Download Antibody Conjugates with Unnatural Amino Acids

Review
pubs.acs.org/molecularpharmaceutics
Antibody Conjugates with Unnatural Amino Acids
Trevor J. Hallam,† Erik Wold,‡ Alan Wahl,§ and Vaughn V. Smider*,‡
†
Sutro Biopharma, 310 Utah Avenue, Suite 150, South San Francisco, California 94080, United States
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
§
Ambrx, Inc. 10975 North Torrey Pines Road, La Jolla, California 92037, United States
‡
ABSTRACT: Antibody conjugates are important in many areas of
medicine and biological research, and antibody−drug conjugates
(ADCs) are becoming an important next generation class of
therapeutics for cancer treatment. Early conjugation technologies
relied upon random conjugation to multiple amino acid side chains,
resulting in heterogeneous mixtures of labeled antibody. Recent
studies, however, strongly support the notion that site-specific
conjugation produces a homogeneous population of antibody
conjugates with improved pharmacologic properties over randomly
coupled molecules. Genetically incorporated unnatural amino acids
(uAAs) allow unique orthogonal coupling strategies compared to those used for the 20 naturally occurring amino acids. Thus,
uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based site-specific conjugation could
also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents.
KEYWORDS: antibody−drug conjugate, tRNA synthetase, unnatural amino acid, drug:antibody ratio, heavy chain, light chain,
Chinese hamster ovary, pharmacokinetics
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INTRODUCTION
Antibodies are highly specific molecules which can bind with
high affinity to their cognate antigens. As tools, biomedical
research has used conjugated antibodies as important reagents
for decades. These molecules encompass secondary antibodies
coupled to enzymes or fluorophores for detection of
biomolecules in multiple different formats like flow cytometry,
ELISA, and fluorescence microscopy.1−3 Common coupling
strategies use N-hydroxysuccinimide (NHS) esters (or other
alkylating or acylating compounds) which react with lysine
amines, or maleimide reactions with cysteine sulfhydryl groups.
Similar strategies enabled creation of the early antibody−drug
conjugates (ADCs) used in the clinic.4−6 Random coupling to
lysines or cysteines produce a mixture of different antibody
species conjugated at disparate amino acid sites. In this
heterogeneous mixture some antibodies might be conjugated
multiple times whereas other antibodies may not be coupled at
all. Because of the random nature of the conjugation process,
optimization efforts have aimed to limit the average number of
conjugations per antibody to between two and four. In the case
of ADCs, this label:antibody relationship is termed the drug to
antibody ratio (DAR). A low DAR (below 2) is expected to be
less potent, whereas an elevated DAR may lead to poorly active
ADCs, potentially due to destabilization, aggregation, increased
metabolism, or disruptive coupling in the antigen binding
pocket. Thus, a small window for optimal ADC activity exists
using random coupling chemistries. The same issues are also
present in conjugates used for research, for example,
fluorophore-labeled antibodies for biodetection. The technologies used to randomly label the natural amino acids have
© XXXX American Chemical Society
successfully generated the marketed ADCs ado-trastuzumab
emtansine (KADCYLA; Genentech/Roche) and brentuximab
vedotin (ADCETRIS; Seattle Genetics) as well as gemtuzumab
ozogamicin (Mylotarg; Pfizer), which was subsequently withdrawn from the market. Despite the success of randomly
coupled ADCs, Junutula et.al. recently showed that specific
engineered cysteine residues could be coupled to a drug sitespecifically, resulting in a homogeneous product that had
improved safety and pharmacokinetic profiles compared to
ADCs which were randomly coupled.7 However, the placement
of cysteine is limited due to the potential for unwanted disulfide
pairing and the varying stability of maleimide linkage used for
conjugation. Genetically encoded unnatural amino acids
provide another route to site-specific coupling and also enable
ADCs with favorable pharmacokinetic, potency, and antigen
binding properties.8,9 Thus, in procedures analogous to
determination of structure−activity relationships in small
molecules, the site-specific aspect of derivitization enables
protein-based medicinal chemistry where several analogues may
be made and tested as homogeneous products (as opposed to
mixtures) which should allow optimization of conjugated
antibodies for multiple uses.
There are several techniques which can modify proteins sitespecifically (Table 1).10−12 Cysteine is a reactive “natural”
Special Issue: Antibody-Drug Conjugates
Received: January 27, 2015
Revised: April 16, 2015
Accepted: April 21, 2015
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Table 1. Commercial Technologies for Site-Specific
Antibody Coupling
company
technology
Sutro Biopharma
Ambrx
Allozyne
Genentech/Roche
GNF/Novartis
Catalent
Pfizer
Meditope Biosciences
cell-free unnatural amino acid incorporation
cell-based uAA incorporation; engineered RS
methionine codon based uAA incorporation
THIOMAB; engineered cysteines
PCL; pyrrolysine analogue incorporation
formylglycine incorporation via FGE
transglutaminase-based conjugation
“Meditope” mediated noncovalent conjugation
amino acid; however, engineered cysteines coupled through
maleimide chemistry can be reversible and unstable, which are
important properties for an ADC. The context of the cysteine is
important, however, as the light chain mutation LC-V205C is
able to conjugate efficiently to produce unique stable ADCs.7,13
Technologies using site-specific enzymatic reactions can also
conjugate proteins in the context of a unique consensus
sequence in the substrate. Specifically, two methods, using
either formylglycine converting enzyme (FGE) or transglutaminase (TGM), can enable coupling to a sequence-specific
cysteine or glutamine, respectively. FGE produces formylglycine, which has an aldehyde side chain that is chemically
orthogonal to the naturally occurring amino acids, and
transglutaminase can directly catalyze a site-specific coupling
reaction. In the formylglycine method (SmarTag, Catalent,
Inc.), the FGE enzyme and antibody are coexpressed; however,
the antibody contains en engineered CXPXR motif which is
recognized by FGE, which catalyzes the conversion of cysteine
to formylglycine. The aldehyde on formylglycine is orthogonally reactive and can participate in several chemical coupling
strategies, including the hydrazino-Pictet−Spengler (HIPS)
reaction described below.14,15 In the transglutaminase system,
acyl acceptors derived with drug payloads are conjugated to the
glutamine residue within the LLQGA consensus sequence.16,17
While these two methods use consensus sequences to drive
site-specific coupling, other strategies have focused on single
side chains.
The proteins of most organisms use only the 20 common
natural amino acids as building blocks; however, certain
archaebacteria incorporate selenocysteine or pyrrolysine as
21st and 22nd natural amino acids.18 Maleimide analogues can
couple site-specifically to selenocysteine,19,20 and pyrrolysine
derivatives that have unique reactivities can be used to create
site-specific bioconjugates.21−23 Besides these unusual “natural”
amino acids, there are a number of non-natural amino acids that
have unique chemical properties that make them useful as
“chemical handles” for bioconjugate creation. These genetically
encoded uAAs can be incorporated recombinantly in in vitro
(cell-free) expression systems or in prokaryotic or eukaryotic
host cells. Here we will summarize the coupling chemistry and
uAA-based recombinant protein expression techniques in each
of these systems, as well as latest research into next-generation
bioconjugates and applications.
Figure 1. Unnatural amino acids and site-specific bioconjugation. (A)
p-Acetophenylalanine (pAcF) and p-azidophenylalanine (pAzF) (left
boxes) contain ketone and azide side chains which can participate in
oxime condensations and click chemistry reactions, both of which are
orthogonal to the side chains of the 20 natural amino acids. (B)
Schematic of the oxime ligation with pAcF encoded in the constant
region of an antibody Fab fragment.
creation of stable conjugates under the relatively mild
conditions needed to maintain protein integrity. While the
engineering and incorporation of these uAAs is discussed
below, the goal of producing homogeneous bioconjugates at a
single amino acid side chain requires unique chemical
reactivities to the uAA compared to the rest of the protein.
In this vein, the two primary chemical reactions used for
conjugation onto uAA containing proteins are the oxime
ligation and the copper-free click reaction. By incorporating the
pAcF amino acid site-specifically into a protein, one can use the
ketone moiety as a bioorthogonal electrophile for reaction with
an aminoxy nucleophile to generate a stable oxime linkage24(Figure 1A, top). This reaction proceeds in high yield in
buffered aqueous solution in the presence of an aniline catalyst,
and has been used to synthesize a large number of ADCs and
other protein conjugates.25−27 One drawback of this method is
that a pH of less than 5.0 is required for the reaction to
proceed, precluding acid sensitive proteins from use with this
method. The pAzF amino acid can be used to incorporate the
azide moiety required to perform the click reaction. The click
reaction, originally described by Sharpless et al., is a copper
catalyzed cycloaddition between an azide and a copper
activated alkyne.28,29 While this reaction is bioorthogonal, and
can be run in aqueous buffer at neutral pH, the presence of
copper can potentially cause oxidative damage to proteins. An
alteration of this reaction, using strained, high energy alkynes,
allows this reaction to proceed without copper catalysis and
broadens the scope of this reaction in biological contexts30,31(Figure 1A, bottom).
A novel variation of the bioorthogonal oxime and hydrazone
ligations was reported by Agarwal et al.14,32(Figure 2A).
Traditionally, oximes have been exclusively used in preference
of hydrazones when making protein conjugates, because,
despite the increased nucleophilicity of hydrazines at neutral
pH over aminoxys, the hydrozones formed by this reaction tend
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CONJUGATION TO UNNATURAL AMINO ACIDS
While well over 50 non-natural amino acids have been
genetically incorporated into recombinant proteins, the most
widely used for producing bioconjugates are p-acetophenylalanine (pAcF) and p-azidophenylalanine (pAzF) (Figure 1). The
side chains of these uAAs provide novel reactivity, enabling
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Using the chemistry described in the two Agarwal et al.
reports, Drake et al. reported the conjugation of maytansine to
a variety of sites on trastuzumab (Herceptin) via the HIPS
ligation.14,32,33 Maytansine was conjugated at three different
sites including in the CH1 and the LC and at the C-terminus,
and the activity and PK of each of the constructs was
investigated in order to determine how the site of conjugation
affected these properties. It was discovered that while there was
no difference in in vitro efficacy or serum stability, the Cterminus labeled species had greater efficacy as well as a longer
in vivo half-life. Importantly, the site-specifically modified ADC
had substantially lower toxicity than its randomly labeled
counterpart.
In a novel variation of the copper-free click reaction, Wallace
et al. report a strain promoted reaction of a phenyl sydnone 1,3dipole with a strained bicyclononyne dipolarophile to generate
a stable pyrazole product34(Figure 2C). Additionally, the sitespecific incorporation of a bicyclo[6.1.0]nonyne amino acid
into GFP is reported in high yields, further broadening the
application of this reaction into bioorthogonal conjugation. In
the context of protein conjugation, they reported quantitative
conjugation between GFP with the incorporated uAA and 25
equiv of phenyl sidnone labeled BODIPY in 6 h at 37 °C, pH
8.0. While the rate of the reaction was reported to be
comparable to that of the strain promoted copper-free click
reaction, the loss of CO2 in this reaction is an entropic driver
that will drive equilibrium of the reaction toward product.
Schmidt et al. report an approach to protein conjugation via
photo-cross-linking utilizing a furanyl uAA35(Figure 2B). With
the use of a photoinitiator and long wavelength light, a singlet
oxygen species is generated which will react via [4 + 2] with the
incorporated furanyl uAA. This transient intermediate will then
react with water to generate a 1,4-enedione which readily reacts
with cytosine-like compounds to generate a stable diazole
linkage. This approach is described as a method to photo-crosslink proteins and DNA to study protein−DNA interactions;
however, this process could potentially be utilized as a
conjugation technique for small molecules, considering the
mildness of the conditions.
Patterson et al. report an improved linker for the conjugation
of proteins via reaction with reduced cysteine residues (e.g.,
THIOMABs).36 While this chemistry is not relevant to
bioconjugation with pAcF or pAzF, it is an alternative sitespecific methodology that could also be applied in principle to
sulfhydryl containing amino acids (natural or unnatural). As
opposed to the traditional cysteine−maleimide reaction, a
sulfone moiety is utilized as the electrophile to react with the
nucleophilic free cysteine (Figure 2D). It is reported that the
sulfone linker has equivalent conjugation specificity and does
not affect ADC activity. The sulfone linker does, however,
result in increased serum stability by a significant margin when
compared to a comparable maleimide linker.
Figure 2. “Next-generation” conjugation approaches for uAA
bioconjugates. (A) Hydrazino-Pictet−Spengler (HIPS), using an
incorporated carbonyl moiety as a bioorthogonal handle, hydrazone
ligation is used as the first step to activate an intramolecular Pictet−
Spengler-like reaction in order to form a more stable C−C bond (as
opposed to relying on the hydrolytically less stable C−N bond). Rate
of reaction is 3-fold faster and serum stability is 5-fold longer than
comparable oxime formation. (B) A photoinitiator and long
wavelength light are used to generate singlet oxygen which reacts via
[4 + 2] with the incorporated furanyl uAA. The resulting intermediate
then reacts with water to generate a 1,4-enedione, which readily reacts
with cytosine-like compounds to generate a stable diazole linkage. This
method has been successful in applications of protein−DNA crosslinking and could potentially be used for conjugate synthesis. (C) A
novel variation of the copper-free click reaction. Strain promoted
reaction of a phenyl sydnone 1,3-dipole with a site-specifically
incorporated strained bicyclononyne dipolarophile uAA to generate
a stable pyrazole linkage. Loss of CO2 is an entropic driver in this
reaction to drive the reaction toward the product. (D) The sulfone
linker is an improved alternative to maleimide linkers for the
conjugation of proteins via reaction with reduced cysteine residues.
The sulfone linker has equivalent conjugation specificity and does not
affect ADC activity, yet shows increased serum stability by a significant
margin when compared to a comparable maleimide linker.
to be less stable in physiological conditions. However, using the
described conjugation chemistry, the oxime or hydrazone
ligation is used as the first step to activate an intramolecular
Pictet−Spengler-like reaction in order to form a more stable
C−C bond (as opposed to relying on the hydrolytically less
stable C−N bond) to create the protein conjugates. Additionally, because of the irreversible nature of this reaction, and
because one can use the hydrazone formation as the initial step
without sacrificing product stability, these reactions can be run
at neutral pH and without an aniline catalyst. Protein
conjugation using the hydrazino-Pictet−Spengler (HIPS)
chemistry on proteins containing site-specifically generated
formylglycine was shown to form at a rate about 3-fold faster
than that of comparable oxime formation. Additionally, serum
stability studies showed that the Pictet−Spengler modified
proteins are stable about 5-fold longer than the comparable
method.
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EXPRESSION OF UAA CONTAINING ANTIBODIES
IN CELLS
Expression of recombinant proteins with genetically encoded
uAAs by cells requires several key components. First, the uAA
should be able to penetrate the cell membrane and accumulate
in the cytosol where it will serve as a substrate for tRNA
synthetases. In this vein, the uAA must be chemically stable in
the cellular milieu and effeciently cross the membrane to
provide intracellular concentrations that exceed the RS Km for
efficient incorporation into the recombinant protein. Second, a
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recombinant tRNA synthestase, engineered to (i) specifically
acylate a tRNA molecule with the uAA and (ii) avoid acylating
the natural tRNAs that are associated with the 20 natural amino
acids, must be efficiently expressed. Third, an engineered tRNA,
which serves as a substrate for the engineered tRNA synthetase,
must also be coexpressed. This orthogonal reactivity of the RS/
tRNA pair is crucial for effective uAA incorporation in the
absence of unwanted background incorporation of natural
amino acids at the same position. Multiple reviews have
described the selection and molecular evolution strategies
involved in engineering the tRNA synthestases,37−39 which will
not be covered here. Lastly, the gene for the recombinant
protein must be modified by a codon recognized by the
anticodon loop of the engineered tRNA. The engineered codon
is often an amber (TAG) triplet but can also be four base pair
codons.40,41 The origin of the tRNA/RS pair is usually from an
organism with a distant evolutionary relationship to the host
cell. For example, the Methanococcus jannaschii tRNATyr/TyrRS
pair has been engineered to recognize several different uAAs in
Escherichia coli.37−39 Thus, the genetic requirements for
producing a recombinant protein in a host cell are genes for
(i) an engineered tRNA, (ii) an engineered tRNA synthetase,
and (iii) the protein of interest which has an engineered codon
recognized by the tRNA (Figure 3).
Well expressing model proteins, including green fluorescent
protein, T4 lysozyme, and myoglobin, have been expressed
with multiple different uAAs in various research systems.37−39
However, a unique phage display system was used to first
demonstrate uAA incorporation into antibody scFv fragments.
Liu et al. encoded sulfotyrosine in the CDR H3s of antibodies
and selected those which specifically bound HIV gp120.42
Antibodies are unique compared to the aforementioned model
proteins. First, the dimeric nature of the antibody requires
appropriate expression of both heavy and light chains;
unbalanced expression of these two components can lead to
aggregation and poor yield. Additionally, antibodies are
naturally secreted through an N-terminal signal peptide, and
their proper folding and assembly requires an oxidative
environment. The parameters of antibody expression with
uAA incorporation conditions in E. coli were studied by
Hutchins et al., who optimized both uAA incorporation and
conjugation using a M. jannaschii pAcF RSTyr/tRNA system
engineered to incorporate pAcF in response to the amber
codon. The trastuzumab (Herceptin) Fab fragment was used as
a model antibody fragment. Five positions were chosen for
evaluation in the light chain (LC). These positions were
spatially separate from one another in the LC constant region
and had good solvent accessibility based on the trastuzumab
crystal structure.43 Fab yields of these light chain mutants
ranged from 69% to 112% of the control wild-type protein.
Later work revealed that pAcF could also be incorporated
efficiently at position 138 in the heavy chain CH1 region.44 To
evaluate coupling efficiency, an alkoxyamine−Alexafluor 488
reaction with the incorporated pAcF was performed for each
mutant. The conjugation efficiency was from 33% (LC
Ser156pAcF) to 81% (LC Lys169pAcF). Thus, the pAcF side
chain is impacted by local effects on the protein surface
resulting in differential efficiencies of oxime formation.
Additionally, protein yield and uAA incorporation were not
correlated with coupling efficiency. Thus, the biological factors
governing uAA incorporation are independent of the chemical
reactions that ultimately impact bioconjugate formation.
Importantly, unnatural amino acid incorporation and con-
Figure 3. Insertion of uAAs into recombinant antibodies. (A) The
basic translation machinery of a ribosome (gray), tRNAs, mRNA, and
growing polypeptide are shown. A tRNA charged with its cognate
amino acid recognizes its specific codon in mRNA and transfers its
amino acid to the growing translation product. An engineered tRNA
and its cognate unnatural amino acid are shown in red, with the tRNA
recognizing the UAG codon. (B) Components and schematic for
production of recombinant antibody fragments encoding uAAs. An
engineered tRNA synthetase (labeled UAA RS; red) is expressed
within cells along with an engineered tRNA that recognizes the UAG
codon. The antibody genes are also recombinantly expressed with the
uAA incorporation site encoded by a TAG. Unnatural amino acid is
added to the cell medium and taken up by cells, and it is a substrate for
charging its cognate tRNA molecule.
jugation did not alter the Fab’s antigen binding properties, but
trastuzumab which was randomly coupled with NHS-biotin on
lysine residues had significantly diminished Her2 binding
activity.43
Pott et al. investigated how the sequence context of the
amber codon can affect the expression of proteins containing
uAAs.45 A 106 member library was generated consisting of
NNK-NNK-TAG-NNK-NNK in a protein to test whether the
sequences near the TAG make a difference on expression levels.
Successive positive and negative selections were performed to
select for sequences with maximal expression levels. They
found that the context sequence does matter, and with proper
selection can get protein expression levels equivalent to that of
the nonamber counterparts. It was identified that a high A
content in the context sequence and, in particular, an A at the
+4 position resulted in significant increases in expression of
uAA containing proteins.
While uAA incorporation and coupling efficiency can be
impacted by uAA positional context, a clear biological
functional impact of uAA position was shown by creating
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pharmakokinetic analysis revealed that the uAA based ADC was
equal to the unlabeled IgG.8
Tian et al. analyzed ADCs produced via different coupling
strategies. They compared (i) a monomethyl auristatin D
(demethyldolastatin 10; MMAD) conjugated site-specifically to
pAcF, (ii) MMAD coupled randomly to cysteines, and (iii)
MMAD coupled site-specifically to an engineered cysteine.9
Importantly, the optimal “thiomab” cysteine, LC-V205C,7 was
not compared in these experiments. Antibodies against two
antigens, 5T4 and Her2, were derivitized using the different
conjugation approaches and tested for biological activity. Equal
activity was observed for the pAcF labeled versus the randomly
coupled cysteine construct in cytotoxicity experiments with
5T4 transformed MDA-MB-435 cells. In cytotoxicity studies on
Her2+ cells using ADCs based on trastuzumab, the pAcF ADC
had up to 12-fold enhanced activity compared to the randomly
coupled cysteine ADC, which itself was 2- to 3-fold improved
over the site-specific cysteine conjugate. The randomly coupled
cysteine ADCs also had higher nonspecific killing activity on
Her2-negative cells. In addition to the linkage amino acid
(cysteine vs pAcF) and multiplicity of conjugation (site-specific
vs random), these ADCs also necessarily used different linker
moieties. Despite this, the stable oxime linkage may provide
more efficient intracellular release of the MMAD in the pAcF
ADCs.9 In orthotopic tumors, the randomly labeled cysteine
anti-5T4 ADC had no detectible therapeutic activity; however,
the pAcF conjugate induced tumor regression using four 10
mg/kg doses. In Her2+ orthograft models all of the constructs
had an antitumor effect; however, only the pAcF ADC
mediated long-term regression.9 These results suggest that
the oxime linkage provided by the pAcF ADC may confer
improved efficacy compared to site-specific or random cysteine
linkages coupled via maleimide chemistry. Broad conclusions
based on this data cannot be reached because these analyses
were done using constructs made with the optimal uAA
position (HC-A118C) as opposed to the optimal THIOMAB
position (LC-V205C).
Tian et al. also analyzed the pharmacokinetics of the pAcF
ADCs and found, quite surprisingly, that a MMAD labeled
HC115 had systemic exposure superior to that of unconjugated
IgG, and both of these molecules were better than a randomly
labeled ADC.9 One would expect that unconjugated IgG should
possess the maximal pharmacokinetics, and the mechanisms
underlying this unusual result are unclear. Perhaps charged or
hydrophobic patches on an ADC alter trafficking or pinocytosis
mechanisms in cells. Further work in elucidating these
principles could be undertaken by varying pAcF position and
linkers and analyzing the context for achieving these superior
PK properties.
While substantial work has been done in optimizing uAA
incorporation in E. coli, efforts are just beginning to yield
improved results in mammalian systems. Schmied et al. report
an optimization of unnatural amino acid incorporation in
mammalian cells via optimized pyrrolysyl tRNA synthetase/
tRNA expression and an engineered eRF1.53 The eRF1 protein
was engineered to enhance unnatural amino acid incorporation
in response to the amber stop codon without increasing readthrough of opal and ochre stops in mammalian cells. This
process is similar to previously reported RF1 deficient E. coli
cell lines for bacterial uAA incorporation into overexpressed
proteins (see below). This engineered eRF1, coupled with
increasing expression levels of the RS, resulted in an increased
protein yield of 17- to 20-fold from controls (wt eRF1 and
tetramers through nutravidin, where differences in tetramer
tertiary structure were controlled by coupling biotin to pAcF at
different positions. Remarkably, multimers made by tetramerization at light chain position 202 could inhibit Her2
phosphorylation in cell-based assays nearly 10-fold more than
tetramers coupled at residues 169 or 156 (EC50 of 0.06 nM
compared to 0.7 and 0.4 nM, respectively). Importantly,
tetramers could only be created by conjugating at a single
position in each Fab; randomly coupled Fab would result in a
multitude of different oligomeric species, and could not
efficiently create defined tetramers. Furthermore, these experiments established the importance of subunit orientation in
multimeric antibody constructs; there are now several bispecific
antibodies in clinical trials, and it is probable that optimization
of fragment orientation could improve the activity of certain
constructs. The use of uAAs to rapidly assess activity based on
different orientations of subunits is a feasible technological
approach to optimize protein multimers.43
With the ease of genetic manipulation and rapid and
standardized growth conditions of E. coli, many applications of
site-specific uAA technology have been realized, including
creation of antibody−fluorophore bioconjugates,43 toxinFabs,46 production of bispecific Fabs,44 small molecule−Fab
conjugates,47 siRNA−Fab conjugates,48 and Fab-oligonucleotide molecules for immuno-PCR.49 As an extension to the
methodology to create Fab-oligonuceotide molecules, the
oligonucleotide bases can be used to hybridize to their
complement in other Fab-oligo molecules, producing a system
where multimers can be made through the complementarity of
base pairs.50 This “oligobody” approach is not limited to
dimers, as unique trimeric or cruciform structures can be
envisioned to create higher order multimers.
Although E. coli has significant advantages, it is not useful for
producing posttranslational modifications in IgG at high yield.
Thus, several eukaryotic systems have been examined and
engineered for uAA incorporation. Many proteins have been
expressed with a multitude of different uAAs in the yeast
Saccaromyces cerevisiae; however, production of appropriately
glycosylated IgG at high yield is problematic. Model proteins
including human albumin have been expressed in Pichia pastoris
with uAA incorporation. Yields were 150 mg/L in shake flasks
with pAcF.51 Since others have engineered Pichia to produce
IgG with human glycosylation,52 it appears possible that uAA
containing IgG with appropriate glycosylation could be made in
this yeast.
The most common systems for IgG production for clinical or
research material are Chinese hamster ovary cells (CHO) and
HEK293 cells, respectively. Axup et al. used CHO cells to
express pAcF containing trastuzumab IgG to produce a sitespecific monomethyl auristatin F ADC.8 Comparison of this
IgG with an E. coli produced Fab-auristatin F revealed
cytotoxicity EC50s of nearly 100× for the IgG on Her2
transformed MDA-MB-435 cells. The Fab could be less active
because of its lower avidity, lower DAR, poorer internalization
due to its monovalency, or a combination of these factors.
Remarkably, the IgG’s EC50 (0.37 nM) is actually better than
that of the small molecule auristatin F in the same cell system
(1.5 nM). MDA-MB-435 cells lacking Her2 were unaffected by
the ADCs, showing clear antigen specificity of the site-specific
conjugate. Further work in mouse orthotopic tumor models
showed complete regression of Her2+ tumors at a single 5 mg/
kg dose, with no impact on Her2− xenografts. Importantly,
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thermophilus, yeast, rabbit reticulocytes, eukaryotic wheat
germ, Leischmanii, and CHO and human HeLa cells.57,58
Cell-free extracts made from E. coli are straightforward to make
and are widely used because prokaryotic ribosomes are highly
active, making about 20 peptide bonds per second in intact
bacteria.59 These rates fall to about 1 per second in vitro,
perhaps because of dilution. This rate is still similar to that for
eukaryotic ribosomes in intact cells, however. Significantly,
since only the protein of interest is being synthesized (whose
encoding DNA is added to the lysate), no resources are given
to host protein production in cell extracts. Modification of E.
coli strains and extracts60 can include engineering for
transcription and translation factors, but additionally for
enzymes enabling energy generation from low-cost substrates
like glutamate. A robust Kreb’s cycle and the presence of
oxidative phosphorylation on inside-out vesicles are important
aspects for commercial scale manufacturing in a cell-free
system.60
An important feature of cell-free extracts with regard to
antibody expression is their ability to support formation of a
quaternary structure that includes appropriately formed interand intrachain disulfide bonds. Normally the oxidizing
environment of the periplasm (in bacteria) and endoplasmic
reticulum (in eukaryotic cells) would provide this milieu, which
also includes various chaperones like prolyl and disulfide
isomerases.61 In this regard, Yin et al. demonstrated high
expression of both antibody fragments and full length IgG by
altering the redox potential as well as adding chaperone
proteins to the cell-free extract.62 Similarly, Groff et al.
engineered the host strain with combinations of chaperones,
and the resulting cell-free extract could produce IgGs at gram
per liter yields at the appropriate redox conditions in 12 h
reactions.63
With the production of large higher-order protein structures
like antibodies in vitro now feasible, the potential of genetically
encoding uAAs would require additional technology modifications to cell-free systems. As in cells, in vitro E. coli extracts
can utilize the UAG stop codon64 or four-base codons65 to
expand the genetic code. Initially, a significant challenge was to
achieve efficient tRNA charging with the uAA. Early experiments used relatively inefficient RNA ligase acylation
methods;66−68 however, recent advances may make this
technology more promising.69 As with cellular systems, the
solution to providing a replenishable source of uAA-charged
tRNA was to utilize engineered tRNA synthetases. Goerke and
Swartz (2009) incorporated pAzF into dihydrofolate reductase
using an M. janaschii RS-tRNA pair in an E. coli extract70 where
the uAA containing protein could subsequently be coupled to a
fluorescent dye. Later, Bundy and Swartz (2010) also
incorporated p-propargyloxyphenylalanine.71 Despite this progress, low expression yields, poor uAA incorporation, and
relatively low conjugation efficiencies through pAzF click
chemistry71 or pAcF based oxime condensation8 revealed the
need for substantial improvements in order for this technology
to be amenable for commercial use. In this vein, the cell-free
transcription/translation system was found to be ideal for the
kinetic analysis of orthogonal tRNA/aminoacyl-tRNA synthetases, as described below.72
uAA Incorporation Efficiency in Cell-Free Extracts. A
major advantage of cell-free systems is that they lack the cell
membrane; the uAA has unobstructed access to the RS and
tRNA to allow efficient tRNA charging and ready access to the
translation machinery. Despite this, however, uAA incorpo-
unoptimized RS expression). A single uAA incorporation
showed no significant difference in expression from wild-type
control protein, and two uAAs could be incorporated at
expression levels of 43% of the nonamber control.
Manufacturing in Cell-Based Systems. With more than
30 therapeutic antibodies on the U.S. market there are now
standard manufacturing processes including cell line development, expression and purification systems, and multiple
analytical techniques. Whereas expression of uAA-linkable
therapeutic proteins that do not require glycosylation has
been clearly demonstrated in E. coli, Chinese hamster ovary
(CHO) cells are the most common host cell line for
commercial recombinant antibody production. In this regard,
Liu et al. used CHO cells to incorporate multiple uAAs into
green fluorescent protein;54 however, uAA incorporation was
not as robust as E. coli based systems. Tian et al. engineered a
CHO cell line containing the RS and tRNA elements for
production of IgGs containing pAcF.9 A stable CHO K1 cell
line was made by electroporating two vectors: one encoding a
CMV-driven tyrosyl RS and a zeocin selectable marker, and the
second, a 40 copy tRNA and a puromycin selectable marker.
The pooled cells that were doubly resistant to zeocin and
puromycin were then further selected for suppression activity
by transiently transfecting a GFP construct that contained a
TAG at position 56, and performing FACS in the presence of
pAcF. The cells selected for high uAA incorporation efficiency
were then adapted for growth in suspension under serum free
conditions, which would provide a cell line with the appropriate
properties for large scale manufacture. This resulting cell line
(named 4E2) was compatible with the common glutamine
synthetase expression system commercialized by Lonza55 to
express pAcF contianing IgGs. This CHO cell line was used by
Axup et al. to encode pAcF at heavy chain position 121.8 The
antibody heavy and light chain genes were transfected and
selected as stable pools with expression yields of 20 mg/L, or as
selected clones with yields of 300 mg/L. All of the IgG
contained pAcF by mass spectral analysis, which was coupled to
alkoxyamine derived auristatin F with an overall yield of >95%
for the two pAcF uAAs (one per heavy chain) in the dimeric
IgG. Using the 4E2 system, 5L fed-batch processes produced
peak cell densities of 7 × 106 cells/mL and >1 g/L of IgG
containing pAcF at position HC 114. Thus, CHO-based
manufacturing of IgGs containing uAAs in the context of fully
glycosylated IgGs is feasible at commercial scale. This system
will enable manufacture of uAA-based ADCs for clinical
studies,9 and a uAA site-specific conjugate of anti-HER2auristatin F is expected to enter clinical trials later this year.
In Vitro Expression of Antibodies Containing Unnatural Amino Acids. Over 50 years ago it was demonstrated
that intact cells are not required for protein synthesis.56 Since
then many advances in elucidating and understanding the
transcription and translational machinery, as well as the
endoplasmic reticular components needed for appropriate
protein folding, have occurred. The major reason to move
away from cells for protein expression is the speed by which
many protein variants can be produced and analyzed using in
vitro extracts. Additionally, the uncoupled nature of a cell-free
extract allows individual components of the reaction to be
added, subtracted, or complemented in defined ways to
optimize production of complex proteins such as antibodies
with encoded uAAs.
Several extract or lysate-derived transcription/translation
systems have been developed from E. coli, Thermus
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per antibody. In this regard, the recently approved trastuzumabDM1 (KADCYLA) contains an average of 3.5 DM1 molecules
per antibody. An average IgG has 80−86 lysines, and 40 are
available for conjugation; therefore over one million different
conjugation products could theoretically be present within a
randomly conjugated mixture using lysine residues.6 Naturally
occurring cysteines may be a better option, as there are far
fewer available cysteines per antibody; however, there are still
several potential variants that could form with a random
conjugation scheme. The biochemical characteristics of the
individual molecules within the conjugation product could vary
substantially depending on the DAR and locations of the
coupling events.7−9,13,80
Obviously, site-specific conjugation enables the ability to
precisely define the coupling position. With an in vitro
translation strategy, one can rapidly vary the number of drug
molecules to conjugate in a defined manner, and also test
several combinations of positions in optimization efforts. This
strategy could allow true structure−activity relationships to be
defined and avoid the problems associated with the
heterogeneous mixtures of random coupling.
Manufacturing Using Cell-Free Systems. Cell-free
extracts provide a novel opportunity to separate cell growth
and extract production from recombinant antibody expression.
In this vein, the lysate is effectively a raw material used for
protein expression and can be used at all stages in the
preclinical path, from discovery via ribosome display to
commercial manufacturing. While cell-based systems can utilize
transient transfection for initial preclinical studies, ultimately
stable clones are required for later development. As stable cell
lines are not necessary for commercial production using in vitro
systems, a savings of effectively 12−18 months can potentially
be obtained from a typical preclinical development timeline.
The rapidity of the process is due to the fact that the cell extract
(and the preceding cell growth) does not depend on the
composition of the expressed protein of interest. Along these
lines, hundreds to thousands of “hits” from a ribosome display
selection, or positional variants of a uAA in an ADC, can be
made within 12 hours. Furthermore, interesting clones can
subsequently be scaled to several grams within days. In contrast
to cellular production for preclinical studies, cell-free processes
do not require production of a stable cell line to manufacture
significant quantities of high-quality antibody for IND-enabling
studies. Thus, the potential for in vitro production systems is
that they could potentially save significant preclinical development time, provided that they can provide the consistency and
quality needed for cGMP (current good manufacturing
practices) manufacturing.
Only recently has the potential for cell-free systems to
produce recombinant protein at gram and kilogram scales been
realized. In this regard, the cGMP processes used to scale
typical microbial production systems to the thousands of liters
were used to produce high yields of E. coli extracts.60 Yin et al.
(2012) successfully produced aglycosylated trastuzumab in IgG
format, a molecule which contains 16 appropriately formed
disulfides.62 In vitro production of antibodies may be specifically
useful for antibody formats where glycosylation is unnecessary,
such as ADCs, bispecific antibodies, or function blocking
antibodies that do not rely on glycosylated Fc receptors for
effector function (e.g., antibody dependent cellular cytotoxicity
or complement dependent cytotoxicity). However, ongoing
work to engineer extracts to synthesize functional glycoproteins
has shown progress,81 and efforts by Wittrup82 and Georgiou83
ration is still negatively affected by competition of the
endogenous release factors at the UAG codon, which is
naturally a stop codon but was co-opted to encode the uAA.
This competition can result in a mixture of recombinant
proteins where the full length protein containing the uAA is
produced along with a truncated variant made as a result of the
release factor preventing suppression at the UAG codon. In
both cell-based and in vitro systems expression of full length
protein containing uAA can be as little as 30% of the wild-type
protein that does not encode a uAA.73 Release factor 1 (RF1)
causes truncation at UAG; unfortunately, however, RF1
knockouts are lethal in both prokaryotic and eukaryotic cells.
Recently, there has been progress in generating RF1 knockout
strains where RF2 is engineered to complement RF1 loss and
allow incorporation of uAAs efficiently.74,75
A unique attribute of cell-free protein expression is that the
process of cell growth and extract production is separated from
protein production. Therefore, it is feasible to remove or
inactivate components from the extract, like RF1 or other
factors required for cell growth, prior to the extract’s utilization
to produce the recombinant protein. For example a polyclonal
antibody targeting E. coli RF1 could improve uAA incorporation in an E. coli cell-free system.76 Additionally, Suga et al,77
used an anti-RF1 RNA aptamer to substantially enhance UAG
suppression in two E. coli derived cell free systems.
Recently an RF1 deficient E. coli strain was made by Jewett et
al.78 where they ensured viability by reassigning the TAG codon
to TAA at 13 positions in the E. coli genome. The S30 lysate
was tested for pPaF (p-propargyloxy-L-phenylalanine) or pAcF
incorporation into sfGFP, and a 2.5-fold improvement in
production was seen compared to the wild-type parental strain.
Hong et al. reported a cell-free protein synthesis protocol for
uAA containing protein expression with cell lysates from RF1
deficient E. coli.79 A significantly better yield was achieved
utilizing S30 protein lysates from an RF1 deficient E. coli strain
than S30 protein lysates from a wildtype E. coli. A 250%
increase in yield from the lysates of the parent strain to the RF1
deficient strain was reported. It was also shown that the uAA
could be incorporated at up to 5 sites while still maintaining
acceptable protein expression levels, and established that the
cell-free protein synthesis system is far more cost-effective at
the production scale than comparable whole cell approaches. In
a unique approach, Thanos and co-workers engineered a
protease cleavable RF1 in E. coli which is proteolyzed by
endogenous OmpT during the extraction process. The resulting
cell-free system, now devoid of RF1, produces uAA containing
proteins to levels similar to the wild-type protein.
uAA Incorporation and DAR. The importance of uAA
incorporation efficiency is revealed when one considers the
drug payload per antibody. A single uAA per antibody gene will
result in two drugs attached per heterotetramer. Therefore,
increasing the DAR will require efficient incorporation of more
than one uAA. In fact, different drugs (and different antibodies)
may warrant different DARs for optimal effects. One of the
major variables in ADCs made through random coupling
technologies is the number of drugs that can be attached per
antibody without causing deleterious biochemical properties.
The currently understood optimal DARs are based on
maytansines and auristatin derivatives using random coupling
chemistries to the natural amino acids. However, coupling to
accessible lysines and cysteines results in imprecise labeling in
regard to both the amount and location of conjugation, yielding
mixtures that can contain between 0 and 9 molecules of drugs
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Review
DEVELOPMENT ISSUES FOR UAA CONTAINING
ANTIBODIES
Manufacturing. Commercial manufacturing of recombinant proteins containing uAAs has additional issues compared
to antibodies composed of only the 20 natural amino acids. As
described above in depth, these challenges relate to (i) ensuring
efficient uAA incorporation by engineering optimal tRNA
synthetase activity and (ii) providing enough tRNA and uAA.
Recent advances allow expression of several tRNA copies,9,73
which can be limiting for effective uAA expression in cells. A
solution for commercial manufacturing utilizing CHO cells
stably engineered with tRNA and RS to produce pAcF
containing antibodies appears viable,9 and similarly engineered
cell lines could potentially be constructed for other uAAs as
well. While pAcF-based proteins are now on the development
path, most engineering and research efforts for other uAAs have
focused on E. coli based incorporation. As not all E. coli based
RS/tRNA pairs are orthogonal in eukaryotic systems, it may be
necessary to select and engineer synthetases up front in
eukaryotic cells. Cell-free systems, on the other hand, take
advantage of the rapid and well-characterized processes derived
from E. coli fermentation.60,62 In both systems new efforts are
likely to continue improving uAA incorporation, removing
natural amber codons from the host genome to eliminate readthrough of genes, optimization of orthogonal ribosomes, and
impoved methods to eliminate RF1 based translation
inhibition.
Potential Immunogenicity. The risk of immunogenicity
exists for any protein therapeutic, including recombinant fully
human antibodies.87,88 Immunogenicity results from a response
by T-cells that were not eliminated during thymic selection by
self-peptides bound to MHC during T-cell maturation. In this
regard, any amino acid change in a self-protein (either natural
or unnatural) could potentially produce a new immunogenic
peptide when presented on MHC molecules to stimulate a Tcell response. Helper T-cells then collaborate with low affinity
self-antigen binding B-cells, with the latter being driven to
produce anti-self antibodies. In theory, certain unnatural amino
acids which are substantially different in chemical composition
or structure may have an increased risk of generating altered
peptides that may induce a T-cell response. In this vein, the side
chains may be so substantially different from the natural amino
acid (or even conservative natural amino acid changes) at the
same position that the resulting peptide−MHC complex would
activate T-cells, and be predisposed to conferring immunogenicity. However, the commonly used pAcF is a structural
analogue of phenylalanine and tyrosine, and it is unclear if it has
any specific tendency to alter MHC binding and stimulate a Tcell response beyond its natural analogues. Importantly for an
ADC, the presented peptide likely would include the uAA as
well as the conjugated linker and drug moiety. In this context, it
is currently unknown what the composition of a final peptide
presented on MHC molecules may include; but this
composition will likely be dependent on the linker, drug, and
their stability through the MHC processing pathway. Similarly,
coupling to the natural amino acids lysine or cysteine could also
generate “nonself” peptides (with an attached linker and/or
drug) capable of being recognized by T-cells. The diversity of
peptides potentially created by random coupling also creates a
theoretical possibility that one or more of the conjugated
residues could create an immunogenic peptide.
demonstrate that certain mutations within the Fc region can
increase or improve effector function in aglycosylated antibodies.82,83 Therefore, although some applications of cell-free
extracts may be currently limited, the potential to apply these
Fc variants to lysate-based protein production may eventually
enable additional Fc function.
Coupling Efficiency with uAAs. As mentioned above in
discussing uAA expression and conjugation in cellular systems,
two major variables in producing antibody conjugates are the
uAA incorporation (suppression efficiency) and the conjugation chemistry efficiency. Several uAAs have been successfully
incorporated in vitro using a M. jannaschii TyrRS, including
pAcF, bipyridyl-F, and, with lower efficiency, pAzF.75,84 As
mentioned previously, p-azidophenylalanine is potentially quite
useful in ADCs as it can participate in click chemistry-based
conjugation.84 Trastuzumab IgG with pAzF has been produced;
however, the kinetics of conjugation are relatively inefficient
with the strained cyclooctyne derivative, dibenzyl cyclooctyne
(DBCO) functional group on the linker.85 In theory, altering
either the uAA or the linker could serve to optimize the
reaction. Using a cell-based system, however, modification of
the uAA would additionally require substantial effort as each
uAA derivative would also require evolution of a new RS. A
potential option for cell-based systems would be to use a cell
line with a polyspecific synthetase86with the caveat that the RS
activity toward the uAA derivatives may not be maintained. As
an additional concern, the modified uAA may have other
deleterious consequences for use in cells such as reduced
capacity to traverse the plasma membrane. Therefore, the focus
in cell-based uAA systems has been to optimize the reaction
through linker modifications as opposed to the uAA itself.
Alternative approaches focusing on the uAA using cell-free
systems are possible and provide a unique opportunity for
optimization.
Zimmerman et al. created a library of pAzF derivatives and
screened for enhanced DBCO reactivity and maintainance of
selectivity.85 They identified pAzMeF as a 7−10-fold improved
variant over pAzF. While improving reactivity for DBCO, the
methylene spacer makes the uAA hydrophilic and potentially
less useful for cell-based systems. To improve incorporation
efficiency of pAzF and pAzMeF, a recombinant 1760 member
library was created in the active site of Mj TyrRS.85 The
individual RS variants were screened for their ability to suppress
a TAG containing GFP using a cell-free extract. Controls for
the screen included GFP expression in the absence of uAA or in
the presence of alternative uAAs to evaluate mischarging.
Multiple synthetase variants were discovered that specifically
and efficiently charged pAzMeF. While the impact of in vitro
protein expression systems for improving protein function
through high throughput screening was shown, similar
approaches could be extended to identify novel synthetases
for multiple other uAAs. In this regard, it seems feasible that,
with a toolbox of uAAs and variant synthetases, multiple uAAs
could be tested for functional activity in a variety of
recombinant proteins at a multitude of positions. Thus, a
combinatorial approach where both uAA composition and
position are varied could be envisioned for testing binding
domains on cytokines, regulatory or active sites in enzymes, or
complementary determining regions in antibodies. Thus, in the
context of cell-free expression systems, protein medicinal
chemistry and SAR are within reach with currently available
techniques, opening exciting avenues for future biochemical
investigation.
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Figure 4. Next-generation bioconjugates enabled by uAA technology. (A) Bispecific Fab fragments targeting T-cells to tumors, small molecule-Fab
conjugates, site-specific drug conjugates, and dual drug conjugates are all unique molecules that have been made using uAA conjugation technology.
(B) Coupling oligonucleotides to antibody fragments enables immuno-PCR, as well as unique multimeric complexes, where novel strategies for
nucleic acid hybridization can be used to effectively couple subunits in defined ways.
seeing extensive use as an approved veterinary medicine in
several countries and to date has shown no evidence of
neutralizing antibody response. Thus, preclinical and clinical
evidence currently support uAAs as a site-specific mechanism
for bioconjugation without enhanced risk of immunogenicity.
Certain unnatural amino acids are known to break tolerance
in a T-cell dependent, site-specific manner.89−92 In this line of
research p-nitrophenylalanine was incorporated into the selfprotein TNF-α in an effort to “haptenize” the self-protein, as
pNO2F is similar to common haptens known to elicit a robust
immune response when coupled to self-proteins. Certain uAAs
may have theoretical concern for binding to naive B-cells;
however, they should only induce an autoimmune reaction if
they also produce a new T-cell epitope.
As a result of drug development activities of therapeutic
proteins containing uAAs, there is now experience in measuring
immunogenicity in preclinical animal models as well as human
clinical trials, and the early results are encouraging. Ambrx’s
PEGylated human growth hormone (ARX201; PEG-hGH)
comprises recombinant human growth hormone with sitespecific substitution of a single pAcF, physically distal to the
receptor binding domain. The chemically reactive carbonyl
group of pAcF is then covalently conjugated with an oxyaminoderivatized 30 kDa polyethylene glycol (PEG) molecule.
Unpublished data from preclinical studies showed, as
expected, that most rats developed an antibody response to
the human ARX201 with approximately 75% having a
neutralizing antibody response by day 29 following repeated
dosing. Notably, however, comparable repeat dosing of
nonhuman primates produced no evidence of an antibody
response to ARX201. All significant toxicology findings were
consistent with pronounced effects of the normal pharmacological activity of hGH. Consistent with this, data from
extensive human clinical studies indicated negligible immunogenicity to ARX201 and no detectable immunogenicity to hGH
over almost 12 patient years of total exposure to ARX201.
By way of partnerships other extended half-life therapeutic
proteins using the Ambrx site-specific linkage technology have
entered or will enter the clinic this year, with no preclinical or
clinical evidence of neutralizing anti-drug immune response.
Ambrx’s PEG-hLeptin is expected to enter clinical trials this
year. In part by virtue of its improved protein stability at neutral
pH and in circulation, preclinical studies of PEG-hLeptin have
shown significantly reduced immunogenicity as compared to
recombinant human leptin alone. Lastly, the Ambrx-engineered
extended half-life biotherapeutic PEG-bovine G-CSF is now
■
FUTURE DIRECTIONS
New Antibody−Drug Conjugates. Now that the
technology and manufacturing principles are in place for
antibody−drug conjugate generation using uAAs, these
techniques can be applied to any recombinant antibody.
While early proof-of-concept studies used well-known model
systems like trastuzumab, other applications have also recently
been reported. The GPCR CXCR4 is known to be overexpressed in many metastatic tumors and is a negative predictor
of survival in some cancers. Kularatne et al. report an antiCXCR4−auristatin antibody−drug conjugate.93 The sitespecific conjugation of the auristatin drug to the anti-CXCR4
antibody was accomplished via oxime ligation between the sitespecifically incorporated pAcF uAA and an aminoxy containing
auristatin-linker compound (the linker used in this study is a
noncleavable PEG linker). In vitro assays showed this ADC to
be highly potent against CXCR4 expressing cancer cell lines
with an EC50 of 80−100 pM. The in vivo efficacy of the ADC
was investigated with an osteosarcoma lung seeding tumor
metastasis model in mice and was shown to eliminate
secondary pulmonary lesions with 3 doses of ADC at 2.5
mg/kg.
Other Unique Bioconjugates. Now that therapeutic
proteins containing uAAs are in the clinic,94 next generation
molecules mediated through uAA conjugation can be
envisioned. The possibility of encoding two different uAAs in
a single protein appears feasible,95 with interesting applications
including coupling to two different drugs or potentially
combinations of drugs and imaging agents. Unnatural amino
acids beyond pAcF or pAzF, such as bipyridyl-alanine for
chelating metals like 64Cu, may have advantages in either
radioimmunotherapy or specialized tumor imaging. As new
applications of click chemistry, uAAs with a ring strained alkyne
can be encoded by an evolved Methanosarcina mazei
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pyrrolysine synthetase, and used to conjugate fluorescent dyes
directly in cells.96 Production of ADCs with this uAA is
certainly feasible.
Beyond ADCs, the use of uAAs to form other types of novel
conjugates is also possible (Figure 4). Bispecific antibodies have
now shown clinical benefit,97 and targeting tumor-specific
antigens with killer T-cells is a broad strategy with likely
application in multiple tumor types. Most strategies to
construct bispecific antibodies rely on genetic fusion of two
antigen binding fragments; however, chemical coupling of
antibodies via uAAs allows for optimization of the steric
properties and orientation of the subunits.43 In proof of
concept studies using this approach, Kim et al. produced an
anti-CD3 Fab UCHT1/anti-Her2 bispecific Fab encoding pAcF
in each constant region, coupled them separately to a
cyclooctyne and azide, respectively, and then conjugated
them together using a copper-free click reaction.44 The
bispecific Fab was able to direct human T-cells to kill Her2+
breast cancer cells in vitro.
Lu et al. report a bispecific anti-CLL1/anti-CD3 antibody for
the delivery of cytoxic T-cells to acute myeloid leukemia cells.98
The bispecific antibody consists of an anti-CLL1 Fab and an
anti-CD3 Fab connected through a bispecific PEG linker that
reacts with the site-specifically incorporated pAcF uAA via
oxime ligation with an aminoxy moiety and contains either an
azide or cyclooctyne at the other end. The linkers are
conjugated to each Fab (each Fab couples to either the azide
or the cyclooctyne linker), and then the two Fabs are joined via
the copper-free click reaction. An anti-CD33/anti-CD3
bispecific Fab was also produced for comparison purposes
(CD33 is the target of Mylotarg). The two bispecific Fabs were
tested for activity with both AML cell lines and patient derived
AML cells and were evaluated in mouse xenograft models. In
every case, the anti-CLL1/anti-CD3 molecule was significantly
more potent than the anti-CD33 counterpart.
Van Dieck et al. engineered bispecific Fabs to more
accurately analyze protein−protein interactions.99 Each Fab
was selected to have a rapid off rate such that if only one
epitope was bound, the bispecific Fab would be lost during
washing. However, only upon simultaneous, cooperative
binding to both epitopes was stable target binding accomplished. Using the engineered bispecific antibodies, specific
detection of the activated Her2/Her3 complex in formalinfixed, paraffin-embedded cancer cells was achieved. Additionally, superior detection specificity was observed for phosphoHer3 compared to the corresponding monoclonal antibody.
In an approach related to the T-cell recruiting bispecific
antibodies, target specific small molecules can be used to
generate bispecific molecules to recruit cytotoxic T-cells to
cancer cells.47,100 Kulutrane et al. reported the synthesis of a
bispecific anti-CD3−folate conjugate for the treatment of a
variety of cancers that overexpress the folate receptor
FOLR1.101 In vitro assays showed this anti-CD3−folate
conjugate to be highly potent against a number of FOLR1
overexpressing cancer cell lines with an EC50 of 10−100 pM
depending on the specific cell line, and concentrations of up to
1000-fold higher had no activity against FOLR1 negative cell
lines. In vivo efficacy of the anti-CD3−folate conjugate was
tested in a KB cell xenograft model in mice. Ten daily doses of
1.5 mg/kg were administered starting the same day as
implantation and shown to be very effective. Additionally,
Cui et al. conjugated folate to selenocysteine on the V9 antiCD3 Fab and could also target the FOLR1 receptor on
tumors.100 Similarly, the same group used LLPA2 conjugated to
anti-CD3 Fab to target the α4β1 integrin.100 LLPA2 specifically
binds the active form of α4β1, which may provide further
specificity in targeting. DUPA, a small molecule which binds
and inhibits the active site of the PMSA protease on prostate
cancer cells, was conjugated to pAcF on an anti-CD3 Fab and
was used by Kim et al. to redirect T-cells to prostate tumors.47
Thus, multiple examples of small molecule−antibody conjugates have shown activity as novel bispecific agents created
through uAA or selenocysteine coupling.
Nucleic Acid Conjugates. While bispecific molecules have
opened up unique mechanisms of action to antibody
therapeutics, the construction of higher order multimers
could enable a wide range of binding and effector functions
to be contained within a single molecule. Chemistries
orthogonal to the 20 natural amino acids are limited, with
click chemistry and the oxime conjugates being the most widely
used. Thus, production of multimers using chemical methods
are potentially limited to only two conjugation strategies.
However, recently Kazane et al. used the base-pairing
properties of nucleic acids to construct Fab multimers.50
Since base-pairing combinatorics are nearly limitless, multiple
different complementary oligonucleotides can be engineered to
enable subunit coupling. In this regard, trimers, tetramers, and
larger complexes could be exquisitely controlled through novel
cruciform and other base-pairing strategies (Figure 4).
Furthermore, analogues of natural nucleic acids such as PNAs
(protein nucleic acids) may have favorable properties in vivo
such as high stability and protease and nuclease resistance.
PNA−uAA conjugates are straightforward to produce; a Fab
fragment containing pAcF could be conjugated to a PNA
molecule with an aminoxy modification, and this Fab-PNA
hybridized to its complement to form a Fab-dimer at high
yield.50
Cross-Linking Biomolecules. A novel approach to answer
certain biological research questions involves the covalent
cross-linking of an antibody to its antigen. Furman et al. report
the incorporation of electrophilic uAAs into the CDR3 of
Herceptin for cross-linking antibody−antigen complexes.102
This methodology was investigated by incorporating three
variably electrophilic uAAs (N-acryloyl-lysine, p-acrylamidophenylalanine, and p-vinylsulfonamido-phenylalanine) into the
CDR3 and attempting to achieve covalent cross-linking of the
antibody−antigen complex. The incorporated p-vinylsulfonamide-F provided the most efficient reaction and resulted in
rapid covalent cross-linking of the anti-ErbB2−ErbB2 complex
at physiological pH with >95% yield, showing this method to
be a robust protein−protein cross-linking strategy for native
target proteins. Additionally, considering the tunable electrophilicity of the Michael acceptor amino acids, one could
envision the described uAAs being used as a toolkit to achieve
conjugation in a variety of pH ranges.
Similarly, Chen et al. report the incorporation of electrophilic
uAA 2-amino-6-(6-bromohexanamido)hexanoic acid (BrC6K)
with a long linear alkyl side chain and terminal bromide as the
leaving group, and application of this incorporated uAA as a
means for protein cross-linking.103 Proteins are cross-linked via
SN2 reaction with a nucleophilic amino acid residue displacing
the primary alkyl bromide in the BrC6K uAA.
Xu et al. describe a computational, structural investigation at
incorporating uAAs into antibody CDRs for covalent crosslinking of antibody−antigen complexes.104 Computational
biology was used to analyze what sites within CDRs of the
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protein than previously described methods. In 2014, Lammers
et al. reported an optimized method for the previously
described system of incorporating multiple uAAs via an evolved
ribosome.107,108 It was identified that the orthogonal aminoacyl-tRNA synthetase was the primary limiting factor of protein
yield in this system. Balancing the expression levels of each of
the individual components significantly improved expression
levels and growth rates. Additionally, a new streamlined and
optimized set of plasmids was created in order to broaden the
potential applications of this method.
A modified and reconstituted cell-free extract (the PURE
system109) has been developed to allow several codons for use
with multiple uAAs. In this regard, natural RS’s have been
deleted from the extract so that endogenous tRNAs can be
repurposed to transfer uAAs.110 However, this system still lacks
efficient charging of the uAA-tRNA necessary for commercial
manufacture.
Diagnostics and Research Reagents. Antibodies have
been extremely successful as therapeutics, diagnostics, and
research reagents. While uAA technology has been applied
commercially to develop therapeutic product candidates, there
is considerable opportunity to generate unique next-generation
diagnostics and research tools. For example, fluorescently
labeled antibodies are widely used in several applications,
including flow cytometry,43,96 and the same issues with random
labeling also affect diagnostic and reagent biomolecules (a
population of molecules with varying and suboptimal activity is
produced). Thus, uAA-based site-specific labeling could
engender greater activity and specificity, and such molecules
have already been made using click or oxime chemistry.43,49
Another area where site-specific antibody labeling could
enable enhanced specificity and activity for biodetection is
immuno-PCR. Recently Kazane et al. coupled oligonucleotides
site-specifically on pAcF in the trastuzumab Fab and utilized
this molecule to perform PCR to detect the Her2 antigen on
breast cancer cells.49 In fact, rare Her2+ cells could be identified
in the background of white blood cells, which mimicks the
situation of circulating tumor cells in patients.49 However, when
the trastuzumab Fab was randomly labeled, substantial
nonspecific PCR amplification occurred on Her2-negative
cells.43,96 Thus, uAA-based oligonucleotide bioconjugates
could expand the use of immuno-PCR methodology.
antiprotective antigen scFv antibody M18, the uAA 3,4dihydroxyphenylalanine, could be incorporated into in order
to achieve the highest antibody−antigen cross-linking. The ten
best computationally derived variants of the 3,4-dihydroxyphenylalanine containing antibody were synthesized and assessed
for covalent antibody−antigen cross-linking, with the best
variant cross-linking to 52% of the available antigen. This
illustrates that choosing incorporation sites via computational
biology is a viable approach.
Combination Bioconjugates. A key parameter in the
success of an ADC is the therapeutic index. Interestingly, the
efficacy of KADCYLA appears to continually increase in low,
medium, and high exposure patients (Immunogen presentation
at PEGS Lisbon, Fall, 2013), suggesting that ADC doses are
likely limited by tolerability to the cytotoxin.105 In this regard,
site-specific antibody−drug conjugates improve the ADC’s
pharmacokinetic properties, enhancing the warhead delivery to
the tumor and decreasing the overall systemic exposure.7,9,12
However, improvements can potentially be made by combining
two different warheads into one ADC, a strategy that could be
enabled by two orthogonally reactive uAAs.
The incorporation of two different uAAs allows the potential
to simultaneously couple two different drugs to one targeting
antibody. There are several potential advantages this type of
molecule could confer, including the following: (i) heterogeneous tumors may have cells with differential resistance to
different drugs (e.g., cancer stem cells and bulk tumor cells),
and all of these cells could be targeted with a single antibody
empowered with a dual payload; (ii) the two drugs could have
mechanistic synergy, thus enhancing activity in a single
molecule. Critical components to incorporating two different
uAAs are optimal suppression efficiency of different RS/tRNA
pairs and the necessity of two different codons to be
suppressed.106 While most uAAs have been incorporated in
response to the amber codon, other stop codons like opal,
ochre, and four base pair codons are potentially available for use
in a dual drug ADC gene.
Xiao et al. combined an amber suppressing polyspecific E. coli
tyrosyl tRNA synthetase (originally evolved to charge Omethyltyrosine) and an ochre suppressing Methanococcus
barkeri pyrrolysyl tRNA synthetase to incorporate pAcF and
azido-lysine (AzK) into trastuzumab in HEK293-F cells. The
pAcF was encoded at light chain position 110 and coupled with
an alkoxyamine-auristatin F, and AzK encoded at heavy chain
position 121 and conjugated to Alexa Fluor 488-DIBO by
copper-free click chemistry. The unique fluorophore-ADC was
used to monitor both binding and cell killing processes in SKBR-3 (Her2+) cells.95 The yield of this dual uAA molecule was
about 10% of that expected for incorporation of a single uAA in
eukaryotic cells.95 Despite the poor expression levels, this work
demonstrates the feasibility and utility of incorporating two
uAAs in a single antibody.
In an in vitro system Neumann et al. reported multiple uAA
incorporation in response to several quadruplet codons and an
amber stop codon by an engineered orthogonal ribosome.107
This system provides multiple blank codons on an orthogonal
mRNA, which the evolved ribosome can translate. By
combining RS/tRNA pairs with the evolved ribosome, the
incorporation of multiple distinct uAAs is encoded by two of
the new blank codons. Both alkyne and azide uAAs were
incorporated into a single protein and could be coupled to one
another using click chemistry. The incorporation of two distinct
uAAs with this method achieves higher yields of recombinant
■
CONCLUSIONS
Proteins containing unnatural amino acids are now in deep
clinical development, with several ADCs also advancing toward
the clinic. Significant data supports the notion that site-specific
conjugates have improved efficacy and pharmacokinetics as a
result of their homogeneous nature. While production
challenges exist, both bench and fed-batch scales have been
successful in producing uAA containing antibodies, and
significant efforts in bringing more uAA-based proteins to the
clinic are sure to advance these manufacturing processes. With
a path to clinical utility now visible, new bioconjugates enabled
by uAA incorporation, including nucleic acid-based multimers
and multidrug conjugates, provide exciting next-generation
possibilities of this technology.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
K
DOI: 10.1021/acs.molpharmaceut.5b00082
Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Review
Molecular Pharmaceutics
Notes
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