aaLccccaeecppctetssp tttyysrrtooyssriionnseein ddeeedrriievvraaivttiiavvteeivss ellisikkee
fflooikrrmmeyyfoll--rttmyyrryoolss-itinnyeero aasssin aae ssauusbbassttsrruaattbees t[[r99a99t]]e.. [FF9oo9rr] .tthhFeeo ruusstehe eiinnu sooetthhienerr oppthrrooettreeiipnnrsso,, tSeSiccnhhsuu,mmScaahcchhueemrr aeectth aaellr.. ellaatbbaeell.leeddla baae 11le33d aaaa
ppaeep1p3ttiiddaeae ((pVVeDDpSStiVVdEEeGG(VEEDEEEESGVGEEEEGE))E,, mEmEiimmGEiiccEkk)ii,nnmgg ttihhmeei cCCk--ittneerrgmmthiinneuuCss o-otffe ααrm--ttuuinbbuuuslliionnf,, wwα-iittthhu bTTuTTlLLin ((,FFwiiggiuuthrreeT 77T))..L TTh(hFiissig ssuoor--eccaa7llll)ee.dd
TTTuuhbbis--TTsaaogg-c awwllaaesds Tiinnuttbrro-oTddauugccweedda siinninttotor oppdrruooctteeediinnissn tffooorrp reeofftffeiicciniieesnnftto rCCe--fttfieercrmmieniinntaaCll -tmmeroomddiiinffiiacclaatmtiiooonnd ioofiffc addtiiioffffneerroeefnndtt ifppferrrooettneeiitnnss
iipnnrccolluuteddiniinnsggi n aaclnnu daainnnttgii--aGGnFFaPPn tnni-aaGnnFooPbboonddayny o [[b11o00d00y]],, [1aa0cc0hh]ii,eeavvciihnnigge vaainllmmgooaslsmtt offuustllllf uccloolnncovvneevrrsseiiroosnnio naaffattfeetrre r555 hhh ooofffi niicnnuccbuuabbtaaiottiinoo.nn..
SSSuuubbbssstttrrraaattteee fffllleeexxxiiibbbiiillliiitttyyy ooofff TTTTTTLLL wwwaaasss fffuuurrrttthhheeerrr i iilllllluuussstttrrraaatteteedddw wwitiihtthh,,,a aammmooonnngggo oothtthhereesrr,ssu,, ununnnanntaauttruuarrlaaall naadnnddb ibboiitoointtii-nnco--ccnootnnaittnaaiiinnngiinngg
aaammmiiinnnooo aaaccciiidddsss bbbrrroooaaadddeeennniiinnnggg ttthhheee lllaaabbbeeellliiinnnggg c cchhheeemmmiisissttrtrryyys sspppeeeccctrtturruummm[ 1[[1010101]1.]]..A AAltllhtthhooouuuggghhhli lmliimmitieittdeeddto ttoom mmodooiddfiiicffaiicctaiaottiniooonnf ootfhf tethhee
CCC---ttteeerrrmmmiiinnnuuusss aaannnddd nnnooottt yyyeeettt uuussseeeddd iiinnn cccooonnnttteeexxxttt ooofff fffuuullllll a aannntttiibibbooodddiieieesss,,,T TTTTTLLLb bbeeeaaarsrrsst htthheeep ppoootettneenntittaiilaafll o ffroorrA AADDDCCCg eggneeennreearrtaaiottiinoo.nn..
TTThhheee tttuuubbb tttaaaggg’’’sss ppprrreeedddooommmiiinnnaaannnttt pppooolllaaarrriiitttyyy cccooouuulllddd p pprrroooooofffa aadddvvvaaannntattaagggeeeooouuusssk kkeeeepeeppiniinngggin iinnm mminiidnnddth ttehheeso sslooullbuuibbliiitllyiittypy rpporrboolebbmlleemsmss
wwwiiittthhh mmmaaaiiinnnlllyyy hhhyyydddrrroooppphhhooobbbiiiccc tttoooxxxiiinnnsss ddduuurrriiinnnggg AAADDDCCC gggeeennneeerrraaatttiiiooonnn...
Antibodies 2018,7,4 8of19
Antibodies 2018, 7, 4 8 of 18
Antibodies 2018, 7, 4 8 of 18

Figure 7. Scheme of an antibody modification by tubulin tyrosine ligase.
FFiigguurree7 7.. SScchheemmee ooff aann aannttiibbooddyym mooddiififcicaattioionnb byyt tuubbuulilnint ytyrroossinineel ilgigaassee..
2.6. Proteases (Trypsiligase and Subtiligase)
22.6.6. .PPrrootteeaasseess ((TTrryyppssiilliiggaassee aanndd SSuubbttiilliiggaassee))
Proteases have been utilized for protein semi-synthesis for a long time and recently also for protein
PPrrootteeaasseess hhaavve ebebeene nutuiltiizleizde fdorf oprroptreointe sinemsei-msyi-nstyhnetshise fsoisr afo lronagl otinmget aimnde raencdenrtelyc eanlstoly foarls porofoterin
labeling. subtiligase, a variant of the serine protease subtilisin, though currently not used for ADC
lpabroetliening. lasubebltiinligg.asseu, bat ilvigaraisaen,ta ovf atrhiaen steorifnteh eprsoetreinaseep sruobtetailsiseinsu, bthtioliusginh, cthuorruegnhtlycu nrroetn utlsyedn oftoru sAeDdC
generation, is able to catalyze peptide formation, in addition to its hydrolytic activity [102]. Subtiligase
gfeonreAraDtiCong,e ins earbalteio tno, ciastaablylezeto pceapttaildyez efoprmepattiidoenf, oinrm adatdioitnio,nin toa ditdsi thiyodnrtoolyittsich aycdtirvoiltyyt i[c10a2ct]i. vSiutybt[i1l0ig2a].se
has been improved by phage display for ligase activity [103]. Nonetheless, the problem of increased
hSausb btieleigna simehparosvbeede nbyim pphraogvee ddibspylpayh afgoer dliigsapslea yacftoirviltiyg a[s1e03a]c.t iNviotnye[t1h0e3l]e.sNs, otnheet hperolebslse,mth oefp irnocbrleeamsed
hydrolysis compared to ligase activity persists. Liebscher et al. engineered trypsiligase, a trypsin variant
hoyfdinroclryesaisse cdomhypdarroeldy stios lcigoamsep aarcetidvittoy lpigearssiestasc.t Livieitbyscpheerrs iestt sa.l.L einebgsincheeerreedt tarly.pesnigliignaeseer,e ad trtryyppssinil ivgaarsiea,nt
capable of terminal modification of proteins by cutting the short recognition sequence YRH and ligating
caaptraybplsei nofv taerrimanintacal pmaobdleifoicfatteiromn ionfa plmrootediinfisc batyi ocnutotfinpgr othteei nshsobryt cruecttoingngitthioens hseoqrtureenccoeg YniRtiHon asnedq luigenatcieng
proteins or peptides with an N-terminal RH moiety under the requirement of Zn2+ (Figure 8) [104]. C-
pYrRotHeinasn dor lpigeapttiindgesp wroittehi nasn oNr-pteerpmtiidneasl RwHit hmaonietNy- utenrdmeirn tahleR rHequmiroeimetyenut nodf eZrnt2h+ e(Friegquureir e8m) [e1n0t4]o. fC-
terminal modification with PEG and fluorescein could also be achieved for an anti-Her2 Fab fragment,
tZernm2+in(aFli gmuordei8fi)c[a1t0io4n]. wCi-tther PmEiGna alnmdo fdluifiocreasticoeninw ciotuhldP EaGlsoa nbde aflcuhoierevsecde ifnorc oaun ladnatil-sHoebre2 aFcahbi efvraegdmfoernt,
as well as coupling of the DM1 toxin with a yield of approximately 70% via a successive click reaction
aasn waenltli -aHs ecro2uFpalbinfgra ogfm theen tD,aMs1w teollxains cwoiuthp lain ygieolfdt hoef DapMp1rotxoixminawteiltyh 7a0y%ie vldiao af asupcpcreosxsiimvea tcelliyck7 0r%eacvtiiaon
[105,106]. Though trypsiligase is only applicable to the C-terminus of the antibody, the only 3 aa long
[a10s5u,1cc0e6s]s. iTvheoculigchk rtreyapctsiiolinga[1se0 5i,s1 0o6n]l.yT ahpopulgichabtrley ptosi ltihgea sCe-tiesromnilnyuasp opfl itchaeb laenttoibtohdeyC, -thteer moninlyu s3 oafa tlhoeng
tag presents an advantage over most other mentioned enzyme strategies.
taangt pibroedseyn,ttsh eano naldyv3anataalgoen ogvtearg mproesste ontthsearn maednvtaionntaegde eonvzeyrmmeo ssttraottehgeirems. entionedenzymestrategies.

Figure 8. Scheme of an antibody modification by trypsiligase.
FFiigguurree8 8.. SScchheemmee ooffa anna annttiibbooddyym mooddiififcicaattioionnb byyt rtryyppssiliilgigaassee..
2.7. Phosphopantetheinyl Transferase
22.7.7. .PPhhoosspphhooppaanntteetthheeininyyl lTTrraannssffeerraassee
Phosphopantetheinyl transferases transfer the phosphopantetheinyl moiety of coenzyme A to a
PPhhoosspphhooppaannteteththeeininyyl ltrtraannssfeferraasseess ttrraannssfeferr tthhee pphhoosspphhooppaanntteetthheeininyyl lmmooiieettyy ooff ccooeennzzyymmee AA ttoo a
specific serine residue in the target protein (Figure 9) [107]. Specifically, the Sfp Phosphopantetheinyl
sapsepciefcicifi scesreinrien reerseisdiudeu einin ththe etatargrgeet tpprrootteeiinn ((FFiigguurree 99)) [[110077]].. SSppeecciifificcaallllyy,,t htheeS fSpfpP hPohsopshpohpoapnatnettehtehineiynlyl
transferase (Sfp) from Bacillus subtilis is valued for protein labeling. Several peptide motives recognized
ttrraannssffeerraassee ((SSffpp)) ffrroomm BBaacciilllluuss ssuubbttiilliiss iiss vvaalluueeddf foorrp prrooteteininl alabbeelilningg..S Seevverearlapl pepeptitdideem motoivtievserse rceocgonginzeizded
by Sfp were found, among others the ybbR-tag (DSLEFIASKLA). The enzyme is able to transfer small
bbyy SSffpp wweerree ffoouunndd,, aammoonngg ootthheerrss tthhee yybbbbRR--ttaagg( (DDSSLLEEFFIAIASSKKLLAA).).T Thheee nenzyzymmeeis isa balbeleto totr atrnasnfesfresrm smallall
molecule CoA conjugates to the tag serine positioned at the N- and C-terminus and also in flexible loops
molecule CoA conjugates to the tag serine positioned at the N- and C-terminus and also in flexible loops
of the target protein [108–110]. Grünewald et al. recently generated antibody–drug conjugates site-
of the target protein [108–110]. Grünewald et al. recently generated antibody–drug conjugates site-
Antibodies 2018,7,4 9of19
AmAnntoitbilboeodcdieiuse sl2 e2001C188,o ,7 A7, ,4 4 c onjugatestothetagserinepositionedattheN-andC-terminusandalsoinflexi9b9 ol oef f1 188
loopsofthetargetprotein[108–110]. Grünewaldetal. recentlygeneratedantibody–drugconjugates
ssspipeteecc-isfiifpciceaacllliylfiy cl aalabllbeyelelledadb a eatl ted ddififfaefterredeninftf tel olroeoonpptss li onino tp htshee icn cootnhnsestatacnnot ntr sretegaginoiotnnr o eofg fit ortranasstoutfuzzturumamsataubbz w uwmitihtah bt hthwee ih thheelpltph o eof fhd deilfipffeferorefennt t
SdSfpfipf ft eatragegsns at anSndfdp a atcachghiseievaveneddd a aatc tlh elieaeasvste t9 d955a%%t lc ecoaonsnvtve9er5rs%sioioncno [ n1[1v11e11r].]s .i on[111].

FFiFiggiuguurreree 99 .9. S.S cSchchheememmeee oo fof faa nann aa nantntiitbbiobododdyyy mm moododdiififiicfciacatatiiotoinonnb b ybyyp p hphohosospsphphohopopapanantnteettethhteheieinniynylylt ltr rtaranansnsfsfeefrerarasasese.e. .
222.8..88. ..S SSpppyyyLLLigiiggaaassseee
ZZZaaakkkeeerrri ii eeett t aaall.l. . eeennngggiininneeeeeerrreeeddd tththheee sssooo--cc-caaallllleeleddd S SSpppyyyTTTaagagg f frrforomomm th thteheeC CnCnanBaaB2B2d2 oddmoomamianainion f oofif fb frifboibrnroeoncneteicncti-tnbin-inb-bidninidndigningg
ppprrroootteteeiniinn ffrfrrooommm SSSttrtrreeepppttotoocccoooccccccuuusss pppyyyooogggeeennneeesss [ [11[1111222]]..] .T TThhheee e enengngiginnineeeeerererededd S SpSpypyCyCCaatatccthchehererr is isias baableblelte o totmo memdeiedaditaieateitse o isipsoeopppetepipdtitedidee
ffofoorrrmmmaaatittoiioonnn o oof ffa aa s sisdiiddeee c chchhaaianiinn l yllysysisniinnee ea anandnd da asaspspaparararagagigniniene e( F(F(iFgigiugurureer 1e 1010)0.) .)T .ThThisihs ip sprropotretoeitnien–i–npp–eepppetitpdidteied l ielgiglaiatgitoaiotnin om nmemeththeotodhd ow dwaass
rwreefaifnisnereded fib bnyye Fd FieiberyreerFr ei eetr ta ealr.l .eb btyya s lsp.plbiltyittitsnipnggl it thtthiene ge entnzhzyeymmenee zi nyintmoto et htihnrreteoee tp hpararertests, p,S SapprytysTT,aSagpg ( yA(TAHaHgIVI(VAMMHVVIDVDAMAYVYKKDPPATTYKKK),) P,K TK-KT-Ta)a,gg, ,
(KA(A-TTTHaHgIKI,K(FAFSTSKKHRRIDKD)F )a SanKnddR S DSpp)yyaLLnigidgaaSsspee,y ,w Lwhighiacichshe i ,sisw a abhblielce ht otoi sf ufausbsele et hthteoe t fwtuwosoe t attahggess. t.Tw Thhoeeytya u gussse.edTd th htehiysis su stsrtreaadtetegthgyyi st otso tp rpoaoltyelygmmyeetrorizizee
apafffoifbliybomoddieeiresis z[ e1[11a13f3]fi,] b,a aonndddi eS Ssiei[eg1ge1em3m]u,uannnddd e eSt tia ealg.l .ea ampppuplnileidedd ei tti taf oflo.rr at phthpee lg igeeednneiertrafatoitroiontnh o eof gfA eADnDeCrCsas t[ i1[o11n144]o.] f.B ABooDththC S sSpp[y1yT1Ta4ag]g. a Banondtdh K K--
TSTapagyg T aaarrgee a ssnmmdaaKlll,l- ,T sasogo tahtrhiesis s smstrtaralatle,tegsgoyy t hccaiasnns tereaaastsieligylyy bcbeae n aapepappsliilleiyeddb teotoa pbbpootlihteh d pptrorootbetoeinitnh tpeterrormmteiniinni.i t. eFFrumurrtihtnhei.errmFmuorortrehe,e ,r tmhtheoe r leolo,ww
rtreheaaecctlitooiownn rteetaemmctpipoeernraatteutumrreepss,e ,r wawthuhirlieele s ,sswtitlihll li lrereeasactichlhlinirnegga c8h800i%n%g c8co0on%nvvecerorsnsioivonen r swiwoitnihthw wiwthhhowolelhe oaalnentitabinbotodidbyoy d ssyuubsbsustbrtrasatterteass,t e, asa,rree
daderesesiridraaebbslielre a fbofolrer AfAoDrDCAC D pprCroodpduruocctditoiuoncnt. i. oTTnhh.ee T ehexxecceeesxsscs eeesnnszzyeynmmzeye m nneeeeenddeeeeddd efofdorr f tohtrheet h cceooncnvovenervrsesioirosnni o rnreemrmeamainiansis n asa taatartraggreget te ftoforr
ofopoptritmiompizitziamatitioizonan.t .i on.

FFiFiggiugurureree 11 010.0. S.S cSchchheememmeee oo fof afa nann aa nantntiitbbiobododdyyy mm moododidififiicfciacatatiiotoinonnb b ybyyS S pSpypyLyLiLiggiagasasese.e. T.T MTMMAAAOOO,, T,T rTrimrimimeetehthtyhylyalalmammininieneNe N N-o-o-xoxixdidiede.e. .
22.9.9. .O Oththeerr S Strtraateteggieiess
TThheerree aarree sseevveerraal l fufurrththeerr eennzzyymmee sstrtraateteggieiess ththaat t wweerree aaddaapptetedd toto aanntitbibooddyy––ddrruugg ccoonnjujuggaatitoionn oorr
ccoouuldld b bee u usseefuful li nin t hthisis c coonntetexxt tb baasseedd o onn t htheeirir v vaalildidaatetedd a abbiliiltiyty t oto l ilgigaatete p prrooteteininss. .L Leeee e et ta al.l .c coonnjujuggaatetedd a ann
aanntit-iE-EGGFFRR r reeppeebbooddyy b byy p prreennyylalatitoionn o of fa a c cyyssteteininee i nin a a C CaaaaXX-m-mootitfi fw witihth g geerraannyyl lk keetotonnee p pyyrroopphhoosspphhaatete
aanndd s suucccceessssivivee o oxximimee l ilgigaatitoionn w witihth a ammininooooxxyylalatetedd M MMMAAFF, ,r reeaacchhiningg a almlmoosst tf ufulll lc coonnvveerrssioionn [ 1[11155].] .L Lipipooicic
aaccidid l ilgigaassee t atagggginingg o of fp prrooteteininss a anndd s sitiete-s-sppeeccifiifcic l alabbeelilningg w witihth b bioiotitnin l ilgigaassee c coouuldld b bee u usseefuful lf oforr a anntitbibooddyy--
Antibodies 2018,7,4 10of19
2.9. OtherStrategies
There are several further enzyme strategies that were adapted to antibody–drug conjugation
or could be useful in this context based on their validated ability to ligate proteins. Lee et al.
conjugatedananti-EGFRrepebodybyprenylationofacysteineinaCaaX-motifwithgeranylketone
pyrophosphate and successive oxime ligation with aminooxylated MMAF, reaching almost full
conversion[115]. Lipoicacidligasetaggingofproteinsandsite-specificlabelingwithbiotinligase
couldbeusefulforantibody-drugconjugationaswell[116–118]. EscherichiacolibiotinligaseBirA
has been used for protein labeling by Chen et al. with yields of up to 50% [117]. Comparably
smallamountsofenzymeareneeded,yetthe15aatagislongerthanthatusedinsimilarstrategies.
Theligasecanattachabiotinanalogtoalysineinthetagandtheketonegroupcanthenbelabeled
specifically. Heller et al. introduced a phosphocholination reaction for protein modification by
AnkX. This Legionella pneumophila enzyme recognizes the peptide sequence TITSSYYR and adds
aphosphocholinemoietyfromtaggedCDP-cholinetothesecondserine[119]. Conversionratesofup
to70%couldbereachedwithAnkX,thoughithasnotbeenusedwithantibodiesorantibodyfragment
andmimeticsthusfar. GlycoConnectisalabelingstrategydirectedatglycostructuresoftheantibody.
DerivatizationisachievedbyfirstlytrimmingN-glycanswithendoglycosidasetoexposeanN-acetyl
glucosamineandsecondlyattachaclickconjugatableN-acetylgalactosaminewithglycosyltransferase.
UsingGlycoConnect,vanGeeletal.couldeffectivelygeneratenearlyfullyconvertedantibody–drug
conjugates[120]. Eliminationoftheantibody’sN-glycosylationmighthaveaneffectonindividual
antibodiesregardingbloodclearanceorADCC,buttheimpactisingenerallessimportantforADCs.
Thelongincubationtimesat37◦Cand30◦Care,however,notidealforADCproduction.
3. Conclusions
Thesheernumberandcontinuousdevelopmentofnewlabelingstrategiesforantibody–drug
conjugates demonstrates the interest in and importance of the issue. Besides the enzyme-based
strategiesdiscussedhere,manyparallelconjugationmethodsarebeingpursued,liketheincorporation
ofunnaturalaminoacidsandthedesignofchemicallyspecificreactionsitesintheantibodyorantibody
mimetic.Duetothevariousenzymesavailableforproteinconjugation,chancesaregoodthatasuitable
approach for a given protein of interest can be found as described in Table 1. Different and often
shorttagsmaybeincorporatedwithoutdisruptingstructureandfunction. Problematicpropertiesof
antibodyconstructs,likestabilityorsolubility,canbeconsideredbytestingvariousmethods,which
differinbuffercomposition,additionalchemicalsteps,oreventheoptiontogeneratetheconjugation
readyantibodiesbyco-expressingtheenzyme. Mostoftheenzymesmentionedherehavealready
been used to generate homogenous antibodies at the laboratory scale. Nonetheless, it should be
mentioned that a perfect or one-size-fits-all strategy remains elusive. Possible drawbacks include
thedemandforexcessenzyme,reversibleandincompletereactions,orexclusivelyterminalligation.
Some of these issues have already been addressed and the different strategies could also go hand
in hand for sequential labeling or labeling at different sites, e.g., with a toxin and a fluorophore
orPEG.Onthetherapeuticside, acombinationofantibodiesandantibodymimetics, duetotheir
individualcharacteristics,isconceivableaswell.WhilecurrentclinicallyinvestigatedADCsaremainly
constructedusingengineeredcysteines,futuregenerationsofantibody–drugconjugateswilllikely
includechemo-enzymaticstrategies.