Synthesis of granulocyte–macrophage colony- stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material

Qiang Zhanga,1, Eric V. Johnstona,1, Jae-Hung Shiehb, Malcolm A. S. Mooreb, and Samuel J. Danishefskya,c,2

aLaboratory for Bio-Organic Chemistry and bCell Biology Program, Sloan-Kettering Institute for Cancer Research, New York, NY 10065; and cDepartment of Chemistry, Columbia University, New York, NY 10027

Contributed by Samuel J. Danishefsky, January 7, 2014 (sent for review December 18, 2013)

Granulocyte–macrophage colony-stimulating factor (GM-CSF) is bearing complex glycan domains at each native position, is illus- a medicinally important glycoprotein, used as an immunostimulant trative of the growing power of chemical synthesis to facilitate following bone-marrow transplant. On the basis of reports of its the study of medicinally relevant biologic targets (12–14). We potential utility as an anticancer vaccine adjuvant, we undertook describe herein the synthesis of homogeneous GM-CSF glyco- to develop a synthetic route toward single-glycoform GM-CSF. We proteins through a convergent route that offers ready access to describe herein a convergent total synthesis of GM-CSF aglycone a menu of glycoforms for further study. and two homogeneous glycoforms. Analytical and biological studies Structurally, GM-CSF consists of 127 aa with multiple sites of 27 confirm the structure and activity of these synthetic congeners. glycosylation (Fig. 1). Two N-linked glycans are located at Asn and Asn37 (15, 16). Interestingly, neither the position nor the glycoprotein synthesis | peptide ligation | alanine ligation extent of O-linked glycosylation has been unambiguously es- tablished. Studies suggest a range from two (Ser7 and Ser9/Thr10) 5 7 9 10 – to four (Ser , Ser , Ser , and Thr ) sites of glycosylation (17). ranulocyte macrophage colony-stimulating factor (GM- The tertiary structure is strongly influenced by two cross-linked GCSF) is a secreted glycoprotein that promotes cellular disulfide bonds. growth and proliferation. GM-CSF signaling initiates a cascade that culminates in the production of white blood cells through Results and Discussion stem-cell stimulation in the bone marrow (Fig. 1) (1). Thera- Our strategy for reaching homogeneous GM-CSF constructs peutically, this glycoprotein is valued for its properties as an relies upon powerful techniques developed for the synthesis of immunostimulant; GM-CSF impacts the production, differenti- complex biologics: namely, solid-phase peptide synthesis (SPPS), ation, and function of dendritic cells through potentiation of the + native chemical ligation (NCL), and metal-free dethiylation CD4 T-cell response (2, 3) and is regularly administered to (MFD). Merrifield’s SPPS method permits stepwise elongation patients undergoing chemotherapy or autologous bone-marrow of a growing peptide (18, 19). Convergence in the construction of CHEMISTRY transplant. GM-CSF is approved in Europe as the aglycone longer polypeptides, including small proteins, may be achieved (Molgramostim) (4) and in the United States as a glycosylated, through the use of the seminal NCL technology of Kent and mutant form (Sargramostim). At present, glycosylated GM-CSF coworkers, whereby fragments are merged at N-terminal cysteine is obtained exclusively via recombinant technologies using residues (20, 21). The possibility of expanding the scope of NCL yeast (Sargramostim) or Chinese hamster ovary (CHO) cell to encompass, for instance, alanine ligation was demonstrated in a key (Regramostim) technologies, which yield complex mixtures of paper by Yan and Dawson (22), who accomplished dethiylation glycoforms. The glycan heterogeneity reflects a lack of specificity in CHO-cell posttranslational glycosylation. The degree of Significance GM-CSF glycosylation has been reported to affect the in vivo properties of the glycoprotein (5, 6); the aglycone may cause As biologically active glycoproteins are increasingly investigated increased adverse side effects, perhaps due to its enhanced as potential therapeutic agents, there is a growing demand for susceptibility to truncation pathways (7). the development of strategies for the synthesis of homoge- In light of our longstanding interest in synthetic anticancer and neous, single-glycoform constructs for the purposes of rigorous HIV vaccines (8), we took note of reports suggesting that GM- evaluation. Currently, most glycoproteins are accessed through CSF might serve as a useful vaccine immunoadjuvant. Admin- recombinant methods as complex mixtures of glycoforms. istration of GM-CSF results in robust potentiation of the im- – mune response, and clinical studies suggest that the glycoprotein Granulocyte macrophage colony-stimulating factor (GM-CSF) is may hold promise as an adjuvant for anticancer vaccines (9, 10). an important glycoprotein therapeutic, used to stimulate the However, studies to date have used recombinantly derived GM- immune system following bone-marrow transplant and che- CSF mixtures, and results have been inconclusive (9); perhaps motherapy. GM-CSF is also being explored as a potential im- such translational issues might be addressed in a more in- munoadjuvant for anticancer vaccines. We have completed formative fashion with homogeneous GM-CSF agents. In a more a chemical synthesis of homogeneous, single-glycoform GM-CSF. speculative line of inquiry, we also wonder whether appendage of Through adaptation of this modular synthetic route, it will be tumor-associated carbohydrate antigens to the protein backbone possible to gain access to a menu of single-glycoform GM-CSF might yield powerful new anticancer vaccine candidates (11). congeners for a wide range of biological studies. Even beyond these fascinating medicinal questions, we recog- nized in GM-CSF a synthetically compelling glycoprotein target. Author contributions: Q.Z., E.V.J., J.-H.S., M.A.S.M., and S.J.D. designed research; Q.Z., E.V.J., With these considerations in mind, we undertook to design a and J.-H.S. performed research; Q.Z., E.V.J., J.-H.S., M.A.S.M., and S.J.D. analyzed data; and modular route to homogeneous GM-CSF glycoforms. In this Q.Z., E.V.J., J.-H.S., M.A.S.M., and S.J.D. wrote the paper. endeavor, we would rely upon key methodological and strategic The authors declare no conflict of interest. advances from many groups, including ours, in the synthesis of 1Q.Z. and E.V.J. contributed equally to this work. complex glycoproteins. The past decade has indeed witnessed 2To whom correspondence should be addressed. E-mail: [email protected]. a dramatic maturation of the field of “biologics” through chemical This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. synthesis. Our recent synthesis of homogeneous erythropoietin, 1073/pnas.1400140111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1400140111 PNAS | February 25, 2014 | vol. 111 | no. 8 | 2885–2890 Downloaded by guest on October 1, 2021 Fig. 1. GM-CSF 3D structure and fully glycosylated GM-CSF sequence (1). The ribbon structure of GM-CSF is based on the X-ray of GM-CSF aglycone expressed by Escherichia coli.

via Raney nickel mediation (22, 23). In 2007, our laboratory de- With the component fragments in hand, methods to accom- scribed mild MFD conditions that enable major expansion of the plish their merger were evaluated (Fig. 4). In the event, coupling menu of NCL beyond cysteine residues (24). Thus, subsequent to of fragments 1a (1) and 2a (2) in pH 7.0 kinetic NCL buffer cysteine-type ligation, the free thiol group may be readily removed afforded 3. The crude reaction mixture was directly subjected to through MFD to reveal either native alanines or a range of amino MFD. Happily, Cys34 was smoothly reduced to Ala34 (3→4)whereas 54 acid residues at the site of ligation (Fig. 2). the Thr -SEt functional group remained intact. NCL of fragments 3a (6) and 4a (7) followed by treatment with MeONH2•HCl Synthesis of the GM-CSF Aglycone provided polypeptide 8 with all of the cysteine residues depro- 4 8 We first set out to accomplish a synthesis of the GM-CSF agly- tected. The NCL-based coupling of and afforded the GM-CSF 9 9 cone. We envisioned dissection of our target into four fragments, aglycone primary sequence ( ). Processing of construct ,asshown 10 each hopefully accessible via SPPS. The key connections would in Fig. 4, produced fully synthetic, folded GM-CSF aglycone . [In one of the few papers that report yield (27), the authors include a kinetic alanine ligation between fragments 1a and 2a, – as well as two cysteine ligations to join fragments 2a, 3a, and 4a disclose a comparably low yield range, from 14 42%. That article (Fig. 3). As a critical design element, we envisioned that kinetic attributes the low yield to the aggregation-prone properties of ligation between fragments 1a and 2a would be followed by MFD the protein. In our estimation, the modest folding yield is due to at the ligation site (Cys34→Ala34) in the presence of a thioester at the aggregation as well as unspecific binding to the folding cassette.] The properties of aglycone 10 are described in Fully Synthetic C-terminal Thr53 residue. The cysteine residues of fragments 3a GM-CSF: Analytical Characterization and Biological Studies (28). and 4a would be protected as t-butyl thioethers; the thiols would be liberated in the course of the ligation. Adoption of this approach Synthesis of Glycosylated GM-CSF Analogs would obviate the need for extensive protection and deprotection of We now directed our attention to our long-term goal of reaching the cysteines slated for “survival” in the final target. single glycoform versions of GM-CSF. Although our central ob- Our synthetic vision for the GM-CSF aglycone was reduced jective was to install glycan domains of a high-complexity order, to practice (Fig. 4). The synthesis commenced with the prepa- 6 54 simulating those likely to be found in glycoprotein cytokines ration of fragment 3a ( ). Thus, protected polypeptide Thr - (29), we thought it prudent to begin with a more modest level of Pro94 (5) was retrieved from the SPPS resin by treatment with 95 glycosylation. In this way, we could field-test various synthetic HOAc/TFE/CH2Cl2; subsequent appendage of the Ser -SEt strategies using more readily available glycans. Moreover, we residue proceeded under known conditions (25). Global depro- hoped to gain early insights into the effect of glycosylation on “ ” tection of the resultant peptide under modified Cocktail B stability and biofunction, while recognizing that our model conditions [replacing phenol with dimethyl sulfide (DMS)] constructs may not be fully comparable with cytokines bearing afforded fragment 3a (6). In our SPPS-based route to fragment complex glycan domains. Thus, as a first but important step, we 4a (7), deblocking was conducted in a solution of 20% (vol/vol) undertook to install chitobiose disaccharides at the two native piperidine in dimethylformamide (DMF) and 0.5 M oxyma pure (26) to avoid problematic aspartimide formation at Asp119-Cys120. Fragment 4a was obtained following global deprotection with cocktail B (2% triisopropylsilane/5% phe- nol/5% H2O/88% TFA). This general approach also provided access to fragments 1a (1) and 2a (2), which were obtained in good yields (SI Appendix).

Fig. 2. Peptide ligation at cysteine and alanine residues. Fig. 3. Synthetic plan for GM-CSF aglycone.

2886 | www.pnas.org/cgi/doi/10.1073/pnas.1400140111 Zhang et al. Downloaded by guest on October 1, 2021 Fig. 4. Synthesis of GM-CSF aglycone (10). (a) 6 M Gn•HCl, 300 mM Na2HPO4, and 20 mM TCEP, pH 6.9; (b) 6 M Gn•HCl, 100 mM Na2HPO4, VA-044, tBu-SH, TCEP, 37 °C, 2 h (49%, 2 steps); (c) H-Ser-SEt, EDC, HOOBt, CHCl3, 3 h; (d) TFA:TIPS:H2O:DMS (90:2.5:2.5: 5), 45 min (46%, 2 steps); (e) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA and 20 mM TCEP, pH 7.8, 15 h; (f) MeONH2•HCl, pH 4, 3.5 h (61%, 2 steps); (g) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA and 20 mM TCEP, pH 7.8, 15 h (50%); (h) Gn•HCl, arginine•HCl, glutathione reduced, glutathione oxidized, NaCl, 3 d, (27%). Amino acid protecting groups for 5 (bold): Trt (H, Q); tBu (E, S, T, Y); Boc (K); Pbf (R).

sites of N-glycosylation: Asn27 and Asn37. It was envisioned that aspartimide formation at the allyl-protected Asp37 residue (30, 31). the target GM-CSF could be assembled from three fragments (1b– Selective Pd(0)-catalyzed deallylation, followed by Lansbury aspar-

3b) (Fig. 5). Fragments 1b and 2b would be prepared with the tylation (32, 33) with chitobiose, afforded glycopeptide 12. Finally, CHEMISTRY glycans installed at the wild-type sites. Notably, in contrast to the global deprotection served to liberate target fragment 2b (13). strategy adopted en route to GM-CSF aglycone, the program Fragment 3b (14) was prepared by SPPS using the oxyma pure/ directed to reaching the glycosylated congeners would offer piperidine deblocking protocol (26). NCL of fragments 2b (13)and enhanced convergence; the connection between the fragments 3b (14), followed by cleavage of the N-terminal Thz blocking group, would be fashioned exclusively via “alanine ligations.” Interest- afforded 16 in good yield (Fig. 7). Finally, coupling of glycopeptide ingly, earlier studies toward the aglycone had revealed the three- 16 with fragment 1b (15) provided the bis N-glycosylated GM-CSF fragment alanine ligation strategy to be unfeasible in that series; congener 17 in excellent yield. In the penultimate step, the two in the absence of N-linked carbohydrate groups, the Acm-pro- cysteine-based thiol group residues (at positions 33 and 81) of 17 tecting groups could not be removed (SI Appendix). were subjected to MFD, thereby revealing the alanine residues of The synthesis of glycosylated GM-CSF began with the preparation the target wild-type motif (18). In an encouraging demonstra- of fragment 2b (Fig. 6). The SPPS synthesis provided polypeptide 11; tion for future work, deprotection of the four Acm-protecting installation of a temporary pseudoproline group at the Glu38-Thr39 position (see underline) served to effectively suppress undesired

Fig. 6. Synthesis of glycosylated fragment 2b. (a) Pd(PPh3)4, PhSiH3, CHCl3, 20 min (50%); (b) chitobiose, HATU, DIEA, DMSO, 2 h; (c) TFA:TIPS:H2O:DMS (90:2.5:2.5: 5), 45 min (21%, 2 steps). Amino acid protecting groups for compounds 11 and 12 (in bold): Trt (Q); tBu (E, S, T, Y); Boc (K); Pbf (R); Mpe Fig. 5. Synthetic plan for N-glycosylated GM-CSF. (D). Underlines, pseudoproline dipeptide.

Zhang et al. PNAS | February 25, 2014 | vol. 111 | no. 8 | 2887 Downloaded by guest on October 1, 2021 Fig. 7. Synthesis of diglycosylated GM-CSF analog (20). (a) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, and 20 mM TCEP, pH 7.8, 9 h; (b) MeONH2•HCl, pH 4, 3.5 h (61%, 2 steps); (c) 6 M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, and 20 mM TCEP, pH 7.8, 15 h (42%); (d) 6 M Gn•HCl, 100 mM Na2HPO4, VA-044, tBu-SH, TCEP, 37 °C, 2 h (68%); (e) AgOAc, HOAc, H2O, DTT, 90 min (60%); (f) Gn•HCl, glutathione reduced, glutathione oxidized, NaCl, 3 d, (23%).

groups was readily accomplished using AgOAc (34), followed readily achieved, as shown in Fig. 8. Thus, by adopting the by quenching with acidic DTT. This finding is in sharp contrast, conditions developed in the diglycosylated series, we accomplished as noted above, to our inability to isolate GM-CSF aglycone fol- ligation of 16 with nonglycosylated peptide 1.SubsequentMFD, lowing Acm removal in the last step. We can only surmise that deprotection, and processing afforded the folded monoglycosylated construct 20, bearing the two glycosidic fragments, is more stable congener, 21. than is its aglycone domain. Processing of the fully synthetic denatured 19, as shown, afforded GM-CSF with homogeneous Fully Synthetic GM-CSF: Analytical Characterization and N-linked glycans at asparagine wild-type sites 27 and 37 (20). Biological Studies To explore potential directions as to how this chemistry might We next sought to confirm that our fully synthetic materials be permuted to assemble libraries of homogeneously glycosylated, possessed the structural and biological properties exhibited by wild-type GM-CSF agents, we adjusted the scheme to include native folded GM-CSF. Accordingly, these properties were only a single chitobiose domain at the Asn37 site. This goal was examined in comparison with relevant commercially available

Fig. 8. Synthesis of monoglycosylated GM-CSF an-

alog. (a) 6 M Gn•HCl, 300 mM Na2HPO4, and 20 mM TCEP, pH 7.8, 12 h (63%); (b) 6 M Gn•HCl, VA-044, tBu-SH, TCEP, 37 °C, 5 h (80%); (c) AgOAc, HOAc,

H2O, DTT, 90 min (54%); (d) Gn•HCl, arginine•HCl, glutathione reduced, glutathione oxidized, NaCl, 3 d, (21%).

2888 | www.pnas.org/cgi/doi/10.1073/pnas.1400140111 Zhang et al. Downloaded by guest on October 1, 2021 A 16.42 C4, 40-55 B 100 GM-CSF-Fold

3.0 2: Diode Array Range 3.889 2.5

2.0 % AU 1.5 1.0 0.5 0.0 0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 Time 100 1448.25 1.2 15.93 GM-CSF Commercial 1207.06 1.0 2: Diode Array 1316.78 Range 1.422 0.8 1609.07 0.6 % AU 1114.45 0.4 0.2 1810.12 904.60 1034.71 2068.63 0.0 968.10 0 m/z 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 Time 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

CD100k CD of GMCSF Samples 75k 60000

50k 40000

37k a b c 20000

25k 0 212 206 200 194 200 206 212 218 224 230 236 242 248 254 260 20k -20000 CHEMISTRY 15k -40000 mean residue ellipticity in ocm^2/dmol

-60000 Wavelength (nm)

recombinant synthetic synthetic synthetic Leukine Leukine GM-CSF GM-CSF GM-CSF GM-CSF two fold Aglycone Aglycone mono di- concentration chitobiose chitobiose

Fig. 9. Comparison between synthetic and commercially available GM-CSFs. (A) LC-MS traces of synthetic and recombinant GM-CSF aglycone (see SI Ap- pendix for the full span spectra). (B) ESI-MS analysis of synthetic and recombinant GM-CSF aglycone (see SI Appendix for the full mass data). (C) SDS/PAGE gel for synthetic and recombinant nonglycosylated and glycosylated GM-CSF samples. (D) CD spectra of synthetic GM-CSFs compared with recombinant GM-CSF aglycone: trace a, synthetic GM-CSF aglycone; trace b, recombinant GM-CSF aglycone; trace c, bis-glycosylated GM-CSF.

samples of biotechnology-derived counterparts (Fig. 9). The cultured with human Kit ligand and in the presence or absence of LC-MS readout of GM-CSF aglycone (10) is well aligned with various doses of recombinant GM-CSF or our synthetic GM-CSF that of the recombinantly derived aglycone sample (Fig. 9 A samples (Fig. 10B). After 14 d, colony-forming cells (CFCs) were and B). Figure 9C depicts an SDS/PAGE gel comparison of our scored. All three synthetic GM-CSF samples were capable of stim- + synthetic samples (10, 20,and21) with recombinant GM-CSF ulating normal human CD34 cells to form hematopoietic myeloid aglycone and recombinant heterogeneously glycosylated GM-CSF colonies in a dose-dependent manner (Fig. 10B), and their specific (Leukine). The multiple bands observed in the Leukine structure biological activity was in a similar dose range (EC50 = 0.1 ng/mL) reflect the mixture of glycoforms present in this material. Finally, compared with recombinant GM-CSF. This key finding provides circular dichroism (CD) spectroscopy offers further validation of confirmation that the material gained through synthetic means the structure of our fully synthetic GM-CSF samples. may be confidently used in preclinical biological studies directed With validated fully synthetic samples of GM-CSF in hand, we toward immunostimulation, particularly in the adjuvant setting. undertook an in vitro biological comparison with analogous recom- binantly derived materials. In these studies, the human GM-CSF– Conclusion dependent leukemia cell line, TF-1, was used for comparison. We In summary, we have synthesized homogeneous, structurally de- were pleased to find that all three synthetic GM-CSF samples man- fined GM-CSF glycoforms through a route that is in keeping with ifest a specific biological activity similar to the commercially available the goals of high convergence and amenability to small-library GM-CSF (EC50 of ∼2.3 pg/mL) (Fig. 10A). To further examine synthesis. We are encouraged to observe that the biological ac- the effect of our synthetic GM-CSF samples on normal human tivity of our fully synthetic materials tracks quite closely with that + hematopoietic cells, purified cord blood CD34 cells were obtained with recombinant GM-CSF. In addition, there are already

Zhang et al. PNAS | February 25, 2014 | vol. 111 | no. 8 | 2889 Downloaded by guest on October 1, 2021 + Fig. 10. Effect of synthetic GM-CSF on proliferation of TF-1 cells and induction of colony formation in CD34 cells. (A) Effect of synthetic GM-CSF on the proliferation of TF-1 cells. Relative fluorescence intensity = (fluorescence intensity of TF-1 cultures with various doses of GM-CSF)/(fluorescence intensity of TF-1 cultures with 125 pg/mL Leukine GM-CSF). The results are expressed as the mean of relative fluorescence intensity ± SD, n = 3. (B) Effect of synthetic GM-CSF + induction of colony formation in human cord blood CD34 cells.

some early indications that glycosylation, even with small domains, ACKNOWLEDGMENTS. We thank Ms. Hui Fan for LC-MS analysis, Mr. Han confers greater chemical stability to the polypeptide although this Guo and Prof. Minkui Luo for performing the SDS/PAGE gel experiment, and property is not yet reflected in enhanced bioperformance. Drs. Ping Wang, Suwei Dong, and Peter Park for helpful discussions. We thank Dr. Lisa Ambrosini Vadola, Ms. Rebecca Wilson, and Ms. Laura Wilson Although new synthetic challenges will undoubtedly arise as for help with the preparation of the manuscript. Support for this research was the complexity level of the glycan is expanded, we are confident provided by National Institutes of Health Grant HL025848. E.V.J. acknowl- that the framework described herein will be adjustable to reach edges the Swedish Research Council, Stiftelsen Olle Engkvist Byggmästare, even more ambitious targets. and Stiftelsen Bengt Lundqvist Minne for a postdoctoral fellowship.

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