available online at · 2014. 6. 28. · acids chan hyuk kim1,4, jun y axup2,3,4 and peter g...

Protein conjugation with genetically encoded unnatural aminoacidsChan Hyuk Kim1,4, Jun Y Axup2,3,4 and Peter G Schultz1,2,3

Available online at www.sciencedirect.com

The site-specific incorporation of unnatural amino acids with

orthogonal chemical reactivity into proteins enables the

synthesis of structurally defined protein conjugates. Amino

acids containing ketone, azide, alkyne, alkene, and tetrazine

side chains can be genetically encoded in response to

nonsense and frameshift codons. These bio-orthogonal

chemical handles allow precise control over the site and

stoichiometry of conjugation, and have enabled medicinal

chemistry-like optimization of the physical and biological

properties of protein conjugates, especially the next-

generation protein therapeutics.

Addresses1 California Institute for Biomedical Research, 11119 N. Torrey Pines

Road, La Jolla, CA 92037, United States2 Department of Chemistry, The Scripps Research Institute, 10550 N.

Torrey Pines Road, La Jolla, CA 92037, United States3 The Skaggs Institute for Chemical Biology, The Scripps Research

Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, United States

Corresponding author: Schultz, Peter G ([email protected])4 Authors contributed equally.

Current Opinion in Chemical Biology 2013, 17:412–419

This review comes from a themed issue on Next generation

therapeutics

Edited by Paul J Carter, Daria Hazuda and James A Wells

For a complete overview see the Issue and the Editorial

Available online 9th May 2013

1367-5931/$ – see front matter, Published by Elsevier Ltd.

http://dx.doi.org/10.1016/j.cbpa.2013.04.017

IntroductionMethods for the site-specific modification of proteinshave become powerful tools for probing protein structureand function, as well as generating proteins with new orenhanced properties. Historically, protein conjugationreactions involve the modification of lysine, cysteine, ornucleophilic serine side chains with electrophilic agents[1]. However, these reactions generally lead to hetero-geneous mixtures of protein conjugates with distinctproperties, especially when modifying large proteins suchas antibodies. The introduction of chemically orthogonalfunctional groups into proteins, on the other hand, hasallowed for precise control of conjugation site and stoichi-ometry. Common methods for this purpose include enzy-matic or chemical reactions that modify specific peptidesequences with chemical tags [2], and semi-syntheticmethods such as expressed protein ligation (EPL) [3].Alternatively, recombinant methods have also been used


to incorporate unnatural amino acids (UAAs) into proteinsas chemical handles for bio-orthogonal conjugation reac-tions [4��]. The latter approach is particularly attractivebecause, in principle, the UAA can be incorporated at anydesired position in any protein; the structure of the wild-type protein is minimally perturbed by UAA incorpora-tion since it does not require any particular sequencecontext to specify the UAA site; and site-specificallymodified proteins are expressed recombinantly in highyields in bacteria, yeast, or mammalian cells, and requirelittle additional manipulation for further conjugation. Inthis review, we discuss several methods for the incorp-oration of UAAs with orthogonal chemical reactivity intoproteins in living cells and recent examples of theirapplication, especially with regards to protein thera-peutics.

Methods for genetically incorporatingunnatural amino acids into proteinsAmong the early methods for the genetic incorporation ofUAAs into proteins was the use of bacterial strains aux-otrophic for a structurally related canonical amino acid.UAAs that are structurally similar to a common amino acidare recognized by that wild-type or engineered aminoa-cyl-tRNA synthetase (aaRS) and incorporated in theabsence of the cognate amino acid. This method hasbeen applied to analogues of Pro, Tyr, Phe, Leu, Val,and most commonly to Met (the latter has low abundancein the proteosome and hence less toxicity when replacedglobally) [5]. For example, the incorporation of seleno-methionine into proteins using methionyl-tRNA synthe-tase (MetRS) has been extensively used for thedetermination of protein structures by X-ray crystallogra-phy [6]. In another example, an engineered phenylalanyl-tRNA synthetase (PheRS) was used to replace Phe withp-acetylphenylalanine to introduce a reactive ketonefunctional group [7]. The low efficiency of UAA incorp-oration in this system was improved by overexpressingthe cognate aaRS. However, this general approach has anumber of drawbacks: the global replacement of canoni-cal amino acids with structural analogues is often too toxicto support exponential growth of cells; wild-type aaRSstypically only mis-acylate close structural analogues of thecanonical amino acid; and in larger proteins this methodtypically leads to the incorporation of UAAs at multiplesites.

In some archaebacteria, nature has used nonsense codonsto encode selenocysteine (Sec) and pyrrolysine (Pyl),which can be used for selective protein conjugation [8].

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Genetically encoded unnatural amino acids Kim, Axup and Schultz 413

Figure 1

CanonicaltRNA

U UU

U UU

U AAAAG

G CAU

UU

UA C

UA C

UA C

CG U

OrthogonaltRNA

OrthogonalAmino-acyl

tRNASynthetase

EF-Tu

Ribosome

UnnaturalAmino Acid

CanonicalAmino Acid

CanonicalAmino-acyl

tRNASythestase

mRNA

Current Opinion in Chemical Biology

Scheme for the genetic incorporation of unnatural amino acids.

Selenol is relatively acidic (pKa � 5.2) compared to thiol(pKa � 8.3), and mostly exists as the selenate anion atphysiological pH. Thus one can selectively modifyproteins by reacting Sec with electrophiles (e.g., malei-mide or a-haloketones) in the presence of free Cys [9].Unlike Sec, Pyl is unreactive. However, Pyl analoguesbearing reactive groups have been genetically encoded bythe corresponding wild-type aaRS (or mutants thereof)and used in protein conjugation reactions [10].

To expand the genetic code beyond the canonical aminoacids, we and others have reprogrammed the cell’s trans-lational machinery by evolving additional tRNA/aaRSpairs that site-specifically incorporate UAAs in responseto either nonsense or 4-base frameshift codons [4](Figure 1). Importantly, these tRNAs and aaRSs areorthogonal to their counterparts in the host cell (i.e., they


do not cross-react with their natural counterparts) in orderto maintain high translational fidelity. The most successfulstrategy for generating orthogonal tRNA/aaRS pairs is toimport them from phylogenetically distant organisms (e.g.,from archea to Escherichia coli, from E. coli to yeast, fromarchea to mammalian cells, etc.). For example, an engin-eered tRNATyr/TyrRS pair derived from Methanococcusjannaschii has been used to incorporate a large numberof UAAs into proteins in E. coli in response to the ambercodon TAG [11]. To genetically encode a specific UAA, anactive site library of aaRS mutants is subjected to multiplerounds of positive selection (to select mutants that incorp-orate any amino acid) and negative selection (to selectagainst mutants that incorporate endogenous host aminoacids). This method has been used to incorporate over 70UAAs in bacteria, yeast, and mammalian cells (with yieldsup to 5 g/L in a 40,000 L bacterial fermentation) [4��].


414 Next generation therapeutics

More recent advancements have expanded the technologyto include orthogonal tRNA/aaRS pairs derived from leucyl[12], tryptophanyl [13], lysyl [14], pyrrolysyl [15], andprolyl [16] pairs to allow increased diversity in UAA struc-ture. To incorporate two or more UAAs, tRNA/aaRS pairshave been developed that recognize other nonsensecodons (TGA or TAA) or frameshift codons [14]. Inaddition, an orthogonal ribosome [17] that only recognizesorthogonal mRNAs (by means of a modified Shine–Dal-garno sequence) has also been engineered to efficientlydecode quadruplet codons with minimal toxicity [18�].Moreover, it has been shown that a reengineered EF-Tu [19] with improved binding properties allows theincorporation of phosphoserine into proteins, and thatdeletion of RF1 with compensating mutations in RF2can improve suppression efficiency [20]. More recently,the TAG nonsense codon has been more rigorously reas-signed to only encode UAAs by genome-wide substitutionof TAG with other synonymous stop codons (Church et al.,submitted). Hence, one can very effectively reprogrammuch of the translational machinery to efficiently incorp-orate UAAs with novel chemical functionality not found inthe canonical 20 amino acids.

Unnatural amino acids with orthogonalchemical reactivitySeveral chemically reactive UAAs have been geneticallyencoded to facilitate the selective conjugation of proteinsto small molecules, oligonucleotides, synthetic polymers,and other proteins. Inspired by recent advances in bio-orthogonal or protein-compatible reactions, the repertoireof genetically encoded chemically reactive amino acidshas grown considerably. Among them, the most widelyused reactions include oxime formation [21], Cu(I)-cat-alyzed and strain-promoted Huisgen 1,3-dipolar cycload-ditions (‘Click’ reaction) [22�], and, most recently, inverseelectron demand hetero Diels–Alder (HDA) reactions[23]. In addition, other chemically reactive amino acidshave also been encoded that allow the selective modifi-cation of proteins by Michael reactions [24], metathesisreactions [25], transition metal catalyzed cross-couplings[26], radical polymerizations [27], oxidative couplings[28], acyl-transfer reactions [29], and photo click reactions[30].

Ketone-containing UAAs react with alkoxy-amine deriva-tives to form a stable oxime bond (whereas adducts withlysine and cysteine side chains are unstable in aqueousbuffer) [31] (Figure 2a). Although the reaction rate isrelatively slow (k �


Figure 2

(a)O

O O

OO

OHN

O

O

O

HNO

O

H

H

N NN N

H

H

O

O

OHN

HN

O

OHN

O

OHN

HN

N

N

N N

N

NNN

pH < ~ 5

+

+

+

+

H2N

H2NO - H2O

- N2

Cu (I)

CO2H

H2N

N3

N3

N3

CO2H

H2N CO2H

H2N CO2H

(b)

(c)

(d)


UAAs used for site-specific protein conjugation.

traditional methods like lysine or cysteine conjugationoften form heterogeneous products with limited controlover conjugation site and stoichiometry. Furthermore, itis likely that individual product species have distinctefficacy, safety, and pharmacokinetic properties that com-plicate the optimization of a therapeutic candidate. Somerecent applications of genetically incorporated UAAs tothe development of protein therapeutics are highlightedbelow (Figure 3).

One of the earliest applications involved site-specificpolyethylene glycol (PEG) conjugation to extend theserum half-life of proteins (Figure 3a). The orthogonalchemical handle allows for the control of conjugation siteand stoichiometry to maximize biological activity, serumhalf-life and stability/solubility. For example, humangrowth hormone [40�] and fibroblast growth factor 21[41] were expressed with pAcF at defined positionsand conjugated to an alkoxy-amine derivatized PEG in


80–97% yield. The resulting conjugate showed excellentpharmacokinetics, minimal loss of biological activity, andno apparent immunogenicity in vivo.

Genetically encoded UAAs have also been used to syn-thesize antibody–drug conjugates (ADCs). ADCs prefer-entially deliver cytotoxic drugs to cells presenting tumor-associated antigens to achieve improved drug efficacy andsafety. For example, brentuximab vedotin (Adcetris/SGN-35; an anti-CD30 antibody conjugated with themicrotubule-disrupting agent monomethyl auristatin E)has been recently approved for Hodgkin’s lymphoma[42], and several other ADCs are currently in late-stageclinical trials. However, the first approved ADC, gemtu-zumab ozogamicin (Mylotarg; an anti-CD33 antibodyconjugated with the DNA cleaving agent calicheamicin)[43], was pulled from the market in 2010 because oftoxicity and lack of efficacy in larger trials, showing theneed for further optimization of this class of drugs.



Figure 3

(a)

Conjugation to... Examples

(b)

(c)

(d)

Protein withsite-specific

unnatural aminoacid

Self or other proteins

Toxin or other small molecules

Linker

PEG linker or polymer Extends pharmakinetic properties

Antibody-drug conjugates

Bispecific antibodies

Bispecific antibodies, multimers

LinkerO

OO

O On

n

Oligonucleotides (DNA, PNA)

UAA


Protein conjugates synthesized with unnatural amino acids and examples of their applications.

Typical synthetic methods for most ADCs involve modi-fication of lysine or cysteine residues which leads to adistribution of zero to eight toxins per antibody at varioussites. The individual species within this mixture are likelyto have distinct affinities, stabilities, pharmacokinetics,efficacies and safety profiles. A site-specific ADC withonly two conjugation sites (generated by THIOMABtechnology in which additional cysteines are introducedinto the antibody for maleimide conjugation [44�])showed similar efficacy to randomly labeled ADCs, buthad an improved therapeutic index and better pharma-cokinetic properties in rodents. This methodology, how-ever, requires complicated reduction/oxidization stepsand still has inherent cysteine–maleimide instability[45]. An anti-Her2 IgG expressed with two geneticallyencoded pAcF residue was conjugated to an auristatinderivative via a stable oxime-linkage in excellent yield[46��] (Figure 3b). This construct showed selective invitro cytotoxicity (EC50 100–400 pM) against Her2

+

breast cancer cell lines as well as complete regressionof Her2+ mammary fat pad tumors in mouse xenograftmodels. Full-length IgGs containing pAcF can beexpressed in mammalian cells at yields over 1 g/L, mak-ing this a commercially viable technology. The ability tosynthesize homogeneous ADCs enables medicinal


chemistry-like optimization of ADCs, and is currentlybeing applied to the synthesis of antibodies conjugated tokinase and phosphatase inhibitors, nuclear hormone re-ceptor agonists and antagonists, and other drug classes fortreatment of autoimmune, cardiovascular, and metabolicdisease.

Bispecific antibodies that can bind two different antigenssimultaneously (on the same or distinct proteins and/orcells) also have gained considerable attention as next-generation immunotherapeutics for cancer. Bispecific anti-bodies consisting of an antibody (or fragments thereof)specific for a tumor associated antigen linked to an anti-human CD3 (cluster of differentiation 3) antibody havedemonstrated impressive efficacy in the clinic [47]. Theseengineered antibodies recruit CD8+/CD3+ cytotoxic Tlymphocytes to tumor cells and form a pseudo-immuno-logical synapse that results in the activation of T lympho-cytes and subsequent lysis of the target cells. A number ofrecombinant methods (including single chain variable frag-ment (scFv) and IgG-based formats) and some chemicalcrosslinking methods have been developed to generatebispecific antibodies [48–51]. However, genetically fusedscFvs can have short serum half-lives and relatively poorstability, and some IgG-based quadroma formats, which



consist of chimeric mouse IgG2a and rat IgG2b, can gen-erate human anti-mouse (HAMA) or human anti-rat(HARA) antibody responses in patients [52]. Moreover,these methods often lead to restricted (and less optimal)orientations of the fusion proteins (e.g., C-terminal to N-terminal fusions). Synthesis of bispecific antibodies usingtraditional chemical methods is challenging since one gen-erates many different orientations of the two antigen bind-ing sites, which may or may not form productiveimmunological synapses.

We have developed a two-step method to synthesizehomogeneous bispecific antibodies using geneticallyencoded UAAs and flexible bifunctional linkers[53��]. This approach involves conjugating the twopAcF-containing Fabs to bifunctional linkers (a shortPEG with an alkoxy-amine on one end and either anazide or cyclooctyne on the other). In a second step, theantibody-linker conjugates are coupled to each other viacopper-free [3 + 2] Huisgen cycloaddition in goodyields. The affinity of anti-Her2 Fab homodimersgenerated by this method toward Her2 was comparableto full-length IgG. Moreover, heterodimers of anti-Her2/anti-CD3 Fabs showed subnanomolar killing(EC50 � 20 pM) of Her2+ cancer cells in the presenceof purified peripheral blood mononuclear cells (PBMCs)in vitro, which is comparable to the most potent bispe-cific antibodies reported to date. Furthermore, the con-jugate has a half-life of �7 hours in rodents and hasshown effective tumor suppression in mouse xenograftmodels. Similar bispecific antibodies against other tar-gets, for example, CD20, CD33, and EGFR, are beingdeveloped.

Proteins have also been conjugated to single-strandedDNAs or peptide nucleic acids (PNAs) and can be sub-sequently hybridized to form homodimers, heterodimers,tetramers or higher order complexes [54] (Figure 3d). Forexample, PNA was conjugated to a pAcF-containing Fabvia oxime ligation in excellent yields. The PNA-linkedanti-Her2/anti-CD3 Fabs showed potent and specificcytotoxicity in vitro, similar to that of the PEG-linkedbispecific Fabs. A tetramer of anti-CD20 Fab was gener-ated utilizing PNAs that form a Holiday junction, andpotently induced apoptosis of CD20+ Ramos cells.Additionally, an anti-Her2 Fab conjugated to single-stranded DNA was used to detect Her2+ cells byimmuno-PCR with exquisite sensitivity [55].

Other applications of selective conjugation with geneti-cally encoded UAAs include the conjugation of fluor-escent dyes and other spectroscopic probes to proteins.Fluorescent dyes have been conjugated both in vitro, suchas for FRET experiments to study protein dynamics [56]and in vivo for specific labeling on cell surfaces [35,57,58].Spin labels also have been conjugated to probe proteinstructure by electron paramagnetic resonance (EPR) [59].


ConclusionAn advantage of using genetically encoded UAAs for bio-orthogonal protein conjugation is the ability to generatechemically defined, homogeneous protein conjugates thatcan be characterized and optimized with respect to theirefficacy, safety and pharmacokinetics. There are clearlymany directions in which the technology and its appli-cations can be further developed. For example, currentlystoichiometry is largely limited to one site per protein (ortwo sites on a full-length antibody); the efficient incorp-oration of two or more identical or different UAAs into asingle polypeptide will allow the synthesis of more-com-plex conjugates. Moreover, the development of stable celllines each encoding a distinct amino acids will simplifymammalian expression of mutant proteins. Different pay-loads for ADCs such as kinase inhibitors, nuclear hor-mone receptor agonists, or HDAC inhibitors will furtherexpand the therapeutic applications of ADCs. Also, novelbi-specific and multi-specific protein conjugates can becreated to bind specific antigens on cells in definedorientations with respect to each other. These and otheradvances promise to enable a new generation of chemi-cally tailored protein therapeutics.

AcknowledgementsThe authors thank the National Institutes of Health (NIH) for support(R01GM062159). This manuscript is number 22069 of The ScrippsResearch Institute.

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This paper describes THIOMAB technology that utilizes reactivecysteines for site-specific conjugation of drugs to an antibody.

45. Shen BQ, Xu K, Liu L, Raab H, Bhakta S, Kenrick M, Parsons-Reponte KL, Tien J, Yu SF, Mai E, Li D et al.: Conjugation sitemodulates the in vivo stability and therapeutic activity ofantibody–drug conjugates. Nat Biotechnol 2012, 30:184-189.


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46.��

Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA,Halder R, Forsyth JS, Santidrian AF, Stafin K, Lu Y et al.:Synthesis of site-specific antibody–drug conjugates usingunnatural amino acids. Proc Natl Acad Sci U S A 2012,109:16101-16106.

This paper introduces the use of genetically encoded unnatural aminoacids for the synthesis of site-specific homogeneous antibody–drugconjugates.

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53.��

Kim CH, Axup JY, Dubrovska A, Kazane SA, Hutchins BA,Wold ED, Smider VV, Schultz PG: Synthesis of bispecific


antibodies using genetically encoded unnatural amino acids. JAm Chem Soc 2012, 134:9918-9921.

This paper describes the generation of bispecific antibodies using unna-tural amino acids and bifunctional linkers.

54. Kazane SA, Axup JY, Kim CH, Ciobanu M, Wold ED, Barluenga S,Hutchins BA, Schultz PG, Winssinger N, Smider VV: Self-assembled antibody multimers through peptide nucleic acidconjugation. J Am Chem Soc 2013, 135(1):340-346.

55. Kazane SA, Sok D, Cho EH, Uson ML, Kuhn P, Schultz PG,Smider VV: Site-specific DNA-antibody conjugates for specificand sensitive immuno-PCR. Proc Natl Acad Sci U S A 2012,109:3731-3736.

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57. Ye S, Köhrer C, Huber T, Kazmi M, Sachdev P, Yan ECY, Bhagat A,RajBhandary UL, Sakmar TP: Site-specific incorporation of ketoamino acids into functional G protein-coupled receptors usingunnatural amino acid mutagenesis. J Biol Chem 2008,283:1525-1533.

58. Lang K, Davis L, Wallace S, Mahesh M, Cox DJ, Blackman ML,Fox JM, Chin JW: Genetic encoding of bicyclononynes andtrans-cyclooctenes for site-specific protein labeling in vitroand in live mammalian cells via rapid fluorogenic Diels–Alderreactions. J Am Chem Soc 2012, 134:10317-10320.

59. Fleissner MR, Brustad EM, Kálai T, Altenbach C, Cascio D,Peters FB, Hideg K, Peuker S, Schultz PG, Hubbell WL: Site-directed spin labeling of a genetically encoded unnaturalamino acid. Proc Natl Acad Sci U S A 2009, 106:21637-21642.


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Protein conjugation with genetically encoded unnatural amino acidsIntroductionMethods for genetically incorporating unnatural amino acids into proteinsUnnatural amino acids with orthogonal chemical reactivityApplications to protein therapeuticsConclusionAcknowledgementsReferences and recommended reading

available online at · 2014. 6. 28. · acids chan hyuk kim1,4, jun y axup2,3,4 and peter g...

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