Extensive Survey of Antibody Invariant Positions for Efficient Chemical

Jul 20, 2017 - Antibody drug conjugates have made significant progress in the oncology field, as illustrated by the two United States Federal Drug Adm...
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Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes Akifumi Kato,† Mitsuo Kuratani,§,⊥ Tatsuo Yanagisawa,§,⊥ Kazumasa Ohtake,∥,⊥ Akiko Hayashi,∥,⊥ Yoshimi Amano,∥ Kaname Kimura,† Shigeyuki Yokoyama,*,§,⊥ Kensaku Sakamoto,*,∥,⊥ and Yasuhisa Shiraishi*,‡ †

Research Core Function Laboratories, Research Functions Unit and ‡R&D Planning DepartmentR&D Division, Kyowa Hakko Kirin Company, Ltd., Tokyo 100-8185, Japan § RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ∥ Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan ⊥ RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan S Supporting Information *

ABSTRACT: The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-L-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer “Variabodies”, a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.

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INTRODUCTION Synthetic amino acids containing non-natural chemical groups have been site-specifically incorporated into proteins and are potentially useful for the chemical conjugation of antibodies. Small molecules, fluorescent probes, peptides, and proteins can be attached to antibodies through bio-orthogonal chemical reactions involving these non-natural amino acids. IgG antibodies have been attached to anticancer drugs,1−4 whereas Fab has been transformed into bispecific antibodies.5 Sitespecific incorporation of these amino acids allows for the definition of conjugation sites in an antibody molecule, thereby enabling the precise design of resulting conjugates. Site specificity is achieved by assigning specific codons to nonnatural amino acids, with the amber-suppression method previously used to translate in-frame UAG amber stop codons into the non-natural molecules.6 A drawback of this method involves variations in incorporation efficiency depending upon the sequence context surrounding the in-frame UAG codon © XXXX American Chemical Society

and which prevent a comprehensive survey of useful conjugation sites for antibody positioning. The vulnerability to sequence context is ascribed mainly to the competition between UAG-translating tRNA and RF-1, the release factor recognizing UAG for termination of protein synthesis.7 Therefore, RF-1-knockout Escherichia coli cells are likely a better host for incorporating non-natural amino acids into antibodies. RF-1, previously thought to be an essential cellular factor, can be eliminated with no significant reduction in bacterial protein productivity, provided that some or all of the genes ending with the UAG codon are engineered to end with other stop codons.8−12 Because RF-1 confers stop-codon status on the codon, UAG is redefined to represent any amino acid Received: May 10, 2017 Revised: June 14, 2017

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DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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tRNAPyl and an o-Az-Z-Lys-specific variant of pyrrolysyl-tRNA synthetase (PylRS).13 The non-natural amino acid supplemented in the medium was then changed from 3-iodotyrosine to o-Az-Z-Lys. This removal of 3-iodotyrosine rendered the existing tRNA−iodoTyrRS pair idle, resulting in the exclusive incorporation of o-Az-Z-Lys at the UAG position. In subsequent experiments, Fab fragments and the o-Az-Z-Lysspecific pair were expressed from the same plasmid as described in the Experimental Procedures section. Identification of Multiple Positions Useful for Chemical Conjugation in the Trastuzumab−Fab Constant Domains. Only a few positions in Fab domains have been used for chemical conjugation using non-natural amino acids,1−5 mainly due to restrictions on conjugation-site selection. One restriction was the vulnerability of the amber-suppression method to the sequence context surrounding the incorporation site for a non-natural amino acid. To avoid this problem, we used RF-1-knockout E. coli W3110 cells to synthesize Fab variants. Another restriction is related to the degree of solvent exposure in a position as represented by the solvent-accessible surface area (ASA). Conjugation sites were previously selected among positions with ASA values of ≥0.4. Here, we examined whether these restrictions could be greatly relaxed by using the engineered E. coli strain and o-Az-Z-Lys. We first replaced lysine residues with o-Az-Z-Lys in the CLκ and CH1 domains of trastuzumab (Tra), which is a humanized antibody containing invariant positions common to other antibodies used clinically. These lysines show various degrees of exposure, with ASA values ranging from 0.08 to 0.73 (Table 1). The yields of the Tra−Fab variants harboring o-Az-Z-Lys in place of lysines were comparable to those of wild-type Tra−Fab (0.3 mg/100 mL culture). Assembly of the two chains was confirmed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), whereas MS analyses confirmed that the changes in masses were consistent with those expected for the replacements of lysines by o-Az-Z-Lys (Figure 2). No signals indicating contamination by other amino acids at the UAG positions were observed. Enzyme-linked immunosorbent assay (ELISA) showed that the incorporation of o-Az-Z-Lys at any site did not impair the antigen binding of Tra−Fab. We then examined o-Az-Z-Lys reactivity in these variants during strain-promoted alkyne−azide cycloaddition (SPAAC) using the DIBO-Alexa Fluor 488 dye as a model payload. Wildtype Tra-Fab showed no reactivity, whereas most of the variants were efficiently labeled, except for the almost-buried CH1-147 position (Table 1). It was particularly encouraging to note the high reactivity of the CLκ-207 position along with a relatively low ASA value (0.2). We subsequently examined positions other than those of lysines and exhibiting ASA values between 0.2 and 0.4, which represented an ASA range not previously explored for chemical conjugation. We examined 27 additional positions in the Tra−Fabconstant domains using similar methods as those used for examining the lysine positions. We confirmed assembly of the two chains and incorporation of o-Az-Z-Lys, with most of the examined positions supporting highly efficient conjugation and exhibiting reactivities ranging from 80% to 90%. Furthermore, 27 of the 42 Tra−Fab variants, including those involving lysine positions, were examined for thermal stability by differentialscanning calorimetry. The melting temperature (Tm) of the wild-type Tra−Fab was thus determined at 82.3 °C, which agreed with a previous report,16 and all of the examined variants

according to the amino acid specificity of UAG-recognizing tRNAs in its absence. With guaranteed efficient and robust UAG translation, the selection of non-natural amino acids is important for achieving efficient conjugation. Pyrrolysine derivatives can contain reactive groups at the end of a long side chain, thereby largely increasing the solvent exposure of these groups (Figure 1).13

Figure 1. Chemical structures of o-Az-Z-Lys and AzF.

Nε-(o-Azidobenzyloxycarbonyl)-L-lysine (o-Az-Z-Lys) contains an azido group located 13 Å away from the Cα atom, whereas p-azido-L-phenyalanine (AzF) positions the group much closer (7 Å) to the main-chain atom.14 This difference highlights an advantage of using a pyrrolysine derivative for chemical conjugation. In this study, we assigned UAG to o-Az-Z-Lys in an RF-1-knockout E. coli W3110 strain and synthesized variants of a Fab fragment containing o-Az-Z-Lys at different positions. The constant region of the antibody was thus extensively explored, resulting in the discovery of multiple novel positions supporting efficient conjugation.

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RESULTS AND DISCUSSION Redefining the UAG Codon for o-Az-Z-Lys in E. coli W3110. Based on our previous use of E. coli W3110 to synthesize Fab fragments, we decided to redefine the UAG stop codon in this strain. The in vivo translation definition of UAG has been successfully changed in various E. coli strains, including K-12 and B, after the re-engineering of eight genes to end with UAA in place of UAG.10 This synonymous replacement allowed these genes, which are important for bacterial growth, to normally express their protein products. UAG-reading tRNA and aminoacyl-tRNA synthetase were also introduced into the cells to translate UAG. We transformed W3110 with two plasmids: one carrying the eight genes harboring UAA in place of UAG and another carrying the genes for the archaeal pair of tRNA and a variant of tyrosyl-tRNA synthetase (iodoTyrRS-mj) specific for 3-iodo-L-tyrosine.15 This tyrosine derivative was then supplemented in the growth medium to support UAG translation, and the prfA gene encoding RF-1 was knocked out in the chromosome. The resulting E. coli cells, designated as W3110 RFzero-iy, translated the UAG codon into the tyrosine derivative, having been deprived of the capability to terminate translation at this codon. The reason for UAG reassignment to 3-iodotyrosine prior to oAz-Z-Lys was that UAG is more efficiently translated to 3iodotyrosine, which increased the chance of successful RF-1 elimination. The assignment of UAG was then changed from 3iodotyrosine to o-Az-Z-Lys by expressing the archaeal pair of B

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Bioconjugate Chemistry Table 1. ASA Ratios of the Indicated Positions and the Tm Values and SPAAC Reactivities of Tra−Fab Variants Containing o-Az-Z-Lys at These Positionsa

197, CLκ-155, CLκ-191, CH1-177, CH1-199, and CH1-131) (Figure 3) according to the following three criteria. First, they should be included in neither an α-helix nor a β-sheet to avoid the risk of distorting the tertiary structure by incorporation of oAz-Z-Lys. Second, they should not be located near the interface between the two chains; the selected CLκ and CH1 positions were separated by >6 Å from the CH1 and CLκ residues, respectively. Third, they should be selected from the Nterminal, middle, and C-terminal domains of each respective chain. We then synthesized Fab fragments of farletuzumab and adalimumab containing o-Az-Z-Lys at these six corresponding positions, observing that each variant exhibited a reactivity of >90%, except for two of the adalimumab−Fab variants (>80%), in the same SPAAC reaction used for evaluating the Tra−Fab variants (Table 2). We then synthesized a full-length antibody, Tra−IgG, containing o-Az-Z-Lys at the six selected positions and using a mammalian-cell-based system. The archaeal tRNA−PylRS molecules specific for o-Az-Z-Lys, the same pair used in W3110 RFzero cells, were expressed in HEK293 cells. We used the conventional amber-suppression method, because mammalian cells contained the only release factor, thereby precluding its elimination. The Tra−IgG variants containing o-Az-Z-Lys at the aforementioned six positions were purified by affinity chromatography on a protein-G column and then analyzed by nonreducing and reducing SDS-PAGE. We found that the light and heavy chains were properly assembled into IgG, forming a single band at a mass of >140 kDa (Figure S1). Additionally, reducing electrophoresis in the presence of 10 mM dithiothreitol produced two bands at ∼35 and ∼50 kDa corresponding to the light chain and the heavy chain with Nlinked glycosylation in the Fc region, respectively. After treatment with peptide-N-glycosidase F at 37 °C overnight to remove the glycan, MS analyses confirmed the incorporation of o-Az-Z-Lys in place of the original amino acids (Figure 4 and Table S1). All of the variants containing o-Az-Z-Lys at the six selected positions exhibited reactivities of >85% according to the model SPAAC reaction (Table 3). Advantage of o-Az-Z-Lys over AzF for Achieving Efficient Conjugation. To compare the reactivities of o-Az-ZLys and AzF, we synthesized Tra−Fab variants with o-Az-Z-Lys and AzF at the same position (CLκ-155) and performed chemical conjugation of these variants using the SPAAC reaction with DIBO-Alexa Fluor 488 fluorescent dye. Our results indicated that the labeling efficiency of Fab_o-Az-Z-Lys (72.4%) was significantly higher than that of Fab_AzF (11.5%) (Figure 5). To reveal the basis for this large difference in reactivity, we first created model structures of Fab_o-Az-Z-Lys and Fab_AzF, based on a reported Tra−Fab structure (PDB code: 1N8Z). The obtained models predicted that AzF made large contacts with the surrounding residues, whereas o-Az-ZLys was solvent-exposed (Figure S2). This result encouraged us to obtain the actual structures of the variants. In the obtained crystal structures, two Fab molecules were found in an asymmetric unit for both Fab_o-Az-Z-Lys and Fab_AzF. In Figure 6A, the light chains are denoted as molecules A and C, and the heavy chains are denoted as B and D. The light chains from the different Fab_o-Az-Z-Lys molecules assumed almost the same main-chain conformation, with a root-mean-square deviation in terms of Cα atoms of only 0.87 Å. The root-mean-square deviation in terms of Cα atoms was 0.50 Å between Fab_o-Az-Z-Lys and Fab_AzF, indicating no significant discrepancy between the main-chain conforma-

light chain ASA ratio (S)

SPAAC reactivity (%)

Tm (°C)

− 0.24 0.65 0.37 0.30 0.64 0.30 0.35 0.21 0.22 0.39 0.35 0.91 0.26 0.48 0.64 0.57 0.38 0.26 0.30 0.20 0.40 0.30 heavy chain

0.0 106.4 63.8 91.1 86.0 90.9 86.9 94.3 106.6 101.5 103.5 103.2 92.6 88.8 98.5 74.5 70.3 104.3 108.1 99.4 104.0 109.7 101.2

82.3 81.6 N/Ab 82.0 N/A 82.0 N/A 82.0 82.2 82.4 81.4 78.8 81.5 81.3 82.2 N/A N/A 82.3 82.3 82.5 82.0 82.5 82.5

residue

ASA ratio (S)

SPAAC reactivity (%)

Tm (°C)

Thr Lys Pro Ser Lys Thr Lys Asp Glu Asn Thr Val Ser Tyr Ser Ile Lys Lys Lys Lys

0.31 0.73 0.22 0.31 0.44 0.24 0.08 0.36 0.29 0.22 0.28 0.26 0.39 0.28 0.35 0.38 0.62 0.66 0.36 0.53

80.6 87.2 90.8 96.1 102.2 96.3 32.0 79.8 81.9 90.4 82.6 93.7 93.8 102.0 83.0 101.2 96.4 91.3 9.7 66.4

N/A N/A 81.2 82.2 82.3 82.3 N/A N/A N/A 81.0 N/A 80.9 81.2 N/A N/A 82.2 82.0 82.2 N/A N/A

positions (EU number)

residue

WT 119 126 138 141 145 147 149 155 158 161 167 169 180 183 188 190 191 195 197 207 210 211

− Pro Lys Asn Pro Lys Gln Lys Gln Asn Glu Asp Lys Thr Lys Lys Lys Val Glu Thr Lys Asn Arg

positions (EU number) 120 121 127 131 133 135 147 148 152 159 169 173 177 180 190 199 205 210 213 214 a

All of the indicated reactivities, calculated as described in the Experimental Procedures section, represent the mean of two experiments. bN/A denotes “not available”.

showed similar stabilities (Table 1). This result indicated that the incorporation of o-Az-Z-Lys at invariant positions had little effect on structural stability. Utility of Six Representative Positions in Different Antibody Contexts. To investigate the utility of the newly identified positions, we selected six invariant positions (CLκC

DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. Quality checks of the synthesized Tra−Fab variants. Representative data are shown. (A) Nonreducing SDS-PAGE analyses of the wild-type Tra−Fab (lane 1) and the variants containing o-Az-Z-Lys at the positions CLκ-197, CLκ-155, CLκ-191, CH1-177, CH1-199, and CH1-131 (lanes 2−7, respectively). (B, C) Mass spectrometric analyses of the wild-type Tra−Fab and the variant containing o-Az-Z-Lys at the CLκ-155 position. (D) ELISA results for Tra−Fab variants containing o-Az-Z-Lys at the indicated positions. Increasing amounts of Fab were added to HER2-GST cells and detected with antihuman IgG horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine substrate.

Figure 3. Locations of the six selected positions in the tertiary structure of Tra−Fab (PDB ID: 1N8Z). The heavy (red) and light (blue) chains are represented by ribbons. The amino acid residues to be replaced with o-Az-Z-Lys are represented in the CPK model. “Lc” and “Hc” represent light and heavy chains, respectively. The six positions selected for further experiments are colored yellow.

Table 2. SPAAC Reactivities (%) of the Different Fab Variants, Each Containing o-Az-Z-Lys at the Six Selected Positionsa positions CLκ-197 CLκ-155 CLκ-191 CH1-177 CH1-199 CH1-131

trastuzumab−Fab 98.1 110.6 105.6 98.3 105.7 104.5

± ± ± ± ± ±

5.8 4.3 7.3 5.0 7.6 9.6

farletuzumab−Fab 94.6 94.0 102.1 92.4 99.7 90.8

± ± ± ± ± ±

15.0 6.9 3.0 6.8 7.4 12.0

Figure 4. MS spectra of wild-type Tra−IgG and the variant containing o-Az-Z-Lys at position CLκ-155.

adalimumab−Fab 83.7 107.3 101.9 82.8 91.1 103.2

± ± ± ± ± ±

6.1 5.8 6.2 3.5 7.3 5.1

that the structure of Fab_o-Az-Z-Lys superimposed well on the reported structures of the Fab fragments of anti-HER2 antibody 4D5 (PDB codes: 5TDP and 5TDN), IgG heavy chain (PDB code: 4UB0), and humanized anti-P185-HER2 antibody 4D5 (PDB code: 1FVD), with Z-scores of 29.6, 29.6, 29.3, and 29.2, respectively. The root-mean-square deviation values in terms of Cα atoms for these structures (5TDP, 5TDN, 4UB0, and 1FVD) were 0.4, 0.4, 0.4, and 0.9 Å, respectively. These comparisons indicated that the incorpo-

a The SPAAC reactivities, calculated as described in the Experimental Procedures, represent the means of three experiments with standard deviations.

tions of these different Fab variants. Furthermore, a DALI search (http://ekhidna2.biocenter.helsinki.fi/dali/) showed D

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Bioconjugate Chemistry Table 3. SPAAC Reactivities of Tra−IgG Containing o-Az-ZLys at the Indicated Positionsa positions CLκ-197 CLκ-155 CLκ-191 CH1-177 CH1-199 CH1-131

SPAAC reactivity (%) 90.6 88.2 85.4 92.7 100.0 92.1

± ± ± ± ± ±

0.9 0.4 0.7 2.3 4.0 1.5

a The SPAAC reactivities, calculated as described in the Experimental Procedures section, represent the means of three experiments with standard deviations.

Figure 6. Crystal structures of Fab variants. (A) Crystal structure of Fab_o-Az-Z-Lys (CLκ-155). Molecules A, B, C, and D in the asymmetric unit are colored yellow, orange, green, and purple, respectively. The o-Az-Z-Lys residues in molecules A and C, the latter of which was partially disordered, are shown as sticks and colored magenta (left). A close-up view of the o-Az-Z-Lys residue in molecule A on the surface model of Fab_o-Az-Z-Lys (right). The azide moiety is encircled in pale blue dots. (B) The Fab_o-Az-Z-Lys (CLκ-155)_MD structure was perturbed with simple molecular dynamics using PHENIX and was modeled by optimizing the rotamer of o-Az-ZLys. The azide moiety of the modeled o-Az-Z-Lys was fully exposed on the protein surface. (C) A close-up view of the AzF residue on the surface model of Fab_AzF (CLκ-155). The AzF (CLκ-155) residue in molecule A is shown as a stick model and colored similarly to o-Az-ZLys (CLκ-155). A total of four amino acid residues (Asn158, Leu181, Asp185, and His189) surrounding the AzF molecule in the Fab_AzF (CLκ-155) structure are shown as green stick models. The azide moiety of AzF (CLκ-155) is buried within the pocket, possibly making it difficult to react with bulky dibenzocyclooctyl (DBCO) derivatives. The azide moiety of AzF (CLκ-155) is shown as a pale blue dotted circle.

Figure 5. Labeling efficiencies (%) of the Fab_AzF (CLκ-155) and Fab_o-Az-Z-Lys (CLκ-155) proteins with DIBO-Alexa Fluor 488.

ration of o-Az-Z-Lys and AzF did not change the overall tertiary structure of Tra−Fab. In terms of the non-natural residues, the o-azidobenzyl moiety was not visible in molecule C of Fab_o-Az-Z-Lys likely due to structural flexibility, whereas o-Az-Z-Lys assumed a definite conformation in molecule A, protruding the azidobenzyl moiety toward the solvent (Figure 6A, right). The conformation of this residue side chain was subjected to energy optimization to minimize the effect of crystal packing, the results of which suggested that the azidobenzyl moiety was possibly further exposed to the solvent (Figure 6B). In contrast, residue AzF at the corresponding position in Fab_AzF was surrounded by Asn158, Leu181, Asp185, and His189, resulting in limited access to the azido group (Figure 6C). Therefore, these crystal structures clearly illustrated the observed differences in reactivity between Fab_o-Az-Z-Lys and Fab_AzF. In Vitro Cytotoxicity of Tra−Fab−Mertansine DM1 Conjugates. Antibody drug conjugates have made significant progress in the oncology field, as illustrated by the two United States Federal Drug Administration approved drugs adotrastuzumab emtansine (KADCYLA) and brentuximab vedotin (ADCETRIS) and clinical studies currently underway.17 Sitespecific conjugation technology enables the production of homogeneous materials and improvement of pharmacokinetic profiles and has been advanced by utilizing unpaired cysteines, enzymatic modifications, and non-natural amino acids.18,19 The major advantage of using non-natural amino acids is that it enables the application of bio-orthogonal-conjugation chemistry, such as alkyne−azide cycloaddition and click chemistry.3,4 To demonstrate the feasibility of site-specific antibody drug conjugates using our approach, two variants containing o-Az-ZLys residues at defined sites (Tra−Fab CLκ-155 and Tra−Fab CH1-177) were each conjugated with DM1, a cytotoxin, via a DBCO−PEG4−maleimide linker. The formation of the Tra− Fab−DM1 conjugates was confirmed by liquid chromatography−mass spectrometry (LC−MS) analysis (Figure S3 and Table S2). We observed that Tra−Fab−DM1 conjugates exhibited potent in vitro cytotoxicity against the HER2-

overexpressing breast cancer cell line SK-BR-3 (Figure 7A) and resulted in minimal antiproliferative activity in MCF-7 breast cancer cells (Figure 7B), which express normal levels of HER2. This observation agreed with a previous report1 and indicated that the new conjugation sites were also useful for developing site-specific antibody drug conjugates. One-Pot Preparation and the Agonistic Activity of Tra−Fab Dimers. Recently, Scheer et al.20 reported that certain Tra−Fab dimers (bis-Fabs) created by site-specific cysteine substitutions exhibited HER2-agonistic activity. In this report, two Tra−Fab molecules were conjugated with each other via a maleimide-functionalized linker to produce bis-Fabs with different connection sites. However, the creation of new cysteine sites in an antibody can cause the formation of mismatched disulfide bonds, thereby posing a limitation on the selection of cysteine-substitution sites. Because our approach can eliminate this difficulty, we wanted to show that some agonistic Fab dimers would be found among the Fab dimer library created by utilizing the six selected positions for o-Az-ZLys. We first developed a method for one-pot preparation of Fab dimer, where a Fab-containing o-Az-Z-Lys was conjugated with a linker (Scheme 1, step 1) and then immobilized on a Ni resin via a hexa-histidine tag (Scheme 1, step 2), followed by conjugation with another Fab containing o-Az-Z-Lys and no tag E

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cell-proliferation assay (Figure S4). We observed that three Fab dimers between Tra−Fabs CH1-199 and CLκ-197, between Tra−Fabs CH1-199 and CH1-177, and between Tra−Fabs CH1-118 and CLκ-205 possessed agonistic activity comparable with that of heregulin (Figure 8). In contrast, Tra−IgG

Figure 8. Preparation and the agonistic activity of Tra-based bis-Fabs. (A) Nonreducing SDS-PAGE analysis of the Tra-based bis-Fabs CH1199/CLκ-197, CH1-199/CH1-177, and CH1-118/CLκ-205 (lanes 1− 3, respectively). (B) Influence of the indicated molecules on proliferation of the HER2-positive breast cancer cell line BT-474.

Figure 7. In vitro cytotoxicity of Tra−Fab−DM1 conjugates. Cell viability is plotted against the concentrations of Tra−Fab (black circles), the two Tra−Fab−DM1 conjugates (blue and red triangles), and free DM1 (open rectangles). (A) Potent cytotoxicity was observed in HER2-overexpressing breast cancer cell line SK-BR-3. (B) No antiproliferative effect was obserbed in MCF-7 with normal levels of HER2 protein.

exhibited antagonistic activity. Agonistic activity was previously reported for bis-Fab CH1-118/CLκ-205,20 whereas the other two Fab dimer variants were found for the first time to be agonistic. Currently one of the most common application of bispecific antibodies is anti-CD3/antitumor antigen bispecific antibody, which is being used to retarget T cells to tumor cells.21 Although bispecific IgG was successfully applied to recruit T cells to tumor cells, the relative orientation and distance of two antigen-binding domains can hardly be changed in this antibody format. Recently the anti-HER2/anti-CD3 bispecfic Fab dimer was reportedly used to link HER2+ cancer cells and CD3+ T cells with one another,5 suggesting that the connection between these two Fabs could be optimized through our technology to allow for improved efficacy.

Scheme 1. One-Pot Preparation of bis-Fabs Using a 96 Well Filter Plate Filled with a Ni Resin

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CONCLUSIONS The incorporation of o-Az-Z-Lys into Fab using UAGreassigned E. coli cells greatly facilitated the screening for antibody positions available for chemical conjugation. Our Xray crystallographic results indicated an advantage of using oAz-Z-Lys with a long side chain for creating chemical conjugates, thereby implying that its use must have increased the number of available positions in the antibody. Experiments utilizing the six representative positions demonstrated the utility of the newly identified positions and opened ways to control the relative orientation and distance between two Fab molecules conjugated to one another. We described Fab-dimer molecules exhibiting large degrees of flexibility in their connections as “Variabodies”, which encompasses not only monospecific Fab dimers but also bispecific Fab dimers. Our

(Scheme 1, step 3) and retrieval of the Fab dimer by elution using an imidazole buffer (Scheme 1, step 4). The eluted Fab dimer was purified by size-exclusion chromatography for the separation from Fab monomers, if necessary. This one-pot method enabled a facile preparation of both Fab dimers and bispecific Fab dimers with various combinations. Therefore, six Tra−Fab molecules, each containing o-Az-ZLys at the aforementioned six positions, were conjugated with one another in a combinatorial manner by SPAAC along with a DBCO−PEG4−DBCO linker, and all of the 36 combinations were analyzed for HER2-agonistic activity using the BT-474F

DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry findings suggested that the Variabody technology would help advance therapy involving antibody conjugates.

CH1 (defined by the EU numbering system) were changed to UAG. Knockout of prfA. Knocking out prfA in W3110 was performed using plasmids BAC8 and piodoTyrRS-MJR1-gent, as previously described.9 E. coli transformed with both plasmids was grown in a medium containing chloramphenicol and gentamicin at concentrations of 17 and 7 mg/L, respectively. A middle portion of prfA was replaced with the zeocin-resistance gene in the chromosome of E. coli W3110 to create RFzero-iy. ASA Ratio. The ASA ratio (S) was calculated using the MOE ASA Calculator program (MOLSIS Inc., Tokyo, Japan) operated by the Molecular Operating Environment 2013.08 (Chemical Computing Group, Montreal Inc., Canada) based on the structural information in the Protein Data Bank for TraFab (PDB: 1N8Z), assuming that the variants containing o-AzZ-Lys had exactly the same tertiary structure as the PDB entry. To determine the percentage values, the ASA ratio (S) of amino acid “X” in the Gly−X−Gly tripeptide was assumed to be 1.0. Synthesis of Fab Variants. Fab was expressed in E. coli strain W3110 RFzero transformed with the plasmid pFLAG− CTS−PylTS−Fab or the aforementioned plasmid to incorporate AzF. For Az-Z-Lys, single colonies of the transformants were grown overnight at 37 °C in 10 mL of an Lauria−Bertani medium containing 0.1 mg/mL Nε-benzyloxycarbonyl-L-lysine (Z-Lys) and 100 μg/mL ampicillin. Overnight cultures were diluted with 200 mL of Super Broth containing 1 mM o-Az-ZLys in place of Z-Lys and incubated at 37 °C until OD600 reached 2.0, followed by further incubation at 22 °C overnight following the addition of 1 mM isopropyl-β-D-thiogalactopyranoside. For AzF, no exchange of the supplemented amino acids was necessary. Cells were pelleted at 7000 rpm and frozen at −80 °C. The purification of Fabs variants by Protein-G affinity chromatography was performed as described previously.22 Synthesizing Tra−IgG Variants Containing o-Az-Z-Lys. The synthesis of Tra-IgG containing o-Az-Z-Lys at specific sites was tested at a 5 mL scale first and then scaled up using the Expi293 expression system (Thermo Fisher Scientific). At the small scale, 12.5 million cells were inoculated for transfection with plasmids for expressing tRNA, PylRS, and the two chains of Tra−IgG, as well as pCEP4 (Invitrogen, Carlsbad, CA) for expressing the EBNA-1 antigen. Plasmids expressing tRNAPyl and the PylRS variants carried the oriP origin of replication as described previously9 and were designated as pOriP− U6tRNAPyl and pOriP−PylRS(Y384F−Y306A), respectively. The antibody chains were cloned with a common leader sequence (METDTLLLWVLLLWVPGSTGD) in separate oriP-carrying plasmids to be expressed under the control of the CMV promoter. The amounts of these plasmids used for transfection were between 5 and 10 μg and 1, 1, 0.5, and 1 μg for pOriP-U6tRNAPyl, pOriP-PylRS(Y384F−Y306A), the plasmids expressing the heavy and light chains, and pCEP4, respectively. All of the plasmids were mixed with 250 μL of Opti-MEM medium and incubated for 5 min, while ExpiFectamine (13.4 μL) was separately mixed with the same volume of the medium for 5 min. The two solutions were then combined and incubated for 20 min before the mixture and the cell culture were added together with o-Az-Z-Lys (0.03 mM, final concentration) in 50% dimethyl sulfide and 0.25 M HCl. After 20 h, two types of enhancer were added to the culture according to the manufacturer instructions, followed by growth of the cells for 6 days. Finally, the medium was separated from

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EXPERIMENTAL PROCEDURES Materials. o-Az-Z-Lys was purchased from GVK Biosciences (Hyderabad, India). Z-Lys was obtained from Bachem AG (Bubendorf, Switzerland). DIBO-Alexa Fluor 488 was obtained from Thermo Fisher Scientific (Waltham, MA). The murine antipenta-His antibody horseradish peroxidase conjugate was obtained from Qiagen (Hilden, Germany). 3,3′,5,5′Tetramethylbenzidine solution was purchased from Dako (Carpinteria, CA,). DBCO−PEG4−maleimide and DBCO− PEG4−DBCO were purchased from Click Chemistry Tools (Scottsdale, AZ). Mertansine was obtained from Santa Cruz Biotechnology (Dallas, TX). The reaction buffer was composed of 20 mM sodium citrate (pH 6.0) and 150 mM NaCl. The HER2−GST fusion protein was synthesized and validated inhouse. His MultiTrap HP plates were purchased from GE Healthcare (Little Chalfont, UK). The E. coli W3110 strain and breast carcinoma cell lines SKBR-3, MCF-7, and BT-474 were obtained from the American Type Culture Collection (Manassas, VA). Cell Titer Glo was purchased from Promega (Fitchburg, WI). Plasmids. BAC8, a plasmid carrying eight genes engineered to end with UAA instead of UAG, was a mini-F plasmid having a resistance to chloramphenicol and described previously.8 The amount of BAC8 was amplified in the E. coli HST08 strain (Takara Bio, Shiga, Japan) grown in the presence of chloramphenicol at a concentration of 17 mg/L. Plasmid piodoTyrRS-MJR1-gent, derived from pACYC184, harbors the archaeal genes for UAG-reading tRNA and a TyrRS variant specific for 3-iodotyrosine as well as resistance to gentamicin. Plasmid pFLAG−CTS−PylTS was created by inserting a DNA fragment carrying the Methanosarcina mazei genes encoding PylRS and tRNAPyl under the control of gln S′ and lpp promoters, respectively, and downstream of the lacI gene in the pFLAG-CTS vector (Sigma-Aldrich, St. Louis, MO) using an In-Fusion HD cloning kit (Clontech, Mountain View, CA). The original version of this DNA fragment was reported previously9 and was modified in the following three ways: (1) the PylRS gene contained two mutations (Y306A and Y384F); (2) the fragment contained three tandem copies of the tRNAPyl gene; and (3) the NdeI, EcoRI, and HindIII sites were removed from the fragment. The genes encoding the light and heavy chains of the Fab fragments were cloned between the NdeI and SalI restriction sites of pFLAG−CTS−PylTS to enable their expression via the tac promoter, which is inducible by isopropyl-β-D-thiogalactopyranoside. The resulting plasmid was termed pFLAG−CTS−PylTS−Fab (Figure S5). For incorporating azidophenylalanine, the fragment carrying the PylRS and tRNAPyl genes was replaced by one carrying genes for UAG-reading tRNA and an archaea TyrRS variant specific for the amino acid; the latter fragment was reported previously.9 A pelB-signal sequence was added at the Nterminus of either chain of a Fab fragment to direct secretion into the periplasm, and a hexa-histidine tag was added at the Cterminus of the heavy chain for detection and affinity purification. The genes of the CH1 heavy-chain-constant region and Cκ light-chain-constant region were each connected to the genes encoding identical amino acid sequences of the variable regions of the therapeutic anti-HER2 antibody Tra− Fab. The codons at the selected positions in the human Cκ and G

DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

MES−NaOH (pH 6.5) buffer containing 15% (w/v) PEG20 000 and 25% glycerol and flash-cooled in liquid nitrogen on a nylon loop. X-ray data sets were collected at beamline BL05A in TPS (Hsinchu, Taiwan) at 100 K and processed with XDS.23 Phases were calculated by the molecular-replacement method using Phaser,24 with Fab_oAz-Z-Lys used as a search model, and refined using the program PHENIX.25 Statistics associated with data collection and refinement are summarized in Table S3. Data Deposition. The atomic coordinates and structure factors for Fab_o-Az-Z-Lys (CLκ-155) and Fab_AzF (CLκ155) have been deposited in the Protein Data Bank (PDB codes: 5XHG and 5XHF, respectively). Binding Analysis by ELISA. HER2-immobilized ELISA was performed as described previously.24 Briefly, the 96 well ELISA plate was coated with HER2-GST fusion protein (1 μg/ mL) and incubated overnight at 4 °C. After washing with Trisbuffered saline and 0.05% Tween-20, 150 μL of 1% bovine serum albumin in PBS was added to each well and incubated for 1 h at room temperature. Samples were prepared in 1% bovine serum albumin in PBS at a range of concentrations, added (50 μL) to each well, and then incubated for 1 h at room temperature. The wells were then washed with Tris-buffered saline and 0.05% Tween-20, followed by incubation with 50 μL of antipenta-His antibody horseradish peroxidase conjugate (diluted 1000 times in 1% bovine serum albumin in PBS) for 1 h at room temperature. The wells were washed again with Trisbuffered saline and 0.05% Tween-20 and developed using 50 μL of 3,3′,5,5′-tetramethylbenzidine. Analytical Methods. o-Az-Z-Lys-incorporated Fabs and IgG were all analyzed by SDS-PAGE and Coomassie staining. LC−MS analysis and differential-scanning calorimetry were performed as described previously.22,27 Preparation of Tra−Fab−DM1 Conjugates. o-Az-Z-Lysincorporated Tra−Fab molecules obtained after Protein G purification were further purified on a cation-exchange column (Mono S 5/50 GL; GE Healthcare) using AKTA fast protein liquid chromatography (GE Healthcare) with mobile phase A [20 mM sodium citrate (pH 6.0)] and mobile phase B [1 M NaCl and 20 mM sodium citrate (pH 6.0)]. A linear gradient of 0% to 100% mobile phase B was used to remove endotoxin from the samples. The buffer exchange into reaction buffer was performed using an Amicon Ultra-0.5 device (Merck Millipore, Billerica, MA). o-Az-Z-Lys-incorporated Tra−Fab molecules were then conjugated with an 8-fold molar excess of DBCO− PEG4−maleimide linker in the reaction buffer overnight at 4 °C. Removal of the free DBCO−PEG4−maleimide and buffer exchange to reaction buffer were conducted using an Amicon Ultra-0.5 device (Merck Millipore). DBCO−PEG4−maleimideconjugated Fab molecules were conjugated with a 10-fold molar excess of thiol-containing DM1. Prior to in vitro cytotoxicity assays, unconjugated toxins were removed using an Amicon Ultra-0.5 device, and formation of the Tra−Fab−DM1 conjugate was confirmed by LC−MS analysis, as described above. In Vitro Cytotoxicity Assays. SK-BR-3 or MCF-7 cells (5000 cells per well) were seeded onto 96 well plates, and a series of diluted wild-type Tra−Fab and Tra−Fab−DM1 conjugates were added. Cells were incubated for 5 days at 37 °C in a humidified atmosphere of 5% CO2. Cell Titer-Glo luminescent cell viability assay (Promega) reagent was added to the wells and incubated at room temperature for 10 min, and

the cells and subjected to antibody purification using a ProteinG Mag Sepharose column (GE Healthcare). The yields of wildtype Tra−IgG and its variants were typically 7 and 1.5 mg/L, respectively. Copper-Free Click-Chemistry Reaction. A copper-free click-chemistry reaction between o-Az-Z-Lys in the Fab or the IgG and DIBO-Alexa Fluor 488 was carried out in Dulbecco’s phosphate-buffered saline (PBS; pH 7.4) using 5 μM Fab or IgG and 200 μM DIBO-Alexa Fluor 488 (40-fold molar excess in screening or a 4-fold molar excess for comparative analysis of Fab containing o-Az-Z-Lys and AzF) overnight at room temperature. Reaction efficiency was determined by separating the conjugate by cation-exchange chromatography on a TSKgel SP-5PW column (Tosoh, Tokyo, Japan) using the Prominence HPLC system (Shimadzu, Kyoto, Japan) with buffer A [20 mM acetate buffer (pH 5.0)] as the initial buffer. Separation was performed at a flow rate of 1 mL/min at 25 °C using a gradient of buffer B [20 mM acetate buffer (pH 5.0) and 1 M NaCl] from 0% to 100% in the mixture of buffer A and buffer B for 20 min. The reaction mixture (20 μL) was mixed with buffer A and injected. The proportion of the protein moiety of the conjugate was calculated based on absorbance at 280 nm, whereas that of the Alexa Fluor 488 moiety was calculated based on absorbance at 495 nm. Reaction efficiency was determined by calculating the molar ratio between these moieties and considering that Fab and IgG contained one and two o-Az-Z-Lys sites, respectively. Crystallization of and Data Collection for the Fab_oAz-Z-Lys Fragment. The Fab_o-Az-Z-Lys protein was concentrated up to 12 mg/mL by ultracentrifugation. Crystals were grown by sitting-drop vapor diffusion at 20 °C by mixing 250 nL of protein solution with a 250 nL of reservoir solution, which consisted of 0.1 M Bis−Tris−HCl buffer (pH 6.5) containing 0.2 M ammonium sulfate and 25% PEG3350, followed by equilibration against 70 μL of reservoir solution. A crystal was transferred to cryoprotectant solution, which consisted of 0.1 M Bis−Tris−HCl (pH 6.5) buffer containing 0.2 M ammonium sulfate, 25% PEG3350, and 25% ethylene glycol. The crystal was flash-cooled in liquid nitrogen on a nylon loop. X-ray data sets were collected at beamline BL15A1 in NSRRC (Hsinchu, Taiwan) at 100 K and processed with XDS.23 The crystal belongs to the space group P1, with unit cell parameters of a = 38.7 Å, b = 79.9 Å, c = 85.8 Å, α = 113.5°, β = 92.8°, and γ = 102.8°. The phase was calculated by the molecular-replacement method using Phaser24 based on Fab coordinates [obtained from the search model (PDB code: 1N8Z)]. The model was refined using the program PHENIX Autobuild25 and manually corrected using Coot.26 The final model included two light (molecules A and C) and heavy (molecules B and D) chains, and the Rfree and Rwork factors converged to 19.9% and 17.3%, respectively. The final model was validated using the program Procheck.24 Graphical images were prepared using the program PyMOL (http://pymol. sourceforge.net/). Statistics associated with data collection and refinement are summarized in Table S3. Crystallization of and Data Collection for the Fab_AzF Fragment. The Fab_AzF protein was concentrated up to 12 mg/mL by ultracentrifugation. Crystals were grown by sittingdrop vapor diffusion at 20 °C by mixing 250 nL of the protein solution with 250 nL of buffer (no. 36) from the NeXtal PEG suite kit (Qiagen), which consisted of 0.1 M 4-morpholinoethanesulfonic acid (MES)−NaOH buffer (pH 6.5) and 15% (w/v) PEG20 000. A crystal was then transferred to 0.1 M H

DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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BL05A (Hsinchu, Taiwan) and the technical services provided by the “Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology” and the “National Synchrotron Radiation Research Center”, a national user facility supported by the Ministry of Science and Technology of Taiwan, ROC. We also thank Editage for assistance with English language editing.

the luminescent signal was measured using the ARVO X3 system (PerkinElmer, Waltham, MA). Preparation of Tra−Fab Dimers. The o-Az-Z-Lysincorporated Tra−Fabs after protein G purification were further purified as described above prior to conjugation with a 20-fold molar excess of the DBCO−PEG4−DBCO linker in the reaction buffer overnight at room temperature. Removal of the free DBCO−PEG4−DBCO and buffer exchange to reaction buffer were conducted using the Amicon Ultra-0.5 device (Merck Millipore). DBCO−PEG4−DBCO-conjugated Tra− Fabs were applied (50 μg/well) into His MultiTrap HP plates (GE Healthcare) and incubated for 10 min at room temperature to capture the Fab molecules by hexa-histidine tag. Following centrifugation, 100 μg/well of another o-Az-ZLys-incorporated Fab molecule in the absence of the hexahistidine tag was added, followed by incubation overnight at room temperature to generate Tra−Fab dimers. Generated Tra−Fab dimers were eluted with 500 mM imidazole in Dulbecco PBS (pH 7.4) after washing with 40 mM imidazole in Dulbecco PBS (pH 7.4). Cell-Proliferation Assays. BT-474 cells (10 000 cells per well) were seeded onto 96 well plates and treated the following day with a series of diluted Tra−Fab dimers. Cells were incubated for 5 days at 37 °C in a humidified atmosphere of 5% CO2. Cell Titer-Glo luminescent cell viability (Promega) reagent was added to the wells and incubate at room temperature for 10 min, and the luminescent signal was measured using the ARVO X3 system (PerkinElmer). The results are expressed as a percentage of growth compared to the control group.

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ABBREVIATIONS ASA, solvent-accessible surface area; bis-Fabs, Tra−Fab dimers; DBCO, dibenzocyclooctyl; ELISA, enzyme-linked immunosorbent assay; o-Az-Z-Lys, Nε-(o-azidobenzyloxycarbonyl)-Llysine; AzF, p-azido-L-phenyalanine; pylRS, pyrrolysyl-tRNA synthetase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPAAC, strain-promoted alkyne−azide cycloaddition; Tra, trastuzumab

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00265. Figures showing SDS-PAGE analysis, models of the residues, MS analysis of Tra−Fab variants, cellproliferative effect of variants, and plasmid construction. Tables showing MS analyses and data collection and refinement statistics. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81-45-503-9196. Fax: +81-45-503-9195. *E-mail: [email protected]. Phone: +81-45-5039459. Fax: +81-45-503-9458. *E-mail: [email protected]. Phone: +81-33282-0985. Fax: +81-3-3282-0087 . ORCID

Yasuhisa Shiraishi: 0000-0002-8235-1976 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Eri Suzuki-Takanami and Seiji Saito for their technical assistance with the LC-MS instrument and Emiko Honma for establishing each o-Az-Z-Lys-containing Fab variant expression vectors. We thank Dr. Toru Sengoku for X-ray data collection. We also thank the staff at beamlines BL15A1 and I

DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.7b00265 Bioconjugate Chem. XXXX, XXX, XXX−XXX