Sortase-Catalyzed Transformations That Improve the Properties of Cytokines

Sortase-catalyzed transformations that improve the properties of cytokines

Maximilian W. Poppa,b, Stephanie K. Dougana, Tzu-Ying Chuanga, Eric Spoonera, and Hidde L. Ploegha,b,1

aWhitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142; and bDepartment of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02142

Edited by Donald F. Steiner, University of Chicago, Chicago, IL, and approved January 3, 2011 (received for review November 9, 2010)

Recombinant protein therapeutics often suffer from short circulat- ing half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortase- mediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety SCIENCES of proteins, provided that their termini are not involved in receptor APPLIED BIOLOGICAL binding or function.

any clinically relevant cytokines share a four-helix bundle Mstructure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues Fig. 1. The sortase reaction scheme. Cyclization of substrates equipped with a C-terminal LPXTG sortase recognition element as well as an N-terminal critical for interaction of a cytokine with its receptor show that glycine by SrtAStaph (left). Substrates lacking the N-terminal glycine and the receptor contacts the sides of the helical bundles (1). This incubated in the presence of exogenous oligo-glycine functionalized PEG mode of interaction positions the N and C termini of the cytokine are site-specifically PEGylated (right). Circular, PEGylated proteins can be away from the receptor. constructed by incubating substrates bearing an N-terminal glycine and a Polyethylene glycol chains attached to therapeutically impor- C-terminal SrtAstrep cleavage site (LPXTA) with an alanine-based nucleophile tant proteins increase circulatory half-life, reduce clearance by carrying an amino oxy group and the SrtAStaph cleavage site (Center). This kidney filtration, reduce proteolysis, and reduce the generation transpeptidation product is cyclized upon incubation with SrtAStaph. Finally, of neutralizing antibodies (2–4). The attachment of PEG com- PEG is attached by aniline-catalyzed oxime ligation with methoxy-capped PEG propionaldehyde. The oxime bond formed is shown (inset). monly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often gen- erates a heterogeneous mixture in which multiple amino acids in sortase accepts di-alanine based nucleophiles, which the S. aureus the target are modified with a PEG chain, necessitating cumber- enzyme does not. This sortase (SrtAstrep) cleaves the LPXTA mo- some separations and characterization (5). Such PEGylated tif between theronine and alanine and allows installation of mod- molecules often show decreased biological activity, likely due to ified alanine-based nucleophiles. SrtAstrep also recognizes and attachment of a PEG chain to a residue important for interaction cleaves LPXTG motifs, albeit with reduced efficiency, however with the receptor (6)-this problem may be overcome by site- the LPXTA motif is refractory to cleavage by SrtAStaph (13, 14). specific PEGylation. Although engineering of a carefully placed Here we present a general strategy for site-specific modifica- unpaired cysteine residue allows site-specific PEGylation (7, 8), tion of therapeutic recombinant proteins at their termini, an this method must be tailored to the specific protein target. approach that is particularly well suited to the four-helix bundle Sortase A from Staphylcoccus aureus (SrtAStaph) is a thiol- cytokines, where the termini are distant from the receptor inter- containing transpeptidase that recognizes an LPXTG motif in action site. We show improvements in thermal stability by cova- multiple structurally unrelated substrates (9). SrtAStaph cleaves the peptide bond between the threonine and glycine residues with Author contributions: M.W.P. and H.L.P. designed research; M.W.P., S.K.D., T.-Y.C., and concomitant formation of a thioacyl enzyme intermediate that E.S. performed research; M.W.P., S.K.D., and E.S. analyzed data; and M.W.P. and H.L.P. involves the catalytic cysteine and the substrate threonine. This wrote the paper. acyl-enzyme is resolved by nucleophilic attack by the N terminus Conflict of interest statement: The authors have applied for a patent based on the tech- of an oligoglycine peptide, resulting in formation of an amide nology used in the paper. bond between the substrate protein and the incoming nucleophile This article is a PNAS Direct Submission. (10). Sortase A tolerates C-terminal extensions of the oligogly- Freely available online through the PNAS open access option. cine nucleophile, allowing diverse functionalized nucleophiles 1To whom correspondence should be addressed. E-mail: [email protected]. to be installed site specifically onto proteins equipped with an This article contains supporting information online at www.pnas.org/lookup/suppl/ LPXTG motif (11, 12). The related Streptococcus pyogenes doi:10.1073/pnas.1016863108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016863108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 lently joining the N and C termini of these proteins (15). We thus unable to undergo intramolecular cyclization) was achieved present a general method for combining both site-specific by incubation with sortase and a GGGK peptide to which a PEGylation and improved thermal stability in a single molecule 10 kDa PEG moiety was affixed via the lysine ϵ-amine (Probe 1, by using a molecular suture produced in two successive rounds of Fig. 2A and Fig. S1A). We confirmed equipotent biological activ- sortase-catalyzed transpeptidation (Fig. 1). ity of all four interferon alpha variants in a Daudi cell proliferation inhibition assay when compared to a commercially available, Results non-PEGylated preparation (Fig. 2D and Table S1). Addition of Modification of IFNα2. We first applied the sortase reaction to a protein-sized PEG module to the C terminus of IFNα2 does human interferon alpha (IFNα2), a four-helix bundle cytokine not appreciably perturb receptor binding and biological potency. where chemical PEGylation results in multiple positional isomers We hypothesized that the circular IFNα2 would be more resistant and a ∼90% decrease in biological potency (16). We expressed to thermal denaturation, because the termini are clamped shut in Escherichia coli two versions of IFNα2, both with an LPETG and thus should not “fray” (15, 17). We measured thermal dena- sortase motif immediately followed by a hexahistidine tag for ease turation with a Thermofluor assay, using the commercially of purification. At the amino terminus, one of the two proteins available Sypro Orange dye (18). Indeed, the circular form of α possesses a diglycine motif for cyclization. This material can IFN 2 had a significantly elevated melting temperature (Tm) either be cyclized or PEGylated at the C terminus (Fig. 2A). compared to the other three variants, all of which exhibited α ’ IFN 2 with an N-terminal diglycine motif and a C-terminal nearly identical Tm s (Fig. 2E, Table S1, and Fig. S2A). Finally, LPETG was successfully cyclized by incubation with SrtAStaph we injected mice with either the linear IFNα2 variant used to con- and purified to homogeneity by ion-exchange chromatography. struct the PEGylated form, or the IFNα2 variant bearing a 10 kDa The mass of the circularized product in a crude sortase reaction PEG chain (Probe 1) at its C terminus and measured the serum differs from a linear hydrolysis product by −18 Da, consistent levels over time by ELISA. The PEGylated form decayed with with an intramolecular transpeptidation reaction (Fig. 2B). We slower kinetics than its non-PEGylated counterpart (Fig. 2F identified the unique junction peptide that arises from intra- and Table S1). molecular cyclization by tandem mass spectrometry (MS/MS) analysis of tryptic digests (Fig. 2C). Site-specific PEGylation of Modification of a Single Protein Preparation by Multiple Nucleophiles. the IFNα2 variant that lacks the N-terminal diglycine motif (and The sortase platform offers the ability to install nearly any A D

E Fig. 2. Synthesis and characterization of human IFNα2 conjugates. (A) GG-IFNα-LPETGGHis6 was used to obtain backbone-cyclized IFNα2, and IFNα- LEPTGGHis6 was used to make IFNα-PEG (10 kDa), bearing a C-terminal PEG chain. Purified proteins B were resolved by 12.5% SDS-PAGE and stained with Coomassie. (B) ESI-MS characterization of IFNα species in crude sortase reactions either before (linear GG-IFNα-LPETGGHis6) or after (cylic IFNα2) overnight incubation at 25 °C. (C) MS/MS identification of a tryptic peptide comprising the IFNα2 C terminus,

followed by the SrtAStaph cleavage site, joined to the N-terminal glycines in the IFNα2 precursor. (D)In vitro bioactivity of IFNα conjugates in a Daudi cell F proliferation inhibition assay. Dose-response curves for Daudi cell proliferation inhibition were measured C by [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium] bromide (MTT) assay, in triplicate, with the standard deviation displayed. (E) Resistance of backbone-cyclized IFNα to thermal denaturation. Change in T m for circular and PEGylated IFNα conjugates, relative to their precursor proteins, were measured by thermal shift assay, with n ¼ 4.(F) Change in serum IFNα levels following tail vein injection of site- specifically PEGylated IFNα as well as its precursor protein. 10 μg of each protein was injected and, at the indicated time points, blood was withdrawn and cytokine concentration in serum was determined by ELISA assay. Data are means standard deviation for three mice in each group.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1016863108 Popp et al. Downloaded by guest on September 27, 2021 nongenetically encoded entity onto the C terminus of a single ∼40% of its mass is composed of bulky, charged N-linked glycans, preparation of recombinant protein. We demonstrate this ability and cyclization provides a stringent test of the sortase method, as by synthesizing a nucleophile that bears a 20 kDa PEG moiety it requires (minimal) modifications at both termini. We replaced (Probe 2 and Fig. S1A), as well as the previously described the endogenous EPO signal sequence with the murine H-2Kb 10 kDa PEG probe (Probe 1), and appended them to the iden- signal sequence, followed by two glycine residues. This signal tical preparation of IFNα2 (Fig. 3A). Both conjugates were tested peptide is cleaved in mammalian cells with concomitant exposure for biological activity (Fig. 3B) and are highly potent (Table S2). of an N-terminal glycine residue. We added the requisite sortase motif followed by a hexahistitdine tag at the C terminus of EPO Superior Properties Endowed upon Sortase Substrates Are Applicable for ease of purification. Human embryonic kidney (HEK)-293T to Multiple Proteins. Sortase allows the installation of the same cell lines stably transduced with this construct yielded prepara- nonnatural group onto different proteins, unrelated in sequence tions that were <50% pure after a single round of Nickel-nitrilo- or amino acid composition. Accordingly, we applied the sortase triacetic acid immobiliized metal affinity chromatography (Ni- reaction to a different four-helix bundle cytokine, GCSF-3, with NTA IMAC) of conditioned medium. Material from such pre- similar results. We first made the linear precursor GCSF variants parations can be cyclized successfully upon incubation with and then cyclized or PEGylated them with probe 1 using sortase sortase (Fig. 5A). Again, we identified the unique junction pep- (Fig. 4A). All variants possess equal or superior in vitro bioactiv- tide that arises from covalent ligation of the N and C termini by ity relative to a commercial non-PEGylated preparation, as MS/MS analysis of cyclized EPO, shorn of all glycans by incuba- assessed by a standard cell proliferation assay with the NFS-60 tion with peptide: N-glycosidase F (PNGaseF), and digested murine myeoblastic cell line (19) (Fig. 4B and Table S3). Like with trypsin (Fig. 5B). Both the linear preparations as well as cyclic IFNα2, cyclic GCSF-3 shows increased thermostability the sortase reaction containing cyclic EPO were tested in a cell (Fig. 4C, Fig. S2B, and Table S3) and PEGylated GCSF-3 has proliferation assay with Ba/F3 cells that stably express the ery- a significantly extended circulatory half-life relative to its non- thropoietin receptor (20) and these compared favorably with PEGylated precursor (Fig. 4D and Table S3) upon injection into commercial EPO preparations. Most importantly, the cyclic form mice. PEGylated GCSF-3 also led to a more robust and pro- is as potent as its linear counterpart (Fig. 5C and Table S4), longed proliferation of granulocytes in injected mice than its indicating that modification of the EPO termini does not affect non-PEGylated analog (Fig. 4E). its biological activity. SCIENCES Modification of Glycoproteins. Many important therapeutics are Two-Step Transacylation Allows Combination of Enhanced Properties. glycosylated proteins that traverse the secretory pathway and are Having shown for multiple examples that sortase-mediated APPLIED BIOLOGICAL produced in mammalian cell culture. Are such proteins equally site-specific modification of the C terminus with PEG increases amenable to engineering using a sortase-mediated transpeptida- circulatory half-life with nearly no loss of biological activity, tion reaction? We chose human erythropoietin (EPO) because and that covalent closure of the N and C termini yield proteins stabilized against thermal denaturation, we executed a scheme ) that combines these desirable properties (Fig. 1). In the first step, A we exploit a previously described sortase from Streptococcus pyogenes (13, 14) that accepts alanine-based nucleophiles to affix a

-His6 GK(PEG10 kD) GK(PEG20 kD peptide containing a nonnatural amino oxy containing amino

GG GG acid, followed by the SrtAStaph cleavage site. We use SrtAStaph to

α-LPET α-LPETGG effectuate covalent closure, as the LPETAA site left by SrtAstrep is

IFN IFNα-LPET IFN resistant to attack by SrtA , to yield a circular protein with the 100 kD Staph 75 kD amino oxy group at the place of suture. Next, the amino oxy group 50 kD is used in a bioorthognal oxime ligation reaction (21–23) with

37 kD commercially available methoxy-capped PEG-propionaldehyde (Fig. S1B). We recombinantly expressed IFNα2 bearing two 25 kD glycines at the N terminus, and at the C terminus the SrtAstrep 20 kD cleavage site, followed by a hexahistidine tag. The purified protein was subjected to the two-step transacylation procedure to 15 kD yield circular IFNα2 bearing the amino oxy group. After ion exchange chromatography, this material was PEGylated and pur- 10 kD ified by cation exchange chromatography (Fig. 6A). Without Daudi Cell Proliferation Inhibition optimization, the final yield of circular PEGylated protein was Standard ∼10%, because of the multiple separate chromatography and pro- B 0.8 αLPETGGHis6 tein concentration steps involved in production and PEGylation. PEG(10kDa) 0.6 The mass of the material before oxime ligation was consistent PEG(20kDa)

570 with its cyclization (Fig. S3A). Digestion with AspN and MS/ 0.4

Abs MS analysis revealed the unique peptide that arises as a conse- 0.2 quence of backbone cyclization consisting of the molecular suture α 0.0 probe stitched between the C and N termini of IFN (Fig. 6B). All three variants yielded virtually indistinguishable IC50 values in -1 0 1 2 3 4 log (pg/ml) an inhibition of cell proliferation assay (Fig. 6C and Table S5) and the circular versions were stabilized against thermal denaturation Fig. 3. Sortase installs multiple probes onto a single preparation of protein. (Fig. 6D, Table S5, and Fig. S2C). The circular versions remained α (A) The same preparation of IFN -LEPTGGHis6 was PEGylated by sortase re- biologically potent after boiling when given the opportunity to action with either a 10 kDa or a 20 kDa PEG nucleophile. Purified proteins refold, whereas we were unable to extract an IC50 value for the were resolved by 12.5% SDS-PAGE and stained with Coomassie. (B) In vitro bioactivity of 10 kDa and 20 kDa PEGylated IFNα conjugates in a Daudi cell boiled linear preparation over the concentration range assayed proliferation inhibition assay. Dose-response curves for Daudi cell prolifera- (Fig. 6E and Table S6). We injected the linear and the circular, tion inhibition were measured by MTT assay, in triplicate, with the standard PEGylated forms of IFNα2 into the tail vein of mice and mea- deviation displayed. sured half life by ELISA. The circular, PEGylated species was

Popp et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 27, 2021 D A 100000 -His6 GCSF-LPETGGHis G 6 GK(PEG10 kD) 10000 PEG(10kDa)

GG-His6 GG LPETG 1000 LPET LPET CSF-

G-G CSF-

G G GCSF- pg/mL 100

100 kD 10 75 kD 50 kD 1 37 kD 0 20 40 60 Time (hours) 25 kD 20 kD

15 kD Fig. 4. Synthesis and characterization of human GCSF-3 conjugates. (A) GG-GCSF-LPETGGHis6 was used 10 kD E PBS to make backbone cyclized GCSF-3, and GCSF- GCSF-LPETGGHis 25 6 LEPTGGHis6 was used to make GCSF-PEG (10 kDa), GCSF-PEG(10kD) bearing a C-terminal PEG chain. Purified proteins 20 NFS-60 Cell Proliferation were resolved by 12.5% SDS-PAGE and stained with B 15 Coomassie. (B) In vitro bioactivity of GCSF-3 conju- 1.5 gates in an NFS-60 cell proliferation assay. Dose-re- 10 1.0 Standard sponse curves for NFS-60 cell proliferation were 570 GG-GCSF-LPETGGHis 6 5 measured by MTT assay, in triplicate, with the stan- Circular % New granulocytes Abs 0.5 dard deviation displayed. (C) Resistance of backbone GCSF-LPETGGHis 0 6 cyclized GCSF-3 to thermal denaturation. Change in PEG(10kDa) 0 0.0 24 44 53 72 93 168 T m for circular and PEGylated GCSF-3 conjugates, re- Time (hrs) -2 0 2 4 lative to their precursor proteins, were measured by n ¼ 4 log (pg/ml) thermal shift assay ( ). (D) Change in serum GCSF levels following tail vein injection of site-specifically 6 PEGylated GCSF-3, as well as its precursor. 5 μg of each C 4.9 protein was injected and, at the indicated time points, 4 blood was withdrawn and cytokine concentration in )

C serum was determined by ELISA assay. Data are means o 2 from three mice in each group, with the standard

Tm( deviation displayed. (E) GCSF, PEGylated via sortase, ∆ -1.6 is highly potent in vivo. C57BL/6 mice (n ¼ 4 per 0 group) were injected intravenously with linear GCSF, GCSF-PEG (10 kD), or saline control (PBS). Peripheral -2 blood was collected at the indicated times and stained with antibodies to Gr-1 and CD11b. Newly generated granulocytes were defined as Gr-1 intermediate Circular PEG(10kDa) CD11b+ cells (31).

cleared significantly more slowly than the linear form (Fig. 6F and Cyclization of the backbone of protein-based therapeutics also Table S5). proceeds efficiently (Fig. S3B) and yields preparations that are more resistant to thermal denaturation. Cyclic proteins are also Discussion resistant to exoproteolytic attack (25, 26), a feature that may en- The sortase transpeptidase reaction allows facile site-specific hance utility of any therapeutic proteins exposed to exoproteases, PEGylation of multiple distinct proteins. In all cases tested, the for example upon receptor mediated internalization. In addition, site-specific C-terminal PEGylation proceeds efficiently (Fig. S3B) cyclization of proteins and peptides has been shown to improve and yields adducts of known stoichiometry that are biologically potency, stability, and oral bioavailability (27–30). equipotent to the non-PEGylated versions, but retain the in- Finally, we have inserted a nontemplate encoded entity in what crease in circulatory half life associated with PEG modification. is topologically internal to a circular protein, a feat that cannot be We attribute these properties to the fact that all enzymatic trans- accomplished genetically by intein-based methods or by any other formations are performed on native proteins, procedures that currently known means. More generally, this dual transacylation should not affect overall conformation or exposure of function- scheme can be used to insert nonnatural groups between fully ally important side chains. Because our approach to PEGylation native proteins expressed separately, or between protein do- is site specific, all preparations are also homogenous and easily mains. We suspect that this approach will find application in purified (5). Although we limit our examples to four-helix bundle the protein engineering field and so further extend the utility cytokines, this platform should be readily extended to structurally of sortases as tools. distinct therapeutic proteins, with the singular requirement that Experimental Procedures the C terminus is not involved in receptor binding. A sortase- Sortase Reactions. Sortase reactions with SrtAStaph were perform- based approach requires the genetic fusion of a very small (five ed as described previously (11). For either cyclization or PEGyla- amino acid) tag to the protein of interest and all transformations μ μ tion, reactions containing 50 M substrate, 50 M SrtAStaph and occur under native conditions. The additional amino acids that 1 mM probe (for PEGylation) were incubated overnight at 25 °C result from fusion to the sortase recognition motif are close to without agitation. Reactions were purified by cation exchange the site of PEG attachment. Immunogenicity of this site is very chromatography on a Mono-S (GE Healthcare). For IFNa2 re- likely reduced by the PEG moiety shielding this area (3). We and actions, continuous gradient chromatography was performed others have labeled proteins with an exposed N-terminal glycine with 20 mM 2-(N-morpholino) ethane sulfonic acid (MES) pH using sortase (14, 24). This approach should therefore be readily 5.0 and 20 mM MES, 1 M NaCl pH5.0, as eluent. For GCSF re- extended to site-specific PEGylation at the N terminus as well. actions, continuous gradient chromatography was performed

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B E B

C F SCIENCES APPLIED BIOLOGICAL

Fig. 5. Synthesis and characterization of cyclic human erythropoietin. (A) GG-EPO-LPETGGHis6 bearing the H-2Kb signal sequence was expressed in HEK-293T cells. Conditioned media was subjected to Ni-NTA IMAC purifi- α μ Fig. 6. Synthesis and characterization of cyclic, PEGylated IFN 2. (A) GG- cation. Eluted material was supplemented with 100 M SrtAStaph and incu- IFNα-LPETAAHis6 was used to make backbone-cyclized IFNα2 with the amino bated at 25 °C. Aliquots were removed at the indicated time points, oxy moiety stitched between the site of closure. This material was then used subjected to 12.5% SDS-PAGE and protein was visualized by silver staining. to generate cyclic, PEGylated IFNα2 by aniline catalyzed oxime ligation. Pur- (B) MS/MS identification of a peptide generated by tryptic digestion contain- ified proteins were resolved by 12.5% SDS-PAGE and stained with Coomassie. ing the C terminus of EPO, followed by the SrtAStaph cleavage site and (B) MS/MS identification of a peptide generated by AspN digestion contain- joined to the N terminus of EPO. (C) In vitro bioactivity of linear and cylic ing the C terminus of IFNα, followed by the alanine-based probe joined to the EPO conjugates in a cell proliferation assay using BaF3 cells stably expressing N terminus of IFNα by SrtA . Note that the amino oxy group was likely lost the erythropoietin receptor (BaF3-EPOR). Eluted material from Ni-NTA IMAC Staph during MS/MS analysis and ESI-MS reconstructions of unfragmented protein purification was incubated in the presence or absence of sortase for 16 h at revealed a mass consistent with installation of the amino oxy group (Fig. S3). 25 °C and these crude reactions were measured for in vitro EPO bioactivity. (C) In vitro bioactivity of linear and cyclic IFNα conjugates in a Daudi cell pro- Dose response curves for BaF3-EPOR cell proliferation were measured by MTT liferation inhibition assay. Dose-response curves for Daudi cell proliferation assay, in triplicate, with the standard deviation displayed. Concentrations inhibition were measured by MTT assay, in triplicate, with the standard de- were not adjusted for impurities in the EPO preperations. viation displayed. (D) Resistance of circular-AOAA and circular-PEGylated α T ’ with 20 mM sodium acetate pH 4.5 and 20 mM sodium acetate, IFN to thermal denaturation. Change in m s for circular-AOAA and circular- PEGylated IFNα conjugates, relative to their precursor protein, were mea- 1 M NaCl pH 4.5, as eluent. Peaks containing the desired product sured by thermal shift assay (n ¼ 4). (E) Circular cytokines remain biologically were pooled, concentrated with Vivaspin 500 centrifugal concen- active after boiling. The indicated variants were diluted to 1 mg∕mL in trators (Sigma) and protein concentration was determined by RPMI∕10% serum, boiled for 4 min, allowed to cool at room temperature Bradford Assay (BioRad). For EPO cyclization, 300 μLof for 16 h, and assayed for inhibition of Daudi cell proliferation. (F) Change Ni-NTA elutate (0.101 mg∕mL total protein) was incubated with in serum IFNα levels following tail vein injection of circular, site-specifically 100 μM SrtA and sortase buffer in 400 μL total volume at 25 °C PEGylated IFNα as well as its linear precursor protein. 10 μg of each protein for 16 h. For two-step transacylation reactions, 150 μM substrate was injected and, at the indicated time points, blood was withdrawn and was incubated with 50 μM SrtA and 2 mM probe in 100 mM cytokine concentration in serum was determined by ELISA assay. Data are strep means from three mice in each group, with the standard deviation displayed. Tris, 150 mM NaCl pH 8.0 for 4 h at 37 °C. SrtAstrep activity was halted by incubation with 500 μM of the protease inhibitor E-64 on ice for 1 h. Crude reaction mixtures were then supple- propionaldehyde, and 100 mM aniline (JT Baker) and incubated mented with 10 mM imidazole and subjected to Ni-NTA chroma- at 30 °C for 3 h without agitation. This reaction mixture was tography (Qiagen) to remove prematurely cyclized material. again purified by cation exchange chromatography, concentrated, Protein was eluted with 50 mM Tris, 150 mM NaCl, 500 mM and protein concentration was determined by Bradford assay. imidazole, and buffer exchanged into SrtAStaph buffer (50 mM Tris, 150 mM NaCl, and 10 mM CaCl2) with a PD10 desalting Thermal Denaturation Assays. Thermal shift assays were performed column (GE Healthcare). For cyclization, this material was in a Roche 480 lightcycler and fluorescence was measured with μ Excitation ¼ 533 nm, Emission ¼ 610 nm. For GCSF-3, 1 μgof incubated overnight with 50 M SrtAStaph and purified by cation exchange as described for other IFNα2a reactions. Concentrated, each variant was mixed with 1 μLof100× Sypro orange in 25 μL purified circular IFNα2 bearing the Aminooxyacetic acid (AOAA) total volume of 50 mM acetate pH 4.0, 150 mM NaCl. n ¼ 4 for group was then diluted with 1 volume of 50 mM sodium acetate all samples with buffer as a blank control and lysozyme as a pH 4.5, 150 mM NaCl containing 2 mM methoxy-capped PEG- positive control. For IFNα2, 1 μg of each variant was mixed with

Popp et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 27, 2021 1 μLof100× Sypro orange (Invitrogen) in 25 μL total volume Institute) were used to measure EPO activity. BaF3 EPO-R cells of 50 mM MES ph 5.0, 150 mM NaCl. n ¼ 4 for all samples were cultured as described for NFS-60 cells and assays were with buffer as a blank control and lysozyme as a positive control. performed essentially as described for G-CSF, except EPO circu- Samples were heated from ambient temperature to 95 °C at larization reactions incubated with or without sortase for 16 h 0.01 °C∕s with 6 acquisitions∕°C. The included Roche software at 37 °C were used. The total protein concentration of the EPO was used to calculate the negative first derivative of the tempera- input material (measured by the Bradford method) was used and ture change as a function of time and the minima were used as the was not adjusted for impurities. Commercial preparations of Tm. Because of the impurities in the cyclic EPO preperations, Tm EPO (eBioscience) were used as standards and readings were was not determined. blanked against BaF3 EPO-R cells cultured with no cytokines. Activities were measured a minimum of n ¼ 3 times. Cell-Based Bioactivity Assays. Daudi cell proliferation inhibition assays to measure the activity of IFNα2 conjugates were per- Circulatory Half-Life Assays. For circulatory half-life assays, mice formed as described (8) and compared to commercial IFNα2 were injected in the tail vein with each protein (10 μg per mouse (PBL Interferon Source) with low passage Daudi cells (ATCC), for IFNα2 conjugates, 5 μg per mouse for G-CSF conjugates) cultured in RPMI medium 1640∕10%Heat inactivated fetal ð Þ∕50 ∕ 50 μ ∕ and subjected to retroorbital eye bleed at the indicated time calf serum IFS units mL penicillin, g mL streptomy- 1 957 cin sulfate. Cell proliferation was measured by MTTassay accord- points. Blood was harvested, centrifuged at ; xg in a tabletop ing to manufacturer’s directions (ATCC). For the denaturation centrifuge, and serum was collected and snap-frozen. Elisa assays assay, samples diluted to 1 μg∕mL in RPMI medium 1640∕10% to measure the quantity of cytokine in serum samples were IFS were boiled for 4 min, allowed to cool at room temperature performed according to the manufacturer’s directions (IFNα for 16 h, and assayed for Daudi cell proliferation inhibition. from PBL Interferon Source, G-CSF from Invitrogen). Each NFS-60 cell proliferation assays to measure G-CSF conjugate conjugate was injected into n ¼ 3 mice. Data was fit to a two activity were performed as follows. NFS-60 cells (a kind gift from phase exponential decay in GraphPad Prism. James Ihle, St. Jude Children’s Research Hospital) were cultured in RPMI medium 1640∕10%IFS∕50 units∕mL penicillin, 50 μg∕ Granulocyte Proliferation Assay. Peripheral blood was collected mL streptomycin sulfate supplemented with murine IL-3 retroorbitally into EDTA collection tubes. Red blood cells were (20 U∕mL, R&D Systems). Cells were washed extensively in lysed in hypotonic lysis buffer and the remaining peripheral complete RPMI medium 1640 medium lacking IL-3 and resus- blood mononuclear cells were stained with anti-myeloid differen- 5 pended at 1 × 10 cells∕mL in complete RPMI medium 1640 tiation antigen-1 (Gr-1) antibody labeled with phycoerythrin lacking IL-3. Titrated GCSF conjugates (50 μL) in complete (PE) (BD Pharmingen), anti-cluster of differentiation 11b RPMI medium 1640 were aliquoted into a flat bottom 96 well protein antibody labeled with FITC (anti-CD11b-FITC) (BD μ 0 5 × 104 ∕ plate and 50 L of cells ( . cells well) were added to each Pharmingen), and 7-Aminoactinomycin D (7-AAD) (ViaProbe, well. Cells were cultured for 3 d and an MTT assay (ATCC) was BD Pharmingen). Cells were analyzed using a FACS Caliber flow ’ performed according to manufacturer s directions. Each plate cytometer (BD). contained a titration of commercial GCSF3 (Peprotech) and readings were blanked against wells containing only NFS-60 ACKNOWLEDGMENTS. We thank Drs. Nick Yoder, John Antos, and Annemarthe cells. The activity of each conjugate was measured a minimum van der Veen, for helpful discussions, and Drs. Harvey Lodish and James Ihle of n ¼ 3 times. BaF3 cells stably expressing the EPO receptor for reagents. This work was supported by grants from the National Institutes (BaF3 EPO-R cells, a kind gift from Harvey Lodish, Whitehead of Health (NIH).

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