Peptide Tags and Unnatural Amino Acids

13 Pages·2016·1.99 MB·English

MolImagingBiol(2016)18:153Y165
DOI:10.1007/s11307-015-0920-y
*WorldMolecularImagingSociety,2016
PublishedOnline:11January2016
REVIEW ARTICLE
Site-Specifically Labeled Immunoconjugates
—
for Molecular Imaging Part 2: Peptide Tags
and Unnatural Amino Acids
Pierre Adumeau,1 Sai Kiran Sharma,1 Colleen Brent,1 Brian M. Zeglis 1,2
1DepartmentofChemistryandBiochemistry, HunterCollegeandtheGraduateCenteroftheCityUniversityofNewYork,413East69th
Street,NewYork,NY,10021,USA
2Department ofRadiology,MemorialSloan KetteringCancerCenter, 1275YorkAvenue,NewYork,NY,10065,USA
Abstract
Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly
becoming a critical component of modern precision medicine. Yet despite this promise, the
vast majority of these immunoconjugates are synthesized via the random coupling of amine-
reactive bifunctional probes to lysines within the antibody, a process that can result in
heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In
an effort to circumvent these issues, the last 5 years have played witness to a great deal of
research focused on the creation of effective strategies for the site-specific attachment of
payloads to antibodies. These chemoselective modification methods yield immunoconjugates
thataremorehomogenousandbetterdefinedthanconstructscreatedusingtraditionalsynthetic
approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to
exhibitsuperiorinvivobehaviorcomparedtotheirrandomlymodifiedcousins.Theover-arching
goalofthistwo-partreviewistoprovideabroadyetdetailedaccountofthevarioussite-specific
bioconjugation approaches that have been used to create immunoconjugates for positron
emission tomography (PET), single photon emission computed tomography (SPECT), and
fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on
themodificationofcysteineresiduesandthechemoenzymaticmanipulationofglycans.InPart
2, we will detail two families of bioconjugation approaches that leverage biochemical tools to
achieve site-specificity. First, we will discuss modification methods that employ peptide tags
either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And
second, we will examine bioconjugation strategiespredicated ontheincorporation of unnatural
or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare
the advantages and disadvantages of the modification strategies covered in both parts of the
reviewandoffer abrief discussionof theoverall directionof thefield.
Keywords:Positronemissiontomography,PET,Singlephotonemissiontomography,SPECT,
Fluorescence imaging, Near-infrared fluorescence imaging, Optical imaging, Click chemistry,
Site-specific conjugation, Site-selective conjugation, Bioconjugation, Bioorthogonal chemistry,
Glycoengineering,Proteinengineering,antibody,Antibodyfragment,Immunoglobulins,Peptide
tags, Unnaturalaminoacids, Non-canonicalaminoacids
Abbreviations:2-keto-Gal,2-Acetyl-2-deoxy-galactose;aaRS,AminoacyltRNAsynthase;ADC,
Antibody-drug conjugate; AGT, O6-Alkylguanine transferase; AzK, Azido-lysine; CEA,
Carcinoembryonic antigen; Db, Diabody; DBCO, Dibenzocyclooctyne; DFO, Desferrioxamine;
Correspondenceto:BrianZeglis;e-mail:[email protected]
154 P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging
DOTA, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid; DTPA,
Diethylenetriaminepentaacetic acid; EGFR, Epidermal growth factor receptor; FGE,
Formylglycine-generatingenzyme;FR-1,Framework-1region;HER2,Humanepidermalgrowth
factor receptor 2; HIPS, hydrazino-Pictet-Spengler; mAb, Monoclonal antibody; Mb, Minibody;
MET,Metallothionein;MMAD,MonomethylauristatinD;MMAF,MonomethylauristatinF;ncAA,
Non-canonicalaminoacid;NCS,Isothiocyanate;NHS,N-hydroxysuccinimide;NIRF,Near-infrared
fluorescence;OI,Opticalimaging;pAcF,p-Aceto-L-phenylalanine;pAzF,p-Azido-L-phenylalinine;PET,
Positronemissiontomography;PSMA,Prostatespecificmembraneantigen;scFv,Single-chain
variable fragment; sdAb, Single-domain antibody; SDS-PAGE, Sodium dodecyl sulfate
polyacrylamide gel electrophoresis; SPECT, Single photon computed tomography; SrtA,
Sortase;TGase,Transglutaminase; uAA,Unnaturalaminoacid
Introduction developmentoftechniquesforthesite-specificbioconjugation
ofcargoestoantibodies[8,11–13].Whileavarietyofdifferent
Overthepastfewdecades,antibodiesandantibodyfragments approacheshavebeendeveloped,allofthemprovidearouteto
have emerged as extremely effective tumor-targeting vectors betterdefinedandmorehomogeneousimmunoconjugates.Itis
for molecular imaging (Fig. 1) [1–5]. However, the importanttonotethatthisquestforchemoselectivityisnotan
bioconjugation strategies used to synthesize these academic issue, as a number of studies have shown that site-
immunoconjugatesleavemuchtobedesired.Thevastmajority specifically modified immunoconjugates exhibit improved in
of traditional bioconjugation approaches rely on reactions vivo behavior compared to their traditionally synthesized
between lysines in the antibody and bifunctional, amine- cousins[14–17].
reactivechelatorsorfluorophores(e.g.,benzylisothiocyanates Our over-arching goal in writing this two-part review is
orN-hydroxysuccinimidylesters;Fig.2a–b)[6,7].Critically, to provide the reader with a broad yet detailed guide to the
the presence of multiple lysines distributed throughout the different bioconjugation methods that have been applied to
immunoglobulin structure makes controlling the site and the creation of site-specifically labeled immunoconjugates
frequency of these conjugation reactions impossible. This for positron emission tomography (PET), single photon
leads—inevitably and unfortunately—to the creation of con- emission computed tomography (SPECT), and fluorescence
structs that are heterogeneous and poorly defined. To wit, a imaging.InPart1,wediscussedsite-specificbioconjugation
traditionally synthesized immunoconjugate with a degree of approaches predicated on the modification of cysteine
labelingof3cargoes/mAbis,inreality,acomplexmixtureof residues and the manipulation of the heavy chain glycans.
~10,000regioisomers,eachwithitsownchemical,biological, In Part 2,we will shiftgears and focus on two types ofsite-
and pharmacological properties [8–10]. Understandably, this specific bioconjugation strategies that rely heavily on
remarkableheterogeneitycanhavedetrimentaleffectsonthein enzymatic transformations. First, we will address a family
vitroandinvivoperformanceoftheimmunoconjugatesbothin of modification methods that are bound by a common
the laboratory and in the clinic. In order to circumvent these theme: the exploitation of peptide tags as either recognition
issues,anincreasingamountofefforthasbeendedicatedtothe sites for enzymatic ligations or as coordination scaffolds for
Fig.1 Detailedstructural schematicofafulllengthIgGaswellasanassortment ofantibody fragments.
P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging 155
Fig.2 Thebasicchemicalreactions underpinningthebioconjugationstrategies discussedinthiswork.
the chelation of radiometals. Subsequently, we will turn our have found a number of reports in which interesting and
attention to modification strategies based upon the incorpo- effective site-specific bioconjugation strategies have been
ration of unnatural or non-canonical amino acids (uAA and used to create antibody-drug conjugates (ADCs) or
ncAA, respectively) into immunoglobulins via genetic radioimmunotherapeutics rather than constructs for PET,
engineering. In addition, at the end of this installment of SPECT, or optical imaging. Strictly speaking, these works
the review, we will discuss the advantages and disadvan- lie outside the scope of this review. However, in these
tagesoftheapproachestobioconjugation coveredinParts1 cases, we have chosen to include them here in order to
and 2 and offer our humble thoughts on the direction of the increase the breadth of this work and, hopefully, facilitate
field as a whole. the use of these approaches in the synthesis of
Finally, before we begin, we feel compelled to make immunoconjugates for molecular imaging. Second, given
two brief logistical notes. First, as the title of the work the rapid growth and burgeoning interest in this field, it is
suggests, this review is focused primarily on allbutinevitable thatwehave inadvertentlymissed atleast
immunoconjugates for molecular imaging. However, we one of the many publications in this area, possibly more.
156 P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging
To the authors of these works, we offer our apologies and primary amines and the acyl functionality of glutamine
our earnest assurance that no slight was intended. residues (Fig. 3). Given this ability, it is not surprising that
this family of enzymes has attracted attention as tools for
Peptide Tags bioconjugation.Mostnotably,SchibliandcoworkersatETH
ZurichhaveexploitedTGasesforchemoenzymaticantibody
Thisnextsectionofthereviewmarksasignificantchangein conjugations [18]. Working primarily with microbial
course. In Part 1, the site-specific conjugation strategies transglutaminase (mTGase), the researchers have found that
discussed involved no protein engineering or, at most, while antibodies possess many glutamine residues, only
changes to single amino acids within the antibody structure. aglycosylated or deglycosylated antibodies can be function-
However, the ability to insert peptide tags or recognition alized appreciably using mTGase [19, 20]. The deglycosyl-
sitesintoantibodiesandantibodyfragmentsopensupanew ation of the antibody at the N297 position increases the
realmofpossibilities(Fig.3).Whileafewofthesestrategies flexibility of the peptide backbone, exposing Q295 for
have yet to be applied to in vivo imaging agents, we have reaction with the enzyme. This process was used to graft a
includedthemhereinordertoencouragetheirapplicationto cadaverine-bearing variant of the Cy3 fluorophore to the
immunoconjugates for PET, SPECT, and fluorescence L1CAM-targeting antibody chCE7 for in vitro fluorescence
imaging. Finally, a brief note to the reader: for the sake of microscopy (Fig. 4; Fig. 5a) [20]. In subsequent investiga-
clarity, this sectionhas beendividedinto sub-sectionsbased tions, the laboratory used mTGase to modify variants of
on the various types of modification strategies. Admittedly, chCE7 and rituximab that had been enzymatically deglyco-
this has resulted in a somewhat choppy narrative; however, sylated with PNGaseF (chCE7degl and RTXdegl). Two
we believe it best serves the educational purpose of the different chelators, desferrioxamine and a cadaverine-
review. bearing analogue of CPTA, were employed, and chelator/
mAb ratios of ~2 were obtained (Fig. 4) [19]. In an elegant
twist, the authors also developed a mutant chCE7
Methods Based on Enzymatic Modifications
(chCE7agl) in which the N297 residues were replaced with
Transglutaminase Transglutaminases(TGase)areenzymes glutamines, abrogating glycosylation and providing two
that catalyze the creation of isopeptide bonds between additional sites for mTGase modification in a single stroke
Fig.3 Selected chelatorsandcargoesusedinthesite-specifically labeledimmunoconjugatesdiscussedinthiswork.
P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging 157
Fig.4 Site-specific bioconjugation strategiesbasedontheuseofpeptidetags.
(Fig. 5b). This mutant antibody was then successfully labeling of ~4 chelators/mAb. Subsequent in vivo studies
modified to create immunoconjugates with degrees of using mice bearing SKOV3ip xenografts revealed that site-
Fig. 5 Schematics of transglutaminase-based strategies for the site-specific modification of a deglycosylated antibodies, b
aglycosylatedantibodies,andcimmunoglobulins bearinganLLQGtransglutaminaserecognition sequence.
158 P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging
between the glycine and threonine residues of an LPETGG
tag sequence on an EGFR-targeting scFv and subsequently
catalyzed the formation of a peptide bond between the
exposed threonine and a GGG-bearing fluorescein (Fig. 4)
[25]. More recently, Donnelly and coworkers employed
SrtA to site-specifically append a GGG-tagged MeCOSar
sarcophagine chelator for Cu-64 to an anti-LIBS scFv
bearing a C-terminal LPETGG-FLAG tag (Figs. 4 and 6)
[28].In2013,aparticularlycreativeSrtA-basedconjugation
strategy was developed to reduce the number of purification
stepsrequiredtoisolateafinalconjugate.Inthisapproach,a
fusion protein is created in which the protein of interest is
attached to a (His) -tagged variant of SrtA by a flexible
6
linkerthatincludestheLPXTGrecognitionmotif[29].After
theimmobilizationoftheprotein-LPXTG-linker-SrtA-(His)
Fig. 6 Small animal PET/CT images collected 60 min after 6
construct on a solid support, Ca2+ and a GGG-tagged cargo
the injection of a site-specifically labeled scFv -LPET-
anti-LIBS
[64Cu]L2(left)andacontrolfragment(scFv -LPET-[64Cu]L2; are added, prompting the site-specific modification of the
mut
right)intoamousemodelofcarotidarterythrombosis.Figure protein of interest and the consequent release of the protein-
adaptedandreprintedwithpermissionof[28]. cargo conjugate, ultimately leaving the His -tagged SrtA
6
enzyme attached to the solid support.
specifically labeled 67Ga-DFO-chCE7agl exhibits signifi-
cantly improved tumoral uptake and tumor-to-background
Polypeptide-α-N-Acetylgalactosaminyltransferase Somewhat
activity ratios than a randomly conjugated [67Ga]DFO-
ironically, peptide tags have also been used to site-specifically
chCE7agl analogue, and PET imaging data obtained using
introduce glycosylation sites into antibodies. Along these lines,
[89Zr]DFO-chCE7agl further supported these claims (Fig. 3).
Qasba and coworkers pioneered a method in which a
An alternative—though admittedly more
peptide tag containing a recognition site for a substrate-
complex—strategy to the use of the endogenous Q295 site
permissive glycoysyltransferase called polypeptide-α-N-
istheincorporationofaspecific,engineeredrecognitionsite
acetylgalactosaminyltransferase (ppGalNAc-T2) is
for enzymatic labeling. For example, Kamiya et al.
appended to the C-terminus of a non-glycosylated
engineered a GGSPLAQSHGGS tag onto the N-terminus
protein. This method can be used to attach the galactose
ofanscFv oftheanti-hen egg-whitelysozyme antibodyand
derivatives GalNAz and 2-keto-Gal to threonine or serine
were thus able to use mTGase to effectively conjugate
residues of a recognition peptide tag [30]. In practice,
various proteins to the engineered fragment [21]. Using a
this approach was used to site-specifically modify an
similar approach, Strop et al. engineered LLQG mTGase
anti-HER2 scFv with 2-keto-Gal and, subsequently, an
recognition tags into several different sites in the anti-M1S1
aminooxy-bearing variant of AlexaFluor® 488 for in
antibody C16 in order to probe the influence of conjugation
vitro fluorescence microscopy (Fig. 2c; Fig. 4) [31].
site on pharmacokinetics (Fig. 5c) [22, 23]. This method
Given the presence of the heavy chain glycans, this
allowed them to site-specifically graft an amine-bearing
strategy is clearly not particularly useful with intact
MMAD construct as well as cadaverine-modified
IgGs; however, it could prove to be an attractive
fluorophores (AlexaFluor® 350 and AlexaFluor® 488) to
alternative for the creation of antibody fragment-based
the engineered antibodies (Fig. 4). Ultimately, the authors
imaging agents.
found that the molecular location of the bioconjugation site
exerts a very strong influence on the in vivo stability,
toxicity, and efficacy of antibody-drug conjugates, though Lysyl Endopeptidase Proteases are most often associated
the causal relationships underlying this phenomenon remain with cleaving peptide bonds, but the enzymes can create
unclear. them as well. Over 20 years ago, Fisch, et al. exploited this
phenomenon to create a site-specifically labeled F(ab′) -like
2
immunoconjugate [32]. Specifically, the authors used a
Sortase Sortases (SrtA) are a family of Ca2+-dependent protease called lysyl endopeptidase, an enzyme capable of
transpeptidases that cleave peptides at a specific LPXTG digesting antibodies by cleaving polypeptide chains on the
(X=D,E,A,N,Q,orK)motifandcatalyzetheformationof carboxyl side of lysine residues. In this case, the enzyme
a peptidic bond between the cleaved peptide and a glycine- was usedtodigestthechimeric anti-TAG72antibodyB72.3
bearing substrate. Consequently, sortases have been via the cleavage of the heavy chain CH2 domain at either
employed to create a number of different fluorophore- Lys240orLys242,ultimatelycreatingaF(ab′) -likefragment.
2
labeled antibodies and antibody fragments [24–27]. For This fragment was then coupled to a carbohydrazide moiety
example, in the work of Madej et al., the enzyme cleaved via the reverse proteolysis reaction catalyzed by the same
P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging 159
enzyme. Finally, the hydrazide-bearing construct was con- [26].TheauthorscreatedED-BvariantswitheithertheGryA
jugated to an aldehyde-modified 55Fe-ferrioxamine through intein or the DnaE intein attached to the C-termini of the
a hydrazone linkage, and the Fe-55 labeled fragment was C 3domains.IntheED-B-GyrAvariant,anintein-catalyzed
H
shown to have immunoreactivity nearly equivalent to the N-S acyl shift was followed by treatment with sodium 2-
unmodified fragment (Fig. 2d). mercaptoethane sulfonate, incubation with a cysteine-
bearing biotin derivative, and a subsequent S-N acyl shift
to produce a site-specifically labeled ED-B-biotin conjugate
Trypsiligase In 2014, Liebscher, et al. applied a very
with a degree of labeling of ~2 (Fig. 4). The antibody
similar concept to the development of a site-specifically
bearingtheDnaEinteinpresentsaslightlymorecomplicated
labeled anti-HER2 fragment [33]. In this work, the authors
case (see Fig. 7 for a detailed schematic). Following a very
designed an anti-HER2, trastuzumab-derived Fab fragment
similar approach, an anti-RhoB scFv was engineered with
with a YRH recognition site appended to the C-terminus of
theGyrAinteinatitsC-terminusandsubsequentlymodified
the heavy chain. Using a trypsiligase enzyme designed to
withacysteine-biotin-fluoresceinconstruct[36].Whilethese
recognize the YRH sequence, the recombinant antibody
strategies are elegant, their inherent complexity may ulti-
fragment was conjugated to fluorescein and coumarin
mately limit their wide-scale use.
fluorophores bearing RHAK tags through the creation of
peptide bonds (Fig. 4). While the authors did not report any
in vitro or in vivo imaging using their fluorophore-labeled Methods Based on In Vitro Translational
fragments,thesite-specificallymodifiedconstructsexhibited Modifications
almostidenticalbindingaffinityforHER2astheunmodified
fragment. In yet another group of strategies, protein engineering
methods can be harnessed to incorporate recognition and
Methods Based on Self-Labeling Antibody-Enzyme bioconjugation sites into immunoglobulins during
Fusion Proteins translation.
All of these peptide tag-based conjugation methodologies
Engineering for Intracellular Glycosylation In one exam-
require modification reactions with enzymes and, conse-
ple,Leung,etal.successfullyidentifiedanaturalasparagine
quently, purification steps after enzymatic treatment. One
glycosylationsitewithanNVTsequenceintheframework-1
way to circumvent these purification steps is to genetically
(FR-1) region of the V domain of the murine anti-B cell
L
engineer fusion proteins in which the protein-of-interest and
lymphoma antibody LL-2. Subsequently, the authors genet-
the modification enzyme are combined.
ically introduced this tripeptide glycosylation sequence into
the FR-1 region of a non-glycosylated, humanized anti-
SNAP-tag SNAP-tag isa widely employed 20 kDa mutant carcinoembryonic antigen (CEA) antibody, hMN-14 [37].
of O6-alkylguanine transferase (AGT) that catalyzes the This protein engineering resulted in the in vitro glycosyla-
transfer of an alkyl group from a guanidine substrate to a tion of the antibody, as illustrated using SDS-PAGE. After
cysteine residue in its active site [34]. This technology was isolation of the glycosylated hMN-14 antibody, a F(ab′)2
recently used to site-specifically attach a chlorin e6 (Ce6) fragment was created, and then a NaIO4-based sugar
photosensitizer to scFv-425, an anti-EGFR scFv (Fig. 4) oxidation strategy was used to attach an amine-bearing
[35]. While the authors successfully illustrated that the variantofDTPAtothefragment(Fig.2e-f).Inthisway,the
homogeneous scFv-425-Ce6 conjugate specifically binds authorsobtaineddegreesoflabelingof~2DTPAperF(ab′)2
EGFR-expressing cells and promotes tumor cell-specific andwereabletolabelthefragmentefficientlywithY-90and
cytotoxicity, they fail to adequately address the influence In-111 (Fig. 4). Importantly, neither the glycosylation nor
that the addition of a 20 kDa SNAP-tag moiety could have the conjugation steps decreased the immunoreactivity of the
on the in vivo pharmacokinetics of the ~28 kDa scFv. F(ab′)2. However, no in vivo data for the site-specifically
radiolabeled hMN-14 F(ab′) were presented in this initial
2
report or subsequent publications.
Inteins The use of intein tags provides a way to take
advantage of fusion protein conjugation approaches while
avoiding the permanent attachment of a large enzyme to the Formylglycine-Generating Enzymes Bertozzi and Rabuka
immunoconjugate. Inteins, in short, are sections of proteins have developed a related strategy based on the site-specific
capable of excising themselves and ligating the remaining introduction of CxPxR recognition sites for formylglycine-
portions ofthepolypeptidetooneanotherthroughapeptide generating enzyme (FGE). After the synthesis of the target
bond. Generally speaking, this process is called protein protein in E. coli or mammalian cells, FGE oxidizes the
splicing. In 2011, Möhlmann and colleagues creatively cysteine residue of the recognition motif to create a
applied this strategy to the creation of site-specifically formylglycine moiety on the protein of interest. The
labeled conjugates of the anti-fibronectin antibody ED-B aldehyde of this formylglycine can then be exploited for
160 P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging
Fig.7 DiagramoftheDNA intein-basedsite-specificmodificationproceduredevelopedbyMöhlmann,etal.Figureadapted
E
andreprintedwithpermissionof[26].
conjugation with aminooxy-labeled substrates (Fig. 2 g) specifically modified conjugates. Indeed, a number of
[38]. In related work, the same researchers developed the laboratories have used peptide tags not as recognition
Pictet-Spengler and hydrazino-Pictet-Spengler (HIPS) liga- sequences to which chelators are appended but rather as
tions, reactions which create more stable linkages than the chelators themselves.
hydrazones and oximes (Fig. 2h–i) [39]. The laboratory-
engineered trastuzumab antibodies with CxPxR motifs at
(His) Tag The ubiquitious hexahistidine affinity tag
various locations, thereby creating a variety of 6
provides the best example of this phenomenon. A number
immunoconjugates with aldehyde tags at specific sites [40].
of laboratories have labeled antibody fragments bearing C-
Subsequently, the HIPS ligation was used to append the
terminal (His) tags with the [99mTc(CO) ]+ synthon for
chemotherapeutic maytansin to the antibodies in order to 6 3
SPECT imaging [43–46]. Kogelberg, et al., for example,
investigatetheroleofconjugationsiteontheinvivoefficacy
labeledtheα β -targetingdiabodyB6.3viathecoordination
andpharmacokineticprofilesoftheimmunoconjugates,with v 6
of [99mTc(CO) ]+ to a C-terminal (His) tag, and it was
the authors demonstrating that the site of bioconjugation 3 6
found that the resultant conjugate retained nanomolar
exerts significant influence on the in vivo efficacy and
affinity for its target and demonstrated tumor-specific
pharmacokinetic behavior of antibody-drug conjugates
targeting in vivo. Kampmeier and coworkers provided a
(ADCs).
clever variation on this theme. This laboratory site-
specifically labeled a PSMA-targeting, J591-derived
ProteinKinases Anotherstrategy unique tothelabeling of diabody with [99mTc(CO) ]+via a C-terminal (His) tag and
3 6
immunoconjugates with P-32 (t ~14.2 days) for therapeu- successfully performed SPECT imaging in mice bearing
1/2
ticapplicationsistheapplicationofproteinkinases.Theuse subcutaneous DU145 prostate cancer xenografts. Interest-
of these enzymes with proteins lacking native phosphoryla- ingly,however,thegroupalsoincorporatedacysteineatthe
tion sites—such as antibodies and antibody end of the (His) tag and used the thiol functionality to
6
fragments—requires the creation of a kinase substrate site modify the diabody with a fluorophore, indicating that this
to which the enzyme can transfer the γ-phosphate from approach could easily be applied to the synthesis of tracers
[32P]ATP. The introduction of this recognition site, ranging for multimodal imaging.
from 5 to 15 amino acids depending on the specific kinase
used,hasbeenachievedusingtwostrategies:thecreationof
(Gly) Cys Tag Other laboratories have used a peptide tag
antibody-based fusion proteins and the genetic engineering x
composed of several glycines and a terminal cysteine to
of the immunoglobulin. Both of these approaches are
create an N S coordination architecture for 99mTC=O
discussed in great detail in an excellent review dedicated to 3
species. For example, almost 20 years ago, George et al.
the topic [41]. In one example, Patrick et al. engineered an
site-specifically labeled an anti-c-erbB-2 scFv bearing a C-
anti-CEA scFv with a LRRASG kinase recognition site and
terminal Gly Cys tag with 99mTc-99m [47]. To this end, the
subsequently used protein kinase A to label the fragment 4
scFv was first reduced with 2-mercaptoethanol and then
with32P-32[42].Theauthorsfoundthatthesite-specifically incubated with 99mTcO −1 in the presence of Sn2+ to
phosphorylatedconstructretainedtheselectivityofitsparent 4
generate an immunoconjugate with Tc-99m in an N S
fragment and, in in vitro assays, exhibited selective 3
coordination environment. Following a similar strategy, a
cytotoxicity and a high internalization rate in CEA-
different laboratory labeled an anti-ED-B fibronectin scFv
expressing LS174T cells.
bearinga(Gly) -Cys-Alatag[48].Thisworkalsoincludeda
3
revealingcomparisontothesamescFvlabeledwithTc-99m
Metal-Coordinating Peptide Tags via a (His) tag. In mice bearing antigen-expressing F9
6
xenografts, the [99mTc]scFv-G CA and the [99mTc]scFv-
3
Up until now, the peptide tags discussed have all served as (His) agents showeddramatically different biodistributions:
6
recognitionsitesforenzymes.Thisisnot,however,theonly while both constructs effectively targeted tumor tissue, the
way that peptide tags can facilitate the creation of site- authors found that the (His) -tagged variant displayed much
6
P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging 161
higheruptakeinnon-targettissuessuchasthekidney.These Incorporation of uAA by engineered cells
data serve as an effective reminder that chelators can play
non-innocent roles in the pharmacokinetics of radiometal- The Schultz laboratory has pioneered the genetic engineer-
based imaging agents. ing of cells for the integration of uAAs and has used this
technology for the production of site-specifically labeled
immunoconjugates [54, 55]. In one example, the laboratory
used engineered E. coli to incorporate the ketone-bearing
Antibody-Metallothionein Fusion Proteins A seldom-used
uAA pAcF into an anti-HER2 Fab based on trastuzumab,
butnonethelessinterestingalternativetothesecoordinating
which could then be modified with an aminooxy-bearing
peptide tags is the use of fusion proteins bearing
AlexaFluor® 488 fluorophore. In an alternate strategy, this
metallothioneins, low molecular weight proteins capable
pAcF-Fab was modified with an aminooxy-bearing variant
of binding both physiological and xenobiotic metals via
multiple cysteine residues. Pietersz et al. used this of biotin and then conjugated to neutravidin to form
approach to site-specifically label a fusion protein com- multimers which were shown to be more potent inhibitors
posed of a metallothionein and an anti-CEA scFv (MET- ofHER2phosphorylationthantheirunmodified,monomeric
scFv)[49].UsingauniqueZn2+transchelationmethod,the parents [54]. In an expansion of this methodology, it is now
authors labeled MET-scFv with Tc-99m and demonstrated possible to introduce aminoacyl tRNA synthetase (aaRS)/
that the Tc-99m labeled construct is both stable and highly tRNA pairs corresponding to two different uAAs and two
immunoreactive. Further, imaging and biodistribution different nonsense codons, thus enabling the incorporation
experiments in mice bearing LS174T xenografts revealed of two different uAAs into a single recombinant protein.
that despite extraordinarily rapid clearance, [99mTc]MET- Using this strategy, Xiao et al. inserted pAcF and azido-
scFveffectivelydelineatedCEA-expressingtumortissuein lysine (AzK) into the sequence of trastuzumab [56]. These
vivo. twofunctionalgroupswerethenusedtomodifytheantibody
Beforemovingon,itisimportanttonotethatanumberof with an aminooxy-bearing variant of auristatin and a
otherchemoenzymaticproteinmodificationapproacheshave cyclooctyne-bearing Alexa Fluor® 488 fluorophore, and
recentlyemergedthathaveyettobeappliedtoantibodiesor the resulting immunoconjugate was subsequently employed
antibody fragments [50]. This knowledge, coupled with the in in vitro assays (Fig. 2c and j).
tremendous variety of peptide-based bioconjugation strate-
gies discussed in this section, underscores that the next few
Incorporation of uAA in cell-free system
years will be an exceptionally exciting time for the
development of site-specifically labeled immunoconjugates
Despite the promise of systems based on genetically
of all kinds.
engineered cells, these methods are hampered by the
expense and complexity of creating stable cell clones as
Unnatural Amino Acids well as the need for the uAA in question to cross the cell
membranerapidlyinordertojointhetranslationalmachin-
Arguablythemostsophisticatedmethodoffacilitatingsite- ery. Cell-free protein expression systems circumvent these
specific conjugation is the incorporation of orthogonally drawbacks.Thesesystemscontainallthemachineryneeded
reactive functional groups during the translation of recom- for protein expression and provide a powerful tool for the
binant antibodies using non-canonical (ncAA) or unnatural incorporationof uAAs after the introduction ofthesuitable
amino acids (uAA). This approach relies on expanding the aaRS/tRNA pair. The principal advantages of cell-free
genetic code of cells to reinterpret nonsense protein expression lie in addition by subtraction: in the
codons—amber (UAG), ochre (UAA), or opal (UGA)—as absenceofacell,alltheavailableresourcesarededicatedto
sequences coding for uAA, leading to the ribosomal theproductionoftheproteinofinterest,andtheaaRSshave
incorporation of the uAA into the protein in question [51, unobstructedaccesstotheuAAs.Unfortunately,thefolding
52]. At present, the number of uAAs that have been of the recombinant proteins and the incorporation of post-
incorporated into recombinant proteins hovers around 70, translational modifications (like glycosylation) are signifi-
with p-aceto-L-phenylalanine (pAcF) and p-azido-L- cant challenges in cell-free systems, as these functions are
phenylalinine (pAzF) among the most often employed normally handled by organelles such as the endoplasmic
(Fig.8)[52].Whilethistechnologyhasbeenusedtocreate reticulumandGolgiapparatus.Recently,however,avenues
ADCs based on recombinant antibodies and antibody around these issues have emerged [57, 58]. Using these
fragments, there are curiously few examples of uAA- methods, Zimmerman, et al. created a pAzF-containing
based immunoconjugates for imaging [53]. Therefore, in variant of trastuzumab, which was subsequently coupled
this section, we will primarily provide examples of how to a DBCO-bearing MMAF drug moiety (Fig. 4) [59].
this technology has been used to create immunoconjugates In addition, Patel and coworkers created both a
for other applications with a view toward inspiring the homopropargyl glycine-containing variant of luciferase
future application of these methods to molecular imaging. and a pAzF-bearing fusion construct of an scFv and the
162 P. Adumeau et al.: Site-Specifically Labeled Immunoconjugates for Molecular Imaging
IM9 protein [60]. The investigators then coupled the IM9- almost certainly due to the intrinsic complexity of these
scFv fusion protein to the luciferase and employed the approaches. However, we are optimistic that the molecular
resulting conjugate for the in vivo detection of B cell imaging community will be able to leverage this technology
lymphoma cells. in the near future.
Non-canonical amino acids
Moving Forward
TheeffortandexpenseofusinguAAshavepromptedsome The development of site-specifically labeled
research groups to revisit the use of natural, though non- immunoconjugates is a rapidly evolving field, as illustrat-
canonical, amino acids for the construction of site- ed by the emergence of a number of new studies during
specificallymodifiedproteins.Inthisregard,selenocysteine the writing of this work [64–66]. In the two installments
(Sec; U) and pyrrolysine (Pyl; O)—the 21st and 22nd of this review, we have covered a wide variety of
naturallyoccurring,geneticallyencodedaminoacids—have approaches, each with distinct advantages and limitations
been the center of attention (Fig. 8). While pyrrolysine has (Fig.9). For example, while strategies based on unnatural
been found exclusively in prokaryotic organisms, amino acids offer exquisite control over the molecular
selenocysteine has been observed in all kingdoms of life. location and stoichiometry of the conjugation reaction,
From a protein modification standpoint, pyrrolysine boasts they also require specialized and complex genetic engi-
unique orthogonal reactivity, while selenocysteine can be neering. On the other hand, methodologies based on the
coupled to maleimide analogues even in the presence of reaction of native cysteines with maleimide-bearing
cysteineduetothedifferenceinthepK ofthetworesidues probes are simple and straightforward but admittedly do
a
(5.2forselenocysteinevs.8.3forcysteine;Fig.2k–m))[61, not readily produce the degree of homogeneity offered by
62]. Along these lines, Li et al. have recently produced a other approaches. Specifics aside, the benefits of all of
scFv-Fc fragment of trastuzumab bearing two engineered these strategies over traditional synthetic methods are
cysteinesandoneselenocysteineintheFcregion[63].After clear: the reproducible creation of better defined and more
reduction, the selenocysteine residue of the fragment was homogeneous immunoconjugates with in vivo perfor-
selectively coupled to a methyl sulfone-bearing variant of mance comparable to—if not superior to—their randomly
biotinatpH5.2,whilethecysteineresidueswereconjugated modified cousins.
toamethylsulfone-containingfluoresceinmoietyatpH7.4. Ultimately, it is our belief that four of these
The resulting immunoconjugate was then successfully bioconjugation approaches—two that require genetic
employedfortheinvitrotargetingofHER2(Fig.2n–o). manipulation and two that do not—hold particular
Over the last ten years, great strides have been made in promise for the future. First, the incorporation of
the production of recombinant proteins bearing unnatural engineered cysteines into antibodies and fragments offers
or non-canonical amino acids. Yet reports of using complete control over the site of conjugation while
these methods to create site-specifically modified leaving the rest of the immunoglobulin structure un-
immunoconjugates are few and far between. Indeed, in touched [67]. However, the development of thiol-reactive
assembling this review, we were wholly unable to find any alternatives to maleimides is essential to this technology’s
examples of site-specifically modified immunoconjugates ongoing success [68]. Second, the use of sortases to
forinvivoimagingsynthesizedusingthesemethods.Thisis modify C-terminal recognition sequences on antibody
Fig.8 Structuresofselectedunnatural andnon-canonicalaminoacids.