Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery

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Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemoenzymatic Approach Stephan Dickgießer, Nicolas Rasche, Daichi Nasu, Stephen Middel, Sebastian Hörner, Olga Avrutina, Ulf Diederichsen, and Harald Kolmar ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00315 • Publication Date (Web): 01 Jul 2015 Downloaded from http://pubs.acs.org on July 2, 2015

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ACS Chemical Biology

Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemoenzymatic Approach Stephan Dickgiesser,§,‡ Nicolas Rasche,†,‡ Daichi Nasu,§ Stephen Middel,∥ Sebastian Hörner,§,⊥ Olga Avrutina,§ Ulf Diederichsen,∥ and Harald Kolmar§,* §

Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany

†

Protein Engineering and Antibody Technologies, Merck-Serono, Merck KGaA, Darmstadt, Germany

∥Institute

for Organic and Biomolecular Chemistry, Georg-August University Göttingen, Göttingen, Germany

ABSTRACT: Over the last decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g. toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA-DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the C-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads.

First reported in 1990,1,2 folded single-stranded oligonucleotides, called aptamers, have emerged as molecules with great biomedical potential due to their unique binding properties.3 Indeed, with respect to their nano- to picomolar affinities against targets of therapeutic interest, they can be considered as an alternative to monoclonal antibodies (mAbs) successfully used for diagnostics and treatment of cancer over decades.3,4 As contrasted to mAbs with their potentially immunogenic antigen-specific combining sites,5 aptamers are non-immunogenic, can be chemically synthesized in a readily scalable process and are easily equipped with desired functions.3 Some aptamers are currently undergoing clinical trials.6,7 However, since these molecules lack a functional unit comparable to the antibody Fc part, they cannot induce natural immune effector mechanisms being in many cases prerequisites for the efficacy of therapeutic antibodies.4 Furthermore, with a molecular weight of 10–30 kDa, the size of aptamers is below the cutoff for renal clearance leading to an in vivo half-life in the range of minutes, while mAbs (mainly due to their size and affinity to the neonatal Fc receptor) exhibit a serum half-life of days or weeks. In order to prolong the residence time of aptamers in the

body, they have been conjugated to polymers, typically polyethylene glycol (PEG).8 Pegaptanib (Macugen®), a nuclease-stabilized RNA aptamer binding the vascular endothelial growth factor and being, to date, the only aptamer on the market, was PEGylated for that reason.9 Additionally, attempts have been made to transfer the concept of antibody-drug conjugates (ADCs) to aptamers.7,10 Indeed, with their mAb counterpart serving as a vehicle to selectively deliver a cytotoxic payload to the target tumor cell, ADCs possess significantly increased efficacy compared to the parent molecules.11,12 Application of this concept led to the generation of aptamer-drug conjugates produced by either covalent coupling or non-covalent assembly of aptamers with cytotoxic agents.7,10 Herein, we report a generic approach allowing to combine the benefits of aptamers with the prolonged serum halflife, immune functions and toxic properties of mAbs and ADCs within a hybrid molecular construct. To this end, an antibody Fc fragment was envisioned as a scaffold for the simultaneous conjugation of aptamers and toxic/reporting payloads. Recently, a preliminary study outlined the decoration of Fc fragments with aptamers, which relied on formation of a covalent linkage between

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Scheme 1. A modular approach to the generation of aptamer-Fc conjugates via a combination of chemoenzymatic methods with PNA-guided self-assembly. An IgG1 Fc scaffold (grey) is depicted in structures 1, 4, 5, 8, 9, 10, and 12.

glutaraldehyde and surface-exposed addressable functionalities, mainly ε-amino groups of lysine, which obviously results in the formation of heterogeneous product mixtures leading to jeopardized bioactivity.13,14 Addressing of accessible amines and/or thiols is a common method to conjugate antibodies with desired cargoes. However, lack of selectivity is a general problem for these reactions as generated constructs possess nonuniform drug-to-antibody ratios leading to species with different pharmacological properties.11,12 Although uniform Fc conjugates can be produced using cysteine as a handle, valuable strategies for site-specific ligation gain a keen interest in the field of ADCs, with the enzymebased approaches being extremely attractive due to their high specificity and mild reaction conditions.11,15,16

and the resulting constructs 4, 5 and 11 are hybridized to the DNA aptamer CLN000319 decorated with the complementary nucleobase sequence (6, Scheme 1). This aptamer has been used in our study as it specifically binds to the receptor tyrosine kinase hepatocyte growth factor receptor (HGF-R or c-Met), which is overexpressed on various cancerous cells.19,20

Our approach to the assembly of hybrid molecular constructs on the platform of a human IgG Fc comprises several orthogonal chemoenzymatic transformations and a non-covalent self-assembly guided by PNA-DNA base pairing (Scheme 1). Following this strategy, the generation of aptamer-Fc conjugates bearing cargoes of interest can be achieved in a modular way, with peptide nucleic acids (PNAs) serving as anchoring units.17

For the covalent linkage between respective counterparts we considered direct transpeptidation catalyzed by sortase A and a two-step conjugation relying on formylglycine-generating enzyme (FGE) with successive oxime ligation. Being recently applied to the covalent modification of antibodies and antibody fragments,21-26 sortase A, an enzyme from Staphylococcus aureus, catalyzes formation of an amide bond between certain amino acids located at two specific sequences.27-29 FGE catalyzes conversion of a cysteine within a specific peptide motif, an aldehyde tag, into an orthogonally addressable formylglycine. If coexpressed with a tagged protein, FGE assures the conversion inside the cell resulting in a protein that can be addressed siteselectively by e.g. aminooxy- or hydrazide-bearing reagents.30-32

2-Aminoethylglycine PNA allows assembly of nucleo amino acids by solid phase peptide chemistry and further, based on the nucleobases attached to the backbone, stable hybridization with complementary DNA sequences.18 In our approach, peptide nucleic acids (PNAs 2 and 3) are enzymatically attached to the Fc C-terminus

To establish delivery to cancer cells, a molecular system was designed that was additionally equipped with a fluorescent cargo (7, Scheme 1). Novel molecular architectures were examined for their binding against individual c-MET and c-MET-overexpressing cells, as well as in cellular internalization and stability studies.

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RESULTS AND DISCUSSION In our study, we addressed the limitations of aptamers themselves (size, half-life, and lack of immune effector functions) and aptamer-involved conjugations (selectivity, specificity, modularity) by using Fc-scaffolded consecutive chemoenzymatic transformations followed by self-assembly. Synthesis of hybrid constructs, affinity against specific target, as well as targeted delivery of a model cargo in cancer cells are discussed. Installation of PNA guiding units into dual tagged Fc framework. To get an access to dual functionalized scaffolds, an Fc fragment comprising two orthogonally addressable sites was produced recombinantly. Thus, an expression plasmid encoding human IgG1 Fc fragment 1 with an N-terminal aldehyde tag (LCTPSRAALLTGR) and a C-terminal sortase A recognition sequence (LPETG) was cloned and used to transiently transfect HEK293-6E cells together with an expression vector encoding human FGE (Supplementary Section 1, 3). Coexpression of both proteins ensured the efficient oxidation of the cysteine residue within the aldehyde tag to a Cα-formylglycine residue, addressable by orthogonal chemistries.32 Since previous experiments in our lab revealed the formation of N-terminal Fc truncations (most likely by inaccurate signal peptidase processing) additionally an N-terminal TEV protease cleavage site was introduced to prevent this issue (Supplementary Scheme S1). The site-selectivity of FGE was confirmed in experiments with an Fc construct 1a bearing an Ala residue in place of Cys within the aldehyde tag (Supplementary Figures S2 and S3). SDS-PAGE, gel filtration and mass spectrometry studies revealed expression as disulfide-linked homodimers of expected size (Supplementary Figures S3 and S12).

Aptamer conjugation to Fc scaffold via PNA-guided hybridization. While numerous aptamers have been developed to date,3 their conjugation with proteins to gain additional functions enabling half-life extension, addressing the complement system, introducing cytotoxicity or enzyme activity are rarely reported and often suffer from the lack of efficient and site-specific conjugation chemistries.7,10,35 The extremely stable biotinstreptavidin interaction was used by Stecker et al. to attach a cancer cell-specific DNA aptamer to the complement protein C1q.36 Furthermore, in a preliminary study, a poly-γ-D-glutamic acid-specific amine-bearing DNA aptamer was cross-linked with murine IgG Fc fragments via glutaraldehyde.13 Although the aptamer-Fc ratio in the reaction products was not reported, formation of heterogenous conjugates due to lack of specificity can be assumed.13,14 An elegant approach to the site-specific aptamer-protein conjugation has been recently reported that relied on a two-step enzyme-catalyzed reaction. This synthetic procedure comprised attachment of Z-Gln-Gly motif to the 3'-OH of a DNA aptamer by terminal transferase followed by transglutaminase-promoted crosslinking with a reactive lysine.35 However, this twostep procedure requires synthesis of a modified nucleotide and, importantly, lack of an undesirable transglutaminase-addressable Gln site. Several other concepts of aptamer conjugations have been reported, among them the use of succinimidyl-3-(2pyridylodithio)propionate connecting primary amines with cysteine thiols37 as well as hybridization with oligonucleotide counterparts, DNA intercalators or photosensitizers.7,10

To attach PNA guiding units on the scaffold 1, thus assuring a platform for aptamer attachment via hybridization, we used efficient and site-specific sortase A-mediated transpeptidation.28 Although this enzyme has been used to obtain cell-penetrating peptide-PNA conjugates possessing dose-dependent PNA-mediated antisense activity,33 no sortase-catalyzed formation of PNA-protein conjugates has been considered so far. In our study, we applied an activity-optimized sortase variant (eSrtA).34 It recognized an LPETG motif of 1 and cleaved between Thr and Gly residues resulting in the formation of a thioacyl intermediate with cysteine at the active site. Upon a nucleophilic attack of the amino group of PNA-bearing oligoglycine substrates (2 or 3), a new amide linkage between LPET of 1 and the pentaglycine elongation of 2/3 was formed (Scheme 1).28 The required Gly5 sequence was N-terminally attached to PNA oligomers by SPPS (Supplementary Section 1 and Schemes S2, S3). eSrtA-mediated transpeptidation proceeded smoothly resulting in constructs 4 and 5 in almost quantitative conversion according to SDS-PAGE analysis (Figure 1A and Supporting Information Figure S1).

Figure 1. Sortase-mediated generation of constructs 4 and 8 and its binding to c-MET-overexpressing cells. (A) Reducing SDS-PAGE gel to monitor eSrtA-catalyzed reaction between 1 and 2 towards 4. Unreacted 2 as well as eSrtA were removed by SEC prior to analysis. Expected molecular masses of reduced 1 and 4 are 29 kDa and 33 kDa, respectively. M: protein molecular mass marker. (B) SEC monitoring of hybridization of 4 to 6 towards 8. 8a: single-hybridized 4. (C) Flow cytometry studies of binding of 8 to c-MET-positive (EBC-1) and c-MET-negative (T47D) cells. Aptamer-lacking 4



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used as negative control. Cells were incubated with 4 and 8 with successive labeling by Fc-specific IgG-phycoerythrin conjugate and analysis of fluorescence. (D) Fitted binding curves of titration of 8 on EBC-1 and T47D cells with calculated KD value.

In our approach, conjugation of c-MET-binding aptamer units relies on their hybridization with respective PNA-comprising Fc scaffolds. Compared to the reported decoration of DNA oligomers with a triglycine motif38 to allow their covalent conjugation to a protein via sortasemediated reaction, a simple elongation of the aptamer by a sequence complementary to the PNA handle requires no costly chemical modification of the aptamer. In order to enable PNA-guided conjugation, constructs 4, 5, and 11 were hybridized to aptamer 6 bearing a nucleotide sequence complementary to that of the used PNA motif at the 3’ end to yield structures 8, 9, and 12 (Scheme 1). To prove the specificity of duplex formation between 6 and 4, an oligonucleotide 6a lacking the PNAcomplementary sequence was used as a control (Supplementary Section 3). SEC analysis revealed the formation of a new species with a higher molecular mass when 4 was incubated with 6. That species was not detected upon incubation with 6a (Supplementary Figure S6). Due to the dimeric nature of the Fc scaffold it carries a PNA on each of the C-termini; therefore the hybridization of two aptamers is possible. To assess if complex 8 contains two copies of the aptamer, we examined the stoichiometry of hybridization by incubating 4 with 6 taken in different molar ratios (Figure 1B). Depending on the used molar excess of 6 over 4, two species were detected by SEC, which most likely corresponded to the single (8a) and double loading of 4 (Supplementary Scheme S4). Table 1. Binding characteristics of 6 and 8 against recombinant c-MET-Fc (determined by BLI measurements) and c-MET-overexpressing cells (determined by titration on EBC-1 cells and flow cytometry). Compound

5

K [nM]

-1 -1

k [10 M s ]

D

a

-4

-1

k [10 s ] d

K [nM] D

+

recombinant receptor

c-MET cells

6

7.8 ± 0.1

1.17 ± 0.0063

9.10 ± 0.10

n.d.

8

12.9 ± 0.3

0.44 ± 0.0041

5.62 ± 0.13

24.5 ± 4.5

Stability and affinity of the hybrid Fc-PNA-aptamer constructs. Obviously, PNA-DNA heteroduplexes formed by Watson-Crick base pairing are more stable compared to the DNA-DNA ones due to the lack of electrostatic repulsion caused by uncharged PNA backbone.39,40 To investigate whether the aptamer-bearing complex 9 (Scheme 1) is stable at physiological pH in respective buffer, it was incubated in 150 mM NaCl, 50 mM Tris-HCl pH 7.5 over 7 days with time-lapse analysis which revealed no significant dissociation within this period (Supplementary Figure S7A).

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The stability of the PNA-DNA duplex was also assessed by UV melting curve using solitary 2 and a complementary DNA oligomer 6b (Supplementary Sections 1, 3 and Figure S6B). A melting temperature (Tm) of 51.2 °C was obtained for the DNA-PNA duplex corroborating formation of a stable complex between the used PNA and DNA strands. Generally, PNA-DNA hybridization as a conjugation step makes this approach very flexible. Indeed, any aptamer bearing a PNA-complementary strand could be attached to the scaffold and the formed hybrid should possess significant stability. In our hands the DNA-PNA complex between 2 and 6b displayed a Tm beyond 50 °C and no dissociation has been observed for Fc-aptamer construct 9 within 7 days. However, this issue needs to be further assessed at physiological conditions but stability could be easily tuned by elongation of the complementary sequences.39,40 For in vivo application the covalent fixation of the PNA-aptamer duplex may appear determinative; this challenge could be addressed by site-specific incorporation of a suitable crosslinker.41 In the following, binding characteristics of produced aptamer-Fc hybrids were assessed. To demonstrate that complex 8 binds c-MET and to exclude that hybridization to 4 impairs the affinity of 6, biolayer interferometry studies were performed using a recombinantly expressed c-MET-Fc fusion protein. Strong c-MET binding was observed for construct 8 with a dissociation constant (KD) of 12.9 nM, which is in the range of solitary 6 (7.8 nM, Table 1 and Supplementary Figure S8). This suggests that the binding activity of aptamer 6 was fully retained upon PNA hybridization (Table 1). Interestingly, the dissociation constant for the parent aptamer CLN0003 was reported being in the subnanomolar range (0.1 nM).19 However, the used aptamer lacked elongation by the PNA complementary sequence and other methods for KD determination were used, which may contribute to the difference in binding constants.19 Binding selectivity was studied in flow cytometry binding assays with c-MET-overexpressing EBC-1 lung cancer cells42 and c-MET-negative T47D breast cancer cells (Supplementary Section 1).43 Incubation of EBC-1 cells with 8 and fluorescently labeled secondary antibody resulted in a strong increase of fluorescence intensity while only minimal binding of 8 to T47D cells was observed (Figure 1C). Construct 4 alone did not show any binding to neither of the cell lines (Figure 1C). Additional affinity titration experiments with varying concentrations of 8 revealed high-affinity binding to EBC-1 cells with an equilibrium binding constant of 24.5 nM (Figure 1D, Table 1, Supplementary Section 1). Internalization of hybrid constructs by c-METoverexpressing cells. The ability to specifically bind a tumor cell followed by rapid internalization is crucial for the efficacy of ADCs and, obviously, aptamer-based conjugates with e.g. toxic payloads.44 Therefore, complexes 8 and 9 were examined in microscopic and flow cytometric studies with c-MET-overexpressing


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cancer cells using confocal fluorescence microscopy and flow cytometry. Thus, c-MET-positive EBC-142 and c-METnegative T47D cells43 were incubated with 9 and cellular localization was followed microscopically using an Fc specific Alexa Fluor® 488 Fab conjugate. Cells were incubated at 4 °C as non-internalizing control or at 37 °C for internalization, and membrane-bound complexes were removed by washing with acidic buffer.45 EBC-1 cells incubated with 9 at 4 °C showed strong membranelocalized fluorescence which disappeared upon low pH washing indicating efficient binding to the cell surface but lack of internalization (Figure 2A, Supplementary Figure S9). In contrast, incubation at 37 °C resulted in intracellular fluorescence, which was not affected by acidic washing thereby confirming successful internalization (Figure 2A, Supplementary Figure S9). Lack of labeling for T47D cells proved the selectivity of internalization (Supplementary Figure S9). Flowcytometry assays confirmed these findings (Figure 2B, Supplementary Figure S10 and Section 2).

Figure 2. Internalization of complexes 8 and 9. (A) Fluorescence microscopy images of EBC-1 cells labeled with 9 and Fc specific Alexa Fluor® 488 Fab conjugate (green) followed by incubation at 37 °C and 4 °C. Lower panel: Membrane-bound complexes were removed by low pH wash; Upper panel: No low pH wash was done. Cells were embedded in mounting medium containing DNA staining DAPI (blue). Merging with differential interference contrast images is shown. (B) Flow cytometry histogram using EBC-1 cells incubated with 8 or 4 (as control) followed by labeling with Fc-specific IgG-phycoerythrin conjugate and incubation at 4 or 37 °C. Membrane-bound complexes were removed by low pH wash.

Generally, development of anti-c-MET antibody therapeutics in case of bispecific antibodies often resulted in agonistic activities due to receptor dimerization. To prevent unwanted biologic effects of c-MET activation, monovalent c-MET antibodies were developed.46,47 Nevertheless, it has been shown that bivalent c-MET binding not necessarily induced activation of the c-Met signaling pathway. Indeed, Emibetuzumab, a bivalent anti-c-MET antibody inhibits ligand-dependent as well as ligand-independent c-MET activation and is currently undergoing clinical trials.48 This antibody readily induces c-MET internalization and degradation only in its bivalent form, as a Fab generated from it was not able to induce internalization.48 As a matter of fact, dimerizationinduced internalization of receptor tyrosine kinases is not necessarily dependent on kinase activation.49

Nevertheless, our strategy could be extended towards monovalent aptamer-Fc-drug conjugates (AFDCs) by using asymmetric Fc molecules that contain only one sortase A recognition sequence by application of the SEED or ‘knobs-into-holes’ antibody technology.50,51 It would be interesting to evaluate, how receptor dimerization induced by bivalent c-MET-binding AFDCs contributes to internalization rates, the c-Met signaling pathway as well as cytotoxicity in comparison with the performance of monovalent variants. Installation of fluorescent cargo and its delivery to c-MET-overexpressing cells. As our long-term goal is to combine the benefits of novel Fc-PNA-aptamer architectures for cell targeting and internalization with the potency of AFDCs, we installed a fluorescent reporter as a model cargo at the opposite end of the scaffold by addressing the FGE-generated aldehyde groups prior to both the sortase-catalyzed reaction and hybridization. Generally, the usage of an aldehyde tag in combination with consecutive ligation step has been already proven as a successful approach to the generation of antibody-drug conjugates.52,53 In our setup, N-termini of the dimeric molecule 1 were decorated by an aldehyde-specific model cargo 7 using oxime ligation.31,32,54 To this end, aminooxymodified fluorescent dye TAMRA 7 (see Supplementary Section 1) reacted with 1 under mild acidic conditions towards 10 (Scheme 1). Oximation was performed by incubating 1 with 7 at pH 4.6 at ambient temperature to yield 10 (Scheme 1). A fluorescent protein band slightly shifted towards higher molecular weight on SDS-PAGE indicated successful protein conjugation (Figure 3A and Supplementary Figure S2). Maximal conversion was obtained within 72 h using 12.5 molar excess of 7 over 1. Incubation of 7 with the control Fc fragment 1a lacking a modifiable cysteine did not result in fluorescent protein species confirming the selectivity of aldehyde tagpromoted oxime ligation (Supplementary Figures S2 and S3). Although oxime ligation is characterized by good performance, other biorthogonal conjugations could be considered. Thus, the Pictet-Spengler reaction52,53,55,56 or recently reported trapped Knoevenagel condensation could become viable alternatives.57

Figure 3. Assembly of 11 and cell delivery studies with 12. (A) Reducing SDS-PAGE gel monitoring reaction steps towards 11. Left panel: coomassie-stained gel. Right panel: In-gel fluorescence excited by broad bandwidth UV light. M: molecular mass marker; 10 and 11 were purified by SEC prior to analysis. (B) Flow cytometry analysis detecting TAMRA-



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mediated fluorescence of c-MET-positive incubated with 12 and 11 as control.

EBC-1

cells

To further functionalize the Fc fragment carrying fluorescent cargo at the N-terminus (1o), we coupled PNA 2 using eSrtA-mediated ligation towards 11. In this case conjugation was less efficient compared to generation of 4 and 5 (Figure 3A). This outcome could be attributed to the nature of chromophore, used enzyme batch or enzyme-substrate ratio. Indeed, eSrtA-mediated conversion of Fc construct 10a (comprising fluoresceinbased fluorophore 7a instead of the rhodamine derivative 7) to the respective construct 11a was significantly more efficient, especially when a higher amount of enzyme was used (Supplementary Figure S4). Hybridization of 11 with aptamer 6 yielded dual-functionalized 12. Successive flow cytometry-based binding assays were conducted with this construct as described for 8, but instead of staining with fluorescently labeled secondary antibody intrinsic TAMRA moiety served as fluorescent marker. Flow cytometry revealed a significant increase in fluorescence for EBC-1 cells incubated with 12, compared to aptamer-lacking 11 (Figure 3B). No fluorescence increase was observed in case of c-MET-negative T47D cells (Supplementary Figure S11) confirming that the desired payload was delivered to a cancer cell selectively. In conclusion, we demonstrated that Fc fragments of a human IgG1 antibody can serve as useful scaffolds for the generation of multifunctional constructs comprising potent binders of therapeutically relevant targets, vehicles for cellular delivery and small-molecular cargoes. This challenge was addressed using a combination of enzymatic and chemical ligation strategies and hybridization-mediated conjugation of complementary guiding units. We developed a modular platform for the straightforward generation of aptamer-Fc conjugates, which also enables site-specific incorporation of other payloads thereby suggesting that this strategy could be beneficial for various biomedical applications. It provides an opportunity to make use of the large pool of currently available aptamers or to apply an efficient in vitro selection strategy towards aptamers with tailored binding characteristics. Since aptamers seem to be nonimmunogenic and immunogenicity problems with antibodies often occur at the antigen-binding sites,3,5 aptamer-Fc conjugates could potentially diminish the problem of neutralizing anti-CDR antibodies. Future studies will include conjugation of cytotoxic payloads followed by evaluation of targeted cytotoxicity as well as further characterization regarding in vivo stability and in particular immunogenicity of the presented constructs.

METHODS Detailed information on used cell lines and cultivation conditions, DNA aptamer 6 and control DNA sequences 6a and 6b, recombinant c-MET receptor, synthesis of PNAs and aminooxy TAMRA derivatives, as well as cloning of Fc expression plasmids and analytical details are available in the Supporting Information.

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Recombinant production of dual-tagged Fc framework. Fc fragment 1 bearing two enzymatically addressable sites as well as control construct 1a lacking a Cys residue at the FGE recognition motif (Supplementary Section 3, Scheme S1) were simultaneously expressed with human formylglycine-generating enzyme in suspensionadapted human embryonic kidney (HEK293-6E) cells transiently cotransfected with Fc fragment-encoding plasmid pEXPR-IBA42 and the FGE-encoding plasmid pcDNA3.1 Neo(+) hSUMF1 (obtained from AddGene (Plasmid ID 24386),32 details described in Supplementary Section 1). Produced Fc fragments were purified using protein A chromatography. Synthesis of covalent conjugates 4, 5, 10, and 11. Conjugates 4, 5 and 11 were produced upon sortase Amediated transpeptidation. An evolved pentamutant of sortase A (referred to as eSrtA) with improved catalytic activity was produced and purified as described elsewhere.34 Peptide nucleic acids 2 and 3 comprising sequence CATCTAGTGA and N-terminally equipped with a pentaglycine moiety were synthesized on solid support (Supplementary Section 1, Schemes S2, S3) and used for sortase-mediated ligation (Figure 1A, Supplementary Figure S1). Thus, 10 µM Fc fragment 1 were supplemented with 250 µM 2 or 3 in sortase reaction buffer (50 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5 adjusted with HCl). Reaction was started by addition of 10 µM eSrtA and incubated for 30 min at 22 °C. Controls included reactions without either Fc fragment 1, eSrtA or counterparts 2/3, respectively. Conjugation using the aldehyde tag towards construct 10 was performed by addition of equal volume of aminooxy-TAMRA 7 (Scheme 1, Supplementary Section 1) in 500 mM NaOAc, adjusted to pH 4.6 with AcOH to 1 (15 µM in PBS) followed by incubation over 72 h at 20 °C. After optimization of reaction conditions 7.5 µM 1 and 12.5 molar equivalents 7 were used for preparative reactions. Fc fragment 1a comprising the cysteine residue within the aldehyde tag replaced by alanine served as a control regarding site selectivity (Figure 3 and Supplementary Figures S2 and S3). Reaction mixtures were concentrated using Amicon centrifugal filter units (MWCO 10 kDa, EMD Millipore) and products 4, 5, 10, 11 were isolated by size exclusion chromatography (SEC) using a Superdex 200 10/300 GL column and an ÄKTA purifier system (GE Healthcare) at a flow rate of 1 mL min–1 with Tris-buffered saline (Supplementary Figure S5). Self-assembly towards hybrid constructs 8, 9, 12 included hybridization of, respectively, 4, 5, and 11 to aptamer 6 upon incubation in Tris-buffered saline for 10 min at 4° C followed by SEC purification. An 80 bases DNA oligonucleotide 6a was used as a control for hybridization selectivity (Supplementary Figures S5, S6 and S7A). Ratios between 6 and 4 were varied to determine stoichiometry and optimize reaction conditions (Figure 1B). Three equivalents of 6 were used on preparative scale. To ascertain stability of the formed


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hybrid constructs, purified 9 was stored at 4 °C and analyzed after 1, 3 and 7 days by SEC (Supplementary Figure S7A). Additionally, melting temperature of the PNA-DNA complex 2-6b was assessed by UV absorption spectroscopy (Supplementary Figure S7B). Biolayer interferometry. BLI experiments addressing the c-MET-binding kinetics of solitary aptamer 6 and conjugate 8 were conducted using the Octet RED system with anti-human Fc (AHC) or streptavidin biosensors (all ForteBio), respectively. (For the details see Supplementary Section 1 and Figure S8.) Cell binding and internalization studies. Flow cytometry experiments were performed using a BD Influx cell sorter and BD FACS Sortware with minimum detection of 2 × 104 events. Washing and incubation steps were, unless stated otherwise, performed at 4 °C using PBS with 1% (w/v) BSA. For cell binding experiments, EBC-1 and T47D cells were labelled with 8 at varying concentrations followed by incubation with an anti-human IgG (Fc gammaspecific) PE conjugate (eBioscience) and measurement of PE-mediated fluorescence by flow cytometry (Figure 1C). Dissociation constant (KD) upon cell binding was determined by nonlinear regression using SigmaPlot software (version 11, Systat Software) (Figure 1C, 1D and Supplementary Section 1). Binding and fluorescence of compound 12 was assessed by incubation with 250 nM 12 and subsequent determination of TAMRA-mediated fluorescence (Figure 3B, Supplementary Figure S11). Internalization studies were performed using fluorescence microscopy and flow cytometry. In the first case, cells were grown on glass coverslips followed by 1 h incubation with 1 µM 9 and 1 h labelling with Alexa Fluor® 488-conjugated AffiniPure Fab Fragment Goat AntiHuman IgG (Jackson ImmunoResearch Laboratories). Afterwards, cells were washed and incubated in the respective medium with 1% (w/v) BSA (without FBS) at 4° C or 37 °C for 1 hour. A subset of each cell line was afterwards washed 3 times with ice-cold low pH buffer (50 mM glycine, 150 mM NaCl, pH 2.7 adjusted with HCl). Finally, cells were fixed with 4% (w/v) paraformaldehyde, mounted with ProLong® Diamond Antifade Mountant with DAPI (Life Technologies) and analyzed using a Leica TCS SP5 confocal microscope equipped with a 100× objective (Leica Microsystems) (Figure 2A, Supplementary Figure S9. To assess internalization using flow cytometry, 6 × 105 cells were allowed to attach to six well plates. The next day, cells were incubated with 500 nM 8 for 1 hour, labeled with Anti-Human IgG (Fc gamma-specific) PE (eBioscience) for 30 min followed by incubation at 4 °C or at 37 °C for 1 hour and washed three times with ice-cold low pH buffer (50 mM glycine, 150 mM NaCl, pH 2.7 adjusted with HCl). Afterwards, cells were detached using trypsin-EDTA and analyzed using flow cytometry as described (Figure 2B, Supplementary Figure S10).

ASSOCIATED CONTENT Supporting Information Available: This material is available free of charge via the Internet.

AUTHOR INFORMATION Corresponding Author *[email protected] Present Addresses ⊥BioNTech

AG, Mainz, Germany

Author Contributions ‡These authors contributed equally.

Funding Sources This work was supported in part by Deutsche Forschungsgemeinschaft through grant KO 1390/10-1 to H.K. and grant DI 542/8-1 to U.D. in the frame of the DFG priority program SPP 1623.

ACKNOWLEDGMENT We thank S. Becker, A. Doerner, B. Piater and C. Sellmann at EMD Serono as well as J. Becker and D. Könning for the support and advice. Furthermore, we thank U. Betz and the Merck Serono Innovation Cup initiative for funding and support.

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