Antibody Conjugates with Unnatural Amino Acids - Molecular

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Antibody conjugates with unnatural amino acids Trevor Hallam, Erik D Wold, Alan Wahl, and Vaughn V Smider Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00082 • Publication Date (Web): 21 Apr 2015 Downloaded from http://pubs.acs.org on April 26, 2015

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Molecular Pharmaceutics

Molecular Pharmaceutics - DRAFT

Antibody conjugates with unnatural amino acids Trevor J. Hallam1, Erik Wold2, Alan Wahl3, and Vaughn V. Smider2 1

Sutro Biopharma, 310 Utah Avenue, Suite 150, South San Francisco, CA 94080 The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037 3 Ambrx, Inc. 10975 N. Torrey Pines Rd., La Jolla, CA 92037 2

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 heterogenous mixtures of labeled antibody. Recent studies, however, strongly support the notion that sitespecific conjugation produces a homogenous 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 thosed used for the naturally occuring twenty amino acids. Thus uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based sitespecific conjugation could also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents. Key Words ADC – antibody drug conjugate RS – tRNA synthetase uAA – unnatural amino acid DAR – drug:antibody ratio HC – heavy chain LC – light chain CHO – Chinese hamster ovary PK - pharmacokinetics

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 fuorophores 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 1 ACS Paragon Plus Environment


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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 heterogenous mixture some antibodies could 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 use, for example fluorophore-labeled antibodies for biodetection. The technologies used to randomly label the natural amino acids have 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 site-specifically, resulting in a homogenous 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 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 analogs may be made and tested as homogenous products (as opposed to mixtures) which should allow optimization of conjugated antibodies for multiple uses. There are several techniques which can modify proteins site-specifically (Table 1)(10-12). Cysteine is a reactive “natural” 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 2 ACS Paragon Plus Environment

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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

Figure 1. Unnatural amino acids and site-specific bioconjugation. A. pacetophenylalanine (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 natural twenty amino acids. B. Schematic of the oxime ligation with pAcF encoded in the constant region of an antibody Fab fragment.

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 common 20 natural amino acids as building blocks, however certain archaebacteria incorporate selenocysteine or pyrrolysine as 21st and 22nd natural amino acids(18). Maleimide analogs 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

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expression techniques in each of these systems, as well as latest research into next-generation bioconjugates and applications.

Conjugation to unnatural amino acids While well over fifty nonnatural amino acids have been genetically incorporated into recombinant proteins, the most widely used for producing bioconjugates are pacetophenylalanine (pAcF) and pazidophenylalanine (pAzF)(Figure 1). The side chains of these uAAs provide novel reactivity, enabling 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 homogenous 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

Figure 2. “Next-generation” conjugation approaches for uAA bioconjugates. A. Hydrazinopictet-spengler (HIPS), using an incorporated carbonyl moiety as a bioortohogonal 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 is 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 cytosinelike 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 towards 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.


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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 linkage(24)(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 contexts(30, 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 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 and showed that the Pictet-Spengler modified proteins are stable about 5-fold longer than the comparable method. 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, 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. 5 ACS Paragon Plus Environment


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In a novel variation of the copperfree click reaction, Wallace et al. reports a strain promoted reaction of a phenyl sydnone 1,3-dipole with a strained bicyclononyne dipolarophile to generate a stable pyrazole product (34)(Figure 2C). Additionally, the site specific incorporation of a bicycle-[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 eq of phenyl sidnone labled BODIPY in 6h at 37 oC, 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 towards product. Schmidt et al. reports an approach to protein conjugation via photo-crosslinking utilizing a furanyl uAA(35)(Figure 2B).

Figure 3. Insertion of uAAs into recombinant antibodies. A. The basic translation machinery of a ribosome (grey), 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 media, taken up by cells, and is a substrate for charging its cognate tRNA molecule.

With the use of a photoinitiator and long wave length 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 photocrosslink 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.


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Patterson et al. reports 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 site-specific 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 sufone linker does, however, result in increased serum stability by a significant margin when compared to a comparable maleimide linker.

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 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 natural twenty 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 (3739), 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 basepair 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 E.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 7 ACS Paragon Plus Environment


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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 was 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 conjugation 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-NNKNNK 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 the context sequence does matter, and with proper selection can get protein expression levels equivalent to that of the non-amber 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.


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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 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 ten-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 contructs; 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), toxin-Fabs (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 basepairs (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 flaskswith 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 site-specific monomethyl auristatin F ADC(8). Comparison of this IgG with an 9 ACS Paragon Plus Environment


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E.coli produced Fab-auristatin F revealed cytotoxicity EC50s of nearly 100x 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 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, pharmakokinetic analysis revealed that the uAA based ADC was equal to the unlabeled IgG(8). Tian et al analyzed ADC 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 non-specific killing activity on Her2negative cells. In addition to the linkage amino acid (cysteine vs pAcF) and multiplicity of conjugation (sitespecific 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 anti-tumor 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 superior systemic exposure than unconjugated IgG, and both of these molecules were better than a randomly labeled ADC(9). One would expect that unconjugated IgG should possess the 10 ACS Paragon Plus Environment

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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, 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. reports 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 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 non-amber 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 forty 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 11 ACS Paragon Plus Environment


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with the common glutamine synthetase expression system commercialized by Lonza (55) 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 7x106 cells/ml and >1g/L of IgG containing pAcF at position HC 114. Thus, CHObased 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-HER2-auristatin F is expected to enter clinical trials later this year.

In vitro expression of antibodies containing unnatural amino acids Over fifty 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 thermophilus, yeast, rabbit reticulocytes, eukaryotic wheat germ, Leischmanii, as well as 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 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 extracts (60), 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).


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An important feature of cell-free extracts with regards to antibody expression is their ability to support formation of a quaternary structure that includes appropriately formed inter and intra-chain 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-hour reactions (63). With the difficulty of producing large higher-order protein structures like antibodies in vitro now feasible, the potential of genetically encoding uAAs would require additional technology modifications to cellfree systems. As in cells, in vitro E. coli extracts can utilize the UAG stop codon (64) or four-base codons (65) 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 extract (70) 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 chemistry (71) or pAcF based oxime condensation (8) 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 incorporation 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 13 ACS Paragon Plus Environment


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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). Similarly, Sando et al (2007) used an anti- RF1 RNA aptamer to substantially enhance UAG suppression in GFP. Suga et al have performed related experiments and extended these studies to other extract systems (77). 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, Hallam et al engineered a protease cleavable RF1 in E.coli which is proteolyzed by endogenous OmpT during the extraction process. The resuting 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 14 ACS Paragon Plus Environment

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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 regards to both the amount and location of conjugation, yielding mixtures that can contain between 0 and 9 molecules of drugs per antibody. In this regard, the recently approved trastuzumab-DM1 (Kadcyla) contains an average of 3.5 DM1 molecules per antibody. An average IgG has 80-86 lysines, and forty 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 heterogenous 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 twelve 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 high15 ACS Paragon Plus Environment


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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 sixteen 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 and efforts by Wittrup (82) and Georgiou (83) 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 chemistrybased 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 synthetase (86)with the caveat that the RS activity towards 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 16 ACS Paragon Plus Environment

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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 is within reach with currently available techniques, opening exciting avenues for future biochemical investigation.

Development issues for uAA containing antibodies

Manufacturing Commercial manufacturing of recombinant proteins containing uAAs has additional issues compared to antibodies comprised of only the natural twenty 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 17 ACS Paragon Plus Environment


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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 in improving uAA incorporation, removing natural amber codons from the host genome to eliminate read-through 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 T-cell response. Helper T-cells then collaborate with low affinity selfantigen 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 analog of phenylalanine and tyrosine, and it is unclear if it has any specific tendency to alter MHC binding and stimulate a T-cell response beyond its natural analogs. 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. Certain unnatural amino acids are known break tolerance in a T-cell dependent, site-specific manner (89-92). In this line of reseearch p-nitrophenylalanine was incorporated into the self protein TNF-α in an 18 ACS Paragon Plus Environment

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effort to “haptenize” the self protein, as pNO2F is similar to common haptens known to illicit a robust immune response when coupled to self proteins. Certain uAAs may have theoretical concern for binding to naïve Bcells, 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’ PEGylated human growth hormone (ARX201; PEG-hGH) comprises recombinant human growth hormone with site-specific 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 oxyamino-derivatized 30 kDa polyethylene glycol (PEG) molecule. Unpublished data from pre-clinical 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 non-human 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’ 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 PEGhLeptin 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 seeing extensive use as an approved veterinary medicine in several countries, and to-date shown no evidence of neutralizing antibody response. Thus, preclinical and clinical evidence currently supports uAAs as a site-specific mechanism for bioconjugation without enhanced risk of immunogenicity.

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-ofconcept studies used well known model systems like trastuzumab, other applications have also recently been 19 ACS Paragon Plus Environment


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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 site specific conjugation of the auristatin drug to the antiCXCR4 antibody was accomplished via oxime ligation between the site

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.

specifically incorporated pAcF uAA and an aminoxy containing auristatin-linker compound (the linker used in this study is a non-cleavable 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 M. mazei 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 Tcells is a broad strategy with likely application in multiple tumor types. Most strategies to construct bispecific 20 ACS Paragon Plus Environment

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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, coupling them separately to a cyclooctyne and azide, respectively, then conjugating 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. reports 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 moeity 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 formalin-fixed, paraffin-embedded cancer cells was achieved. Additionally, superior detection specificity was observed for phospho-Her3 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 21 ACS Paragon Plus Environment


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be very effective. Additionally, Cui et al conjugated folate to selenocysteine on the V9 anti-CD3 Fab and could also target the FOLR1 receptor on tumors(100). Similarly, the same group used LLPA2 conjugated to antiCD3 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 natural twenty 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 basepairing properties of nucleic acids to construct Fab multimers (50). Since basepairing 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 basepairing strategies (Figure 4). Furthermore, analogs 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).

Crosslinking biomolecules A novel approach to answer certain biological research questions involves the covalent crosslinking of an antibody to its antigen. Furman et al. report the incorporation of electrophilic uAAs into the CDR3 of Herceptin for crosslinking antibody-antigen complexes (102). This methodology was investigated by incorporating 3 variably electrophilic uAAs (N-acryloyl-lysine, p-acrylamido-phenylalanine, and pvinylsulfonamido-phenylalanine) into the CDR3 and attempting to achieve covalent crosslinking 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% 22 ACS Paragon Plus Environment

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yield, showing this method to be a robust protein−protein crosslinking 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 toolkit to achieve conjugation in a variety of pH ranges. Similarly, Chen et al. reports 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 crosslinking(103). Proteins are crosslinked via Sn2 reaction with a nucleophilic amino acid residue displacing the primary alkyl bromide in the BrC6K uAA. Xu et al. describes 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 antiprotective antigen scFv antibody M18, the uAA 3,4-dihydroxyphenylalanine should be incorporated into in order to achieve the highest antibody-antigen crosslinking. The ten best computationally derived variants of the 3,4-dihydroxyphenylalanine containing antibody were synthesized and assessed for covalent antibody-antigen crosslinking, with the best variant crosslinking 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: (i) heterogenous 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, as well as the necessity of two different codons to be suppressed (106). While most uAAs



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have been incorporated in response to the amber codon, other stop codons like opal, ochre, as well as 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 O-methyltyrosine) 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 SK-BR-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 are 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 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 system; (109)) 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 24 ACS Paragon Plus Environment

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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 non-specific PCR amplification occurred on Her2-negative cells (43, 96). Thus, uAAbased oligonucleotide bioconjugates could expand the use of immuno-PCR methodology.

Conclusions Proteins containing unnatural amino acids are now in deep clinical development, with several ADCs also advancing towards the clinic. Significant data supports the notion that site-specific conjugates have improved efficacy and pharmacokinetics as a result of their homogenous 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 multi-drug conjugates, provide exciting next-generation possibilities of this technology.

Figures and Tables Figure 1. Unnatural amino acids and site-specific bioconjugation. A. p-acetophenylalanine (pAcF) and pazidophenylalanine (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 natural twenty amino acids. B. Schematic of the oxime ligation with pAcF encoded in the constant region of an antibody Fab fragment.



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Figure 2. “Next-generation” conjugation approaches for uAA bioconjugates. A. Hydrazino-PictetSpengler (HIPS), using an incorporated carbonyl moiety as a bioortohogonal 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 is 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 proteinDNA 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 towards 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.

Figure 3. Insertion of uAAs into recombinant antibodies. A. The basic translation machinery of a ribosome (grey), 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 media, taken up by cells, and is a substrate for charging its cognate tRNA molecule.

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. 26 ACS Paragon Plus Environment

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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 analog incorporation Formylglycine incorporation via FGE Transglutaminase-based conjugation “Meditope” mediated non-covalent conjugation

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