DOI: 10.1002/chem.201404013
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& Antitumor Agents
Covalent Bond or Noncovalent Bond: A Supramolecular Strategy for the Construction of Chemically Synthesized Vaccines Yue Gao, Zhan-Yi Sun, Zhi-Hua Huang, Pu-Guang Chen, Yong-Xiang Chen, Yu-Fen Zhao, and Yan-Mei Li*[a] Abstract: A novel noncovalent strategy to construct chemically synthesized vaccines has been designed to trigger a robust immune response and to dramatically improve the efficiency of vaccine preparation. Glycosylated MUC1 tripartite vaccines were constructed through host– guest interactions with cucurbit[8]uril. These vaccines elicited high levels of IgG antibodies that were recognized by transformed cells and induced the secretion of cytokines. The antisera also mediated complement-dependent cytotoxicity. This noncovalent strategy with good suitability, scalability, and feasibility can be applied as a universal strategy for the construction of chemically synthesized vaccines.
Many peptides, carbohydrates and glycopeptides expressed on the surface of transformed cells can act as targets for immune recognition and immunotherapy.[1, 2] However, target molecules with weak immunogenicity and/or immunotolerance cannot elicit strong immune responses. To enhance their immunogenicity, antigens have been covalently conjugated with immune activators such as foreign carrier proteins. These chemically synthesized vaccines, especially the antigen-carrier protein vaccines, have triggered robust immune reactions and have eliminated undesirable side effects caused by irrelevant ingredients of bacterial and viral vaccines.[3–16] However, foreign carrier proteins may elicit a strong B-cell response which may suppress immune reaction against saccharide and glycopeptide epitopes.[17, 25] The development of fully synthesized multipartite vaccines has circumvented the use of foreign carrier proteins. Some useful components such as T-helper cell epitopes and Toll-like receptor ligands have been used for constructing chemically synthesized vaccine.[18–25] Present methods require the introduction of a covalent linkage for each active component of a multipartite vaccine. Two-, three-, and four-component covalent vaccines have elicited strong antibody-based immune responses.[26–37] These multipartite vaccines with no [a] Y. Gao, Z.-Y. Sun, Z.-H. Huang, P.-G. Chen, Dr. Y.-X. Chen, Dr. Y.-F. Zhao, Dr. Y.-M. Li Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry Tsinghua University (P.R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404013. Chem. Eur. J. 2014, 20, 13541 – 13546
unnecessary components have very good application prospects, but they are limited by several synthesis challenges. Not all components lend themselves to the ready formation of a covalent bond. Vaccines containing covalent linkages are also limited by the need for time-consuming purification and characterization and by the use of linkers that may suppress the immune response. It is worth noting that a simple mixture of the individual components of the vaccine cannot trigger a strong immune response.[26, 27] Only a well-organized, integrated structures of the components can achieve a robust and efficient immune response. Our group has concentrated on developing a new, noncovalent strategy for vaccine design in which vaccine components are combined through host–guest interactions. The self-assembly process may provide great convenience for vaccine preparations and overcome the difficulties of the covalent linkage strategy. We have employed cucurbit[8]uril (CB[8]) as a “supramolecular handcuff” to join together two different macromolecular units in a noncovalent fashion. All vaccine components were functionalized with either an electron-deficient first guest methyl viologen or an electron-rich second guest naphthalene for the formation of the CB[8] ternary complex.[38–43] This conveniently prepared handcuff showed high binding affinity and sufficiently low toxicity for pharmaceutical use.[44, 45] To test the effect of our noncovalent strategy, we constructed a MUC1 glycopeptide multipartite antitumor vaccine containing full-length MUC1 VNTR domains with different glycosylations as the B epitopes, TT830-843 from tetanus toxoid as the T-helper (Th) cell epitope, and TLR2 ligand lipopeptide Pam3CSK4.[14, 35, 37] The B-epitope–Th-epitope structure and Pam3CSK4 were separately functionalized with naphthalene and methyl viologen (MV2 + ) and were bound together with noncovalent interactions instead of covalent bonds. We compared the immune effects of the novel noncovalent vaccines with simple mixtures of vaccine components and with the aforementioned covalently linked vaccines, with all the preparations containing the same vaccine components. To facilitate synthesis, methyl viologen and naphthalene were each modified by the addition of a carboxyl terminus so that they could be treated as an amino acid “building block” for solid-phase peptide synthesis (SPPS). The B-epitope–Th-epitope structures were modified with a small molecule, 2-naphthylacetic acid, to synthesize the assembly components 1, 2, 3 and 4. We added a polar and nonimmunogenic amino acid spacer between the B epitope and the T-helper epitope to sep-
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Figure 1. Design and synthesis of noncovalent structure of vaccines.
arate these two components.[7, 14] The TLR2 ligand Pam3CSK4 was also connected with MV2 + to prepare the assembly component 9 (Supporting Information Figure S51). Once the noncovalent vaccine candidates, which contained equimolar 1/2/ 3/4, CB[8] and 9, were formed in PBS buffer, they readily underwent a subsequent self-assembly step into spherical structures (Figure 1). After successfully preparing the vaccines, we confirmed the formation of the supramolecular 1:1:1 ternary complex by diffusion ordered NMR (DOSY NMR), isothermal titration calorimetry (ITC), and 1H NMR spectroscopy. DOSY NMR assay of aqueous solutions (0.25 mm in D2O) of 1, 2, 5, 6, 7, 8 and 1 + 9·CB[7] yielded the diffusion coefficient (D). We observed the expected reduction of D in the formed ternary complex and all three components of compound 5 displayed the same D, thus indicating the formation of a supramolecular complex (Figure 2 d). The CB[7] can only accommodate the MV2 + guest molecule which makes sure that the ternary complex cannot be formed, so the solution of equimolar 9·CB[7] and 1 was prepared as control. Two different sets of D were observed and this result further proved the formation of the ternary complex (Figure 2 e). Besides, the ternary complex may self-assemble to other structures because of its amphiphilic property so that it represents a D of 10 or higher. Similar findings were observed with 6, 7 and 8 (Supporting Information Table S1). ITC experiments, conducted in a solution of equimolar 9·CB[8] and 1 in PBS buffer (10 mm; pH 7.0), provided a value for N of 1.15 0.00607, thus confirming the high affinity of 1 for 9·CB[8] (Figure 2 a). The 1H NMR spectra (Supporting Information Figure S48) exhibited an upfield chemical shift for the MV2 + proton peaks, which confirmed the formation of the 1:1:1 complex. Chem. Eur. J. 2014, 20, 13541 – 13546
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To study the shapes and sizes of the vaccine candidates, transmission electron microscope (TEM) and dynamic light scattering (DLS) assays of aqueous solutions (0.05 mm) were carried out and the results were fully identical. The 1:1:1 complex and the individual vaccine components displayed distinctly different features. The TEM image of 5 (Figure 2 b) suggested that these three components formed a spherical structure, indicating the formation of the ternary complex. The produced complex was about 150 nm in diameter. The amphiphilic property of 5, 6, 7 and 8 may be the driving force for the formation of the spherical structure. The TEM image of 9·CB[8] shows a fibrous structure (Supporting Information Figure S44). As indicated by Figure 2 c, the hydrodynamic diameter of compound 5 was determined to be around 240 nm and the hydrodynamic diameter of compound 9 was about 25 nm. The count rate of compound 1 was below 100 kcps. This spherical vaccine structure may be recognized readily by the immune system and vaccines with this self-assembled structure may contribute to sustained-released effects and achieve high local concentrations when injected into the body. The relatively large size may also promote the induction of the immune response. After successfully preparing the noncovalent vaccine candidates, we carried out the immunological experiments to evaluate our noncovalent vaccine strategy. We prepared four types of noncovalent vaccine candidates with different glycosylation patterns (compound 5, 6, 7, 8) for immunological evaluations. To verify that the noncovalent linkage strategy can significantly improve the immune response compared to the simple mixture of each active component, the mixtures of 1 + 9, 2 + 9, 3 + 9, 4 + 9 were also prepared as control groups. We also investigated the adjuvant effect on the noncovalent vaccine candidates by adding control groups
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Communication obvious enhancement in immune response was observed when Freund’s adjuvant was included. Perhaps this lack of effect is caused by the presence of the oil in Freund’s adjuvant which disrupted the chargetransfer (CT) interaction between the electron-rich and electrondeficient guest molecules. We also performed isotype analyses to determine whether the vaccine candidates induced a T-cellmediated immune response, which is critical for cancer therapy. The IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies of G1 appear in Figure 3 c. The results point to the triggering of a blended Th1/Th2 response. Similar findings were obtained for G5, G9 and G13. Isotype assays of all other groups are shown in the Supporting Information Figures S16–S31. We examined the binding of the sera to MUC1-expressing MCF-7 human breast tumor cell line by flow cytometry to evaluate cell surface reactivity (Figure 3 f). Native MUC1 antigens present on the cancer cells were strongly recognized by the mouse antiserum of G1, whereas no binding occurred with preimmune sera. Similar results were observed in the presence and Figure 2. Noncovalent vaccine candidates successfully self-assembled and formed spherical structures. a) ITC data. absence of Freund’s adjuvant At the top are the raw data for power versus time. At the bottom are the integrated enthalpy values versus the (Supporting Information Figurmolar ratio of 1:9·CB[8]. b) TEM image of 5 (scale bar: 200 nm). c) DLS measurements of 9 and 5 aqueous solution. d) DOSY NMR spectroscopy result for 5. e) DOSY NMR spectroscopy result of the equimolar 1 (top line) and es S32–S38). We next investigat9·CB[7] (bottom line). ed the interaction between TLR2 on the surface of the mononuclear phagocytes and Pam3CSK4 with Freund’s adjuvant. The detailed grouping information of through cytokine induction analyses using dendritic cells incuimmunization is shown in Figure 3 a. We compared the antibated with 5, 6, 7, 8, 9 and CB[8]. Evaluation of the cell superMUC1 antibody titers of the noncovalent vaccine candidates natants showed that the noncovalent vaccine candidates effecwith simple mixtures of the components by ELISA (Figure 3 b). tively induced the secretion of TNF-a, IL-6, IL-12/IL-23, and ILThe noncovalently assembled vaccine candidates elicited 10 cytokines. Moreover, similar efficacies of 5, 6, 7, 8 and 9 in a high level of IgG antibody while the simple mixture of all inducing the secretion of each cytokine indicated that the selfcomponents failed to trigger a robust immune response. The assembly process did not interfere with the activity of marked difference of titers verified that the noncovalent strucPam3CSK4 (Figure 3 e and Supporting Information Figures S39– tures of vaccines were sufficiently stable in vivo and truly conS41). tributed to the induction of high titers of IgG antibodies as Moreover, the induced antibodies bound to the cancer cell compared with the simple mixture of active components. The line efficiently initiated the killing of the recognized tumor critical role of the formation of the 1:1:1 complex for improved cells by activation of the complement-dependent cytotoxicity immune response was also verified. We expected the use of (CDC) complex. Sera diluted 1:25 were examined for mediating Freund’s adjuvant to promote the immune effect. However, no lysis by complement in a 4 h MTT assay (Figure 3 d). The antiChem. Eur. J. 2014, 20, 13541 – 13546
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Figure 3. Noncovalent vaccine candidates triggered robust immunological effects compared with simple mixtures of the same components. a) Detailed grouping information of immunization. b) ELISA anti-MUC1 IgG total antibody titers after five immunizations with each immune group. Each data point represents the titer for an individual mouse and the straight lines indicate the mean for the group of four mice. c) ELISA anti-MUC1 IgG1, IgG2a, IgG2b, IgG3 and IgM antibody analyses after five immunizations with G1. The negative control of preimmune sera collected before immunization was deducted. d) Complement-dependent cytotoxicity tests for antisera of G1, G5, G9 and G13. Preimmune sera were measured as blank control. The data are reported as the means SD of triplicate treatments. e) TNF-a cytokine secretion by dendritic cells (DCs) after stimulation with 5, 6, 7, 8, CB[8] and 9. Supernatant before stimulation was assayed as blank control (Pre). The data are reported as the means SD of triplicate treatments. f) FACS analysis on MCF-7 cells for specific anti-MUC1 antibodies. Fluorescence intensity of serum of G1 (1:25 diluted) was assessed before (1) and after five immunizations (3), preimmune sera (2) are shown as negative control.
sera of G1, G5, G9, and G13 effectively mediated CDC for MCF-7 cell lysis. The use of Freund’s adjuvant did not improve the cytotoxic efficacy and the mixture of vaccine components showed negligible cytotoxic effects (Supporting Information Figures S42–S43). In conclusion, we have constructed a multipartite antitumor vaccine through a host–guest interaction with cucurbit[8]uril. The vaccine molecule assembled into a sphere due to the hydrophobic interactions between the hydrophilic and hydrophobic components. Vaccine candidates with this novel structure elicited a strong antibody-based immune response. The immunological effect was dramatically enhanced when the mixture of vaccine components assembled into a 1:1:1 ternary complex. These noncovalent vaccines showed immunological effects comparable to covalently linked vaccines from our previous studies.[35, 37] These results indicate that this noncovalent strategy for constructing chemically synthesized vaccines is effective. This methodology markedly reduces the difficulties of vaccine preparation and may promote mass production and further clinical applications. Additionally, this simple self-assemChem. Eur. J. 2014, 20, 13541 – 13546
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bly method may provide great convenience for the evaluation of new immune components with different properties and permit further screening for optimum combination forms. This noncovalent strategy displays good suitability, scalability, and feasibility that may be applied to the construction of other chemically synthesized vaccines.
Experimental Section General experimental protocol for vaccine preparations Glycopeptides were prepared by standard solid-phase peptide synthesis method of Fmoc chemistry with Na-Fmoc-protected amino acids, on a Liberty CEM microwave peptide synthesizer (Supporting Information Figure S50). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate (HBTU, 6.0 equiv) and N-hydroxybenzotriazole (HOBt, 6.0 equiv) were employed as activators and N,N-diisopropylethylamine (DIEA, 12.0 equiv) was employed as activator base for coupling of natural amino acids, 13, 15 and 14. 2-(7-Aza1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 2.0 equiv) and 1-hydroxy-7-azabenzotriazole (HOAt,
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Communication 2.0 equiv) were used as activators and N-methylmorpholine (NMM, 5.0 equiv) was used as activator base for coupling glycoamino acids. The peptide was then cleaved from the resin with TFA/TIS/ H2O = 15:0.9:0.9 (v/v/v) over 2.5 h. After removing TFA, peptides were precipitated two times with diethyl ether and centrifuged at 11 200 g for 20 min. Then the peptides were dried and purified by HPLC using semipreparative CN column. The synthesis scheme for 14 is shown in the Supporting Information Figure S49. Details of the chemical synthesis and analytical data of the compounds are given in the Supporting Information.
Animals and vaccinations Groups of four female BALB/c mice (4–6 weeks of age; fed at the Animal Facility of Center of Biomedical Analysis, Tsinghua University) were immunized with G1–G16 (20 mg) with or without Freund’s adjuvant (100 mL). Mice were immunized on days 1, 14, 28, 42, 56 and antisera were collected on day 66. Sera were taken before immunization, as negative control. The animal experiments were conducted in accord with the ethical guidelines of Tsinghua University.
Isothermal calorimetry (ITC) measurements Titration experiments were performed on a Microcal VP-ITC apparatus at 298.15 K in sodium phosphate buffer (10 mm; pH 7.4). The binding equilibriums were carried out using a 9·CB[8] concentration of typically 0.06 mm to which the 0.6 mm 5 was titrated. The data were analyzed with Origin 7.0 software, using the one set of sites model.
NMR measurements Each compound was dissolved in D2O at 0.5 mm. 1H NMR spectra was recorded on a Joel JNM-ECA 300 apparatus (300 MHz). Diffusion assays were carried out with a Bruker Avance 600 NMR spectrometer under the same conditions. All samples were tested under the same conditions.
Transmission electron microscopy analyses All samples were dissolved in PBS buffer (pH 7.4) with the concentration of 0.05 mm at room temperature. Each sample (10 mL) was added to copper grids with carbon support films, negatively stained with tungstophosphoric acid (15 mg mL 1, pH 6, 10 mL), and imaged on a Hitachi H-7650B transmission electron microscope after being dried, overnight.
Serological analyses For titer measurements, ELISA plates were separately coated with the corresponding B epitope (Figure 1) in carbonate buffer (0.1 m; pH 9.6) at 2 mg per well, overnight, at 4 8C. Each well was blocked with 0.25 % gelatin in PBS for 3 h and then washed four times with 0.5 % Tween-20 in PBS solution. Then, serially diluted antiserum was incubated in each well for 1 h at 37 8C. After four washes, the second antibody, anti-mouse IgG (whole molecule)–peroxidase produced in rabbit, was diluted 1:2000 and incubated (100 mL per well) with each well for 1 h at 37 8C. After a final wash, 1 mg mL 1 o-phenylenediamine in critic acid buffer (0.1 m; pH 5) with 30 % H2O2 (1.5 mL) was added to the wells (100 mL per well) as substrate. After incubation for 15 min at 37 8C, the optical density was measured at 450 nm. Titers are defined as the highest dilution yielding an optical absorption of 0.1 or greater than the normal control mouse sera. All assays were repeated three times. For subtype Chem. Eur. J. 2014, 20, 13541 – 13546
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assays, ELISA plates were separately coated with the corresponding B epitope (Figure 1) in carbonate buffer (0.1 m; pH 9.6) at 2 mg per well, overnight, at 4 8C. Each well was blocked with 0.25 % gelatin in PBS for 3 h and then washed four times with 0.5 % Tween-20 in PBS solution. Then, 1:25 diluted antiserum (100 mL) was incubated in each well for 1 h at 37 8C. After four washes, the second antibody (Mouse Monoclonal Antibody Isotyping Reagents kit including goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM (Sigma) was diluted 1:1000 and incubated at 100 mL per well for 1 h at 37 8C. After a final wash, the third antibody, anti-goat IgG (whole molecule)–peroxidase produced in rabbit was diluted 1:1000 and incubated at 100 mL per well for 1 h at 37 8C. After incubation for 15 min at 37 8C, the optical density was measured at 450 nm. All assays were repeated two times.
Cell surface reactivity determined by FACS Pre- and postimmunization sera were diluted 25-fold and incubated with MCF-7 single-cell suspensions of 2 105 cells per tube, washed in PBS with 1 % fetal calf serum for 1 h on ice. Next, the cells were washed and incubated with FITC-conjugated rabbit antimouse IgG antibody (1:50 diluted, DAKO) for 1 h on ice. After a final wash, FACS analysis was conducted on a BDC FACS Calibur flow cytometer. All assays were performed in triplicate.
Cytokine induction Dendritic cells were obtained by an established method[27] and incubated with each sample (0.5 mm, 100 mL) for 24 h at 37 8C. After that supernatants were collected and tested with mouse IL-6 precoated ELISA kit, mouse IL-12/IL-23 precoated ELISA kit, mouse TNF-a precoated ELISA kit, and mouse IL-10 precoated ELISA kit (Dakewe). All experiments were repeated three times.
Complement-dependent cytotoxicity assay Complement-dependent cytotoxicity (CDC) was assayed at a serum dilution of 1:50 with MCF-7 cells cultured for 12 h at 37 8C in 96well cell culture plates with the density of 5000 cells per well. Next, pre- and postimmunization sera were diluted 25-fold and added to the plate (50 mL per well). After incubation for 30 min at 37 8C, rabbit complement (1:2 diluted in culture medium, AbD seroTec, 50 mL per well) was added to the plate and incubated for 8 h, at 37 8C. Next, MTT solution (0.5 % MTT in PBS solution) was added to the plate (20 mL per well). The culture medium was removed after incubation for 4 h, formazan, violet crystal, was dissolved in DMSO (150 mL), and the optical density was measured at the wavelength of 490 nm. Cells incubated in culture medium were assayed as control. All assays were performed in triplicate. The cytotoxicity was calculated according to the formula: cytotoxicity [%] = [1 (experimental OD/control OD)] 100.
Acknowledgements We thank Prof. Oren Scherman and Prof. Horst Kunz for helpful discussions. We thank Prof. Xi Zhang for providing a Microcal VP-ITC apparatus and beneficial discussions. This work was supported by the Major State Basic Research Development Program of China (2013CB910700) and (2012CB821600), the National Natural Science Foundation of China (21332006, 91313301) and the Research Project of Chinese Ministry of Education (113005A).
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Communication Keywords: antitumor vaccines glycopeptides · host–guest systems
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Received: June 18, 2014 Published online on August 25, 2014
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& Antitumor Agents
Covalent Bond or Noncovalent Bond: A Supramolecular Strategy for the Construction of Chemically Synthesized Vaccines Yue Gao, Zhan-Yi Sun, Zhi-Hua Huang, Pu-Guang Chen, Yong-Xiang Chen, Yu-Fen Zhao, and Yan-Mei Li*[a] Abstract: A novel noncovalent strategy to construct chemically synthesized vaccines has been designed to trigger a robust immune response and to dramatically improve the efficiency of vaccine preparation. Glycosylated MUC1 tripartite vaccines were constructed through host– guest interactions with cucurbit[8]uril. These vaccines elicited high levels of IgG antibodies that were recognized by transformed cells and induced the secretion of cytokines. The antisera also mediated complement-dependent cytotoxicity. This noncovalent strategy with good suitability, scalability, and feasibility can be applied as a universal strategy for the construction of chemically synthesized vaccines.
Many peptides, carbohydrates and glycopeptides expressed on the surface of transformed cells can act as targets for immune recognition and immunotherapy.[1, 2] However, target molecules with weak immunogenicity and/or immunotolerance cannot elicit strong immune responses. To enhance their immunogenicity, antigens have been covalently conjugated with immune activators such as foreign carrier proteins. These chemically synthesized vaccines, especially the antigen-carrier protein vaccines, have triggered robust immune reactions and have eliminated undesirable side effects caused by irrelevant ingredients of bacterial and viral vaccines.[3–16] However, foreign carrier proteins may elicit a strong B-cell response which may suppress immune reaction against saccharide and glycopeptide epitopes.[17, 25] The development of fully synthesized multipartite vaccines has circumvented the use of foreign carrier proteins. Some useful components such as T-helper cell epitopes and Toll-like receptor ligands have been used for constructing chemically synthesized vaccine.[18–25] Present methods require the introduction of a covalent linkage for each active component of a multipartite vaccine. Two-, three-, and four-component covalent vaccines have elicited strong antibody-based immune responses.[26–37] These multipartite vaccines with no [a] Y. Gao, Z.-Y. Sun, Z.-H. Huang, P.-G. Chen, Dr. Y.-X. Chen, Dr. Y.-F. Zhao, Dr. Y.-M. Li Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry Tsinghua University (P.R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404013. Chem. Eur. J. 2014, 20, 13541 – 13546
unnecessary components have very good application prospects, but they are limited by several synthesis challenges. Not all components lend themselves to the ready formation of a covalent bond. Vaccines containing covalent linkages are also limited by the need for time-consuming purification and characterization and by the use of linkers that may suppress the immune response. It is worth noting that a simple mixture of the individual components of the vaccine cannot trigger a strong immune response.[26, 27] Only a well-organized, integrated structures of the components can achieve a robust and efficient immune response. Our group has concentrated on developing a new, noncovalent strategy for vaccine design in which vaccine components are combined through host–guest interactions. The self-assembly process may provide great convenience for vaccine preparations and overcome the difficulties of the covalent linkage strategy. We have employed cucurbit[8]uril (CB[8]) as a “supramolecular handcuff” to join together two different macromolecular units in a noncovalent fashion. All vaccine components were functionalized with either an electron-deficient first guest methyl viologen or an electron-rich second guest naphthalene for the formation of the CB[8] ternary complex.[38–43] This conveniently prepared handcuff showed high binding affinity and sufficiently low toxicity for pharmaceutical use.[44, 45] To test the effect of our noncovalent strategy, we constructed a MUC1 glycopeptide multipartite antitumor vaccine containing full-length MUC1 VNTR domains with different glycosylations as the B epitopes, TT830-843 from tetanus toxoid as the T-helper (Th) cell epitope, and TLR2 ligand lipopeptide Pam3CSK4.[14, 35, 37] The B-epitope–Th-epitope structure and Pam3CSK4 were separately functionalized with naphthalene and methyl viologen (MV2 + ) and were bound together with noncovalent interactions instead of covalent bonds. We compared the immune effects of the novel noncovalent vaccines with simple mixtures of vaccine components and with the aforementioned covalently linked vaccines, with all the preparations containing the same vaccine components. To facilitate synthesis, methyl viologen and naphthalene were each modified by the addition of a carboxyl terminus so that they could be treated as an amino acid “building block” for solid-phase peptide synthesis (SPPS). The B-epitope–Th-epitope structures were modified with a small molecule, 2-naphthylacetic acid, to synthesize the assembly components 1, 2, 3 and 4. We added a polar and nonimmunogenic amino acid spacer between the B epitope and the T-helper epitope to sep-
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Figure 1. Design and synthesis of noncovalent structure of vaccines.
arate these two components.[7, 14] The TLR2 ligand Pam3CSK4 was also connected with MV2 + to prepare the assembly component 9 (Supporting Information Figure S51). Once the noncovalent vaccine candidates, which contained equimolar 1/2/ 3/4, CB[8] and 9, were formed in PBS buffer, they readily underwent a subsequent self-assembly step into spherical structures (Figure 1). After successfully preparing the vaccines, we confirmed the formation of the supramolecular 1:1:1 ternary complex by diffusion ordered NMR (DOSY NMR), isothermal titration calorimetry (ITC), and 1H NMR spectroscopy. DOSY NMR assay of aqueous solutions (0.25 mm in D2O) of 1, 2, 5, 6, 7, 8 and 1 + 9·CB[7] yielded the diffusion coefficient (D). We observed the expected reduction of D in the formed ternary complex and all three components of compound 5 displayed the same D, thus indicating the formation of a supramolecular complex (Figure 2 d). The CB[7] can only accommodate the MV2 + guest molecule which makes sure that the ternary complex cannot be formed, so the solution of equimolar 9·CB[7] and 1 was prepared as control. Two different sets of D were observed and this result further proved the formation of the ternary complex (Figure 2 e). Besides, the ternary complex may self-assemble to other structures because of its amphiphilic property so that it represents a D of 10 or higher. Similar findings were observed with 6, 7 and 8 (Supporting Information Table S1). ITC experiments, conducted in a solution of equimolar 9·CB[8] and 1 in PBS buffer (10 mm; pH 7.0), provided a value for N of 1.15 0.00607, thus confirming the high affinity of 1 for 9·CB[8] (Figure 2 a). The 1H NMR spectra (Supporting Information Figure S48) exhibited an upfield chemical shift for the MV2 + proton peaks, which confirmed the formation of the 1:1:1 complex. Chem. Eur. J. 2014, 20, 13541 – 13546
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To study the shapes and sizes of the vaccine candidates, transmission electron microscope (TEM) and dynamic light scattering (DLS) assays of aqueous solutions (0.05 mm) were carried out and the results were fully identical. The 1:1:1 complex and the individual vaccine components displayed distinctly different features. The TEM image of 5 (Figure 2 b) suggested that these three components formed a spherical structure, indicating the formation of the ternary complex. The produced complex was about 150 nm in diameter. The amphiphilic property of 5, 6, 7 and 8 may be the driving force for the formation of the spherical structure. The TEM image of 9·CB[8] shows a fibrous structure (Supporting Information Figure S44). As indicated by Figure 2 c, the hydrodynamic diameter of compound 5 was determined to be around 240 nm and the hydrodynamic diameter of compound 9 was about 25 nm. The count rate of compound 1 was below 100 kcps. This spherical vaccine structure may be recognized readily by the immune system and vaccines with this self-assembled structure may contribute to sustained-released effects and achieve high local concentrations when injected into the body. The relatively large size may also promote the induction of the immune response. After successfully preparing the noncovalent vaccine candidates, we carried out the immunological experiments to evaluate our noncovalent vaccine strategy. We prepared four types of noncovalent vaccine candidates with different glycosylation patterns (compound 5, 6, 7, 8) for immunological evaluations. To verify that the noncovalent linkage strategy can significantly improve the immune response compared to the simple mixture of each active component, the mixtures of 1 + 9, 2 + 9, 3 + 9, 4 + 9 were also prepared as control groups. We also investigated the adjuvant effect on the noncovalent vaccine candidates by adding control groups
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Communication obvious enhancement in immune response was observed when Freund’s adjuvant was included. Perhaps this lack of effect is caused by the presence of the oil in Freund’s adjuvant which disrupted the chargetransfer (CT) interaction between the electron-rich and electrondeficient guest molecules. We also performed isotype analyses to determine whether the vaccine candidates induced a T-cellmediated immune response, which is critical for cancer therapy. The IgG1, IgG2a, IgG2b, IgG3 and IgM antibodies of G1 appear in Figure 3 c. The results point to the triggering of a blended Th1/Th2 response. Similar findings were obtained for G5, G9 and G13. Isotype assays of all other groups are shown in the Supporting Information Figures S16–S31. We examined the binding of the sera to MUC1-expressing MCF-7 human breast tumor cell line by flow cytometry to evaluate cell surface reactivity (Figure 3 f). Native MUC1 antigens present on the cancer cells were strongly recognized by the mouse antiserum of G1, whereas no binding occurred with preimmune sera. Similar results were observed in the presence and Figure 2. Noncovalent vaccine candidates successfully self-assembled and formed spherical structures. a) ITC data. absence of Freund’s adjuvant At the top are the raw data for power versus time. At the bottom are the integrated enthalpy values versus the (Supporting Information Figurmolar ratio of 1:9·CB[8]. b) TEM image of 5 (scale bar: 200 nm). c) DLS measurements of 9 and 5 aqueous solution. d) DOSY NMR spectroscopy result for 5. e) DOSY NMR spectroscopy result of the equimolar 1 (top line) and es S32–S38). We next investigat9·CB[7] (bottom line). ed the interaction between TLR2 on the surface of the mononuclear phagocytes and Pam3CSK4 with Freund’s adjuvant. The detailed grouping information of through cytokine induction analyses using dendritic cells incuimmunization is shown in Figure 3 a. We compared the antibated with 5, 6, 7, 8, 9 and CB[8]. Evaluation of the cell superMUC1 antibody titers of the noncovalent vaccine candidates natants showed that the noncovalent vaccine candidates effecwith simple mixtures of the components by ELISA (Figure 3 b). tively induced the secretion of TNF-a, IL-6, IL-12/IL-23, and ILThe noncovalently assembled vaccine candidates elicited 10 cytokines. Moreover, similar efficacies of 5, 6, 7, 8 and 9 in a high level of IgG antibody while the simple mixture of all inducing the secretion of each cytokine indicated that the selfcomponents failed to trigger a robust immune response. The assembly process did not interfere with the activity of marked difference of titers verified that the noncovalent strucPam3CSK4 (Figure 3 e and Supporting Information Figures S39– tures of vaccines were sufficiently stable in vivo and truly conS41). tributed to the induction of high titers of IgG antibodies as Moreover, the induced antibodies bound to the cancer cell compared with the simple mixture of active components. The line efficiently initiated the killing of the recognized tumor critical role of the formation of the 1:1:1 complex for improved cells by activation of the complement-dependent cytotoxicity immune response was also verified. We expected the use of (CDC) complex. Sera diluted 1:25 were examined for mediating Freund’s adjuvant to promote the immune effect. However, no lysis by complement in a 4 h MTT assay (Figure 3 d). The antiChem. Eur. J. 2014, 20, 13541 – 13546
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Figure 3. Noncovalent vaccine candidates triggered robust immunological effects compared with simple mixtures of the same components. a) Detailed grouping information of immunization. b) ELISA anti-MUC1 IgG total antibody titers after five immunizations with each immune group. Each data point represents the titer for an individual mouse and the straight lines indicate the mean for the group of four mice. c) ELISA anti-MUC1 IgG1, IgG2a, IgG2b, IgG3 and IgM antibody analyses after five immunizations with G1. The negative control of preimmune sera collected before immunization was deducted. d) Complement-dependent cytotoxicity tests for antisera of G1, G5, G9 and G13. Preimmune sera were measured as blank control. The data are reported as the means SD of triplicate treatments. e) TNF-a cytokine secretion by dendritic cells (DCs) after stimulation with 5, 6, 7, 8, CB[8] and 9. Supernatant before stimulation was assayed as blank control (Pre). The data are reported as the means SD of triplicate treatments. f) FACS analysis on MCF-7 cells for specific anti-MUC1 antibodies. Fluorescence intensity of serum of G1 (1:25 diluted) was assessed before (1) and after five immunizations (3), preimmune sera (2) are shown as negative control.
sera of G1, G5, G9, and G13 effectively mediated CDC for MCF-7 cell lysis. The use of Freund’s adjuvant did not improve the cytotoxic efficacy and the mixture of vaccine components showed negligible cytotoxic effects (Supporting Information Figures S42–S43). In conclusion, we have constructed a multipartite antitumor vaccine through a host–guest interaction with cucurbit[8]uril. The vaccine molecule assembled into a sphere due to the hydrophobic interactions between the hydrophilic and hydrophobic components. Vaccine candidates with this novel structure elicited a strong antibody-based immune response. The immunological effect was dramatically enhanced when the mixture of vaccine components assembled into a 1:1:1 ternary complex. These noncovalent vaccines showed immunological effects comparable to covalently linked vaccines from our previous studies.[35, 37] These results indicate that this noncovalent strategy for constructing chemically synthesized vaccines is effective. This methodology markedly reduces the difficulties of vaccine preparation and may promote mass production and further clinical applications. Additionally, this simple self-assemChem. Eur. J. 2014, 20, 13541 – 13546
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bly method may provide great convenience for the evaluation of new immune components with different properties and permit further screening for optimum combination forms. This noncovalent strategy displays good suitability, scalability, and feasibility that may be applied to the construction of other chemically synthesized vaccines.
Experimental Section General experimental protocol for vaccine preparations Glycopeptides were prepared by standard solid-phase peptide synthesis method of Fmoc chemistry with Na-Fmoc-protected amino acids, on a Liberty CEM microwave peptide synthesizer (Supporting Information Figure S50). 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyl hexafluorophosphate (HBTU, 6.0 equiv) and N-hydroxybenzotriazole (HOBt, 6.0 equiv) were employed as activators and N,N-diisopropylethylamine (DIEA, 12.0 equiv) was employed as activator base for coupling of natural amino acids, 13, 15 and 14. 2-(7-Aza1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 2.0 equiv) and 1-hydroxy-7-azabenzotriazole (HOAt,
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Communication 2.0 equiv) were used as activators and N-methylmorpholine (NMM, 5.0 equiv) was used as activator base for coupling glycoamino acids. The peptide was then cleaved from the resin with TFA/TIS/ H2O = 15:0.9:0.9 (v/v/v) over 2.5 h. After removing TFA, peptides were precipitated two times with diethyl ether and centrifuged at 11 200 g for 20 min. Then the peptides were dried and purified by HPLC using semipreparative CN column. The synthesis scheme for 14 is shown in the Supporting Information Figure S49. Details of the chemical synthesis and analytical data of the compounds are given in the Supporting Information.
Animals and vaccinations Groups of four female BALB/c mice (4–6 weeks of age; fed at the Animal Facility of Center of Biomedical Analysis, Tsinghua University) were immunized with G1–G16 (20 mg) with or without Freund’s adjuvant (100 mL). Mice were immunized on days 1, 14, 28, 42, 56 and antisera were collected on day 66. Sera were taken before immunization, as negative control. The animal experiments were conducted in accord with the ethical guidelines of Tsinghua University.
Isothermal calorimetry (ITC) measurements Titration experiments were performed on a Microcal VP-ITC apparatus at 298.15 K in sodium phosphate buffer (10 mm; pH 7.4). The binding equilibriums were carried out using a 9·CB[8] concentration of typically 0.06 mm to which the 0.6 mm 5 was titrated. The data were analyzed with Origin 7.0 software, using the one set of sites model.
NMR measurements Each compound was dissolved in D2O at 0.5 mm. 1H NMR spectra was recorded on a Joel JNM-ECA 300 apparatus (300 MHz). Diffusion assays were carried out with a Bruker Avance 600 NMR spectrometer under the same conditions. All samples were tested under the same conditions.
Transmission electron microscopy analyses All samples were dissolved in PBS buffer (pH 7.4) with the concentration of 0.05 mm at room temperature. Each sample (10 mL) was added to copper grids with carbon support films, negatively stained with tungstophosphoric acid (15 mg mL 1, pH 6, 10 mL), and imaged on a Hitachi H-7650B transmission electron microscope after being dried, overnight.
Serological analyses For titer measurements, ELISA plates were separately coated with the corresponding B epitope (Figure 1) in carbonate buffer (0.1 m; pH 9.6) at 2 mg per well, overnight, at 4 8C. Each well was blocked with 0.25 % gelatin in PBS for 3 h and then washed four times with 0.5 % Tween-20 in PBS solution. Then, serially diluted antiserum was incubated in each well for 1 h at 37 8C. After four washes, the second antibody, anti-mouse IgG (whole molecule)–peroxidase produced in rabbit, was diluted 1:2000 and incubated (100 mL per well) with each well for 1 h at 37 8C. After a final wash, 1 mg mL 1 o-phenylenediamine in critic acid buffer (0.1 m; pH 5) with 30 % H2O2 (1.5 mL) was added to the wells (100 mL per well) as substrate. After incubation for 15 min at 37 8C, the optical density was measured at 450 nm. Titers are defined as the highest dilution yielding an optical absorption of 0.1 or greater than the normal control mouse sera. All assays were repeated three times. For subtype Chem. Eur. J. 2014, 20, 13541 – 13546
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assays, ELISA plates were separately coated with the corresponding B epitope (Figure 1) in carbonate buffer (0.1 m; pH 9.6) at 2 mg per well, overnight, at 4 8C. Each well was blocked with 0.25 % gelatin in PBS for 3 h and then washed four times with 0.5 % Tween-20 in PBS solution. Then, 1:25 diluted antiserum (100 mL) was incubated in each well for 1 h at 37 8C. After four washes, the second antibody (Mouse Monoclonal Antibody Isotyping Reagents kit including goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM (Sigma) was diluted 1:1000 and incubated at 100 mL per well for 1 h at 37 8C. After a final wash, the third antibody, anti-goat IgG (whole molecule)–peroxidase produced in rabbit was diluted 1:1000 and incubated at 100 mL per well for 1 h at 37 8C. After incubation for 15 min at 37 8C, the optical density was measured at 450 nm. All assays were repeated two times.
Cell surface reactivity determined by FACS Pre- and postimmunization sera were diluted 25-fold and incubated with MCF-7 single-cell suspensions of 2 105 cells per tube, washed in PBS with 1 % fetal calf serum for 1 h on ice. Next, the cells were washed and incubated with FITC-conjugated rabbit antimouse IgG antibody (1:50 diluted, DAKO) for 1 h on ice. After a final wash, FACS analysis was conducted on a BDC FACS Calibur flow cytometer. All assays were performed in triplicate.
Cytokine induction Dendritic cells were obtained by an established method[27] and incubated with each sample (0.5 mm, 100 mL) for 24 h at 37 8C. After that supernatants were collected and tested with mouse IL-6 precoated ELISA kit, mouse IL-12/IL-23 precoated ELISA kit, mouse TNF-a precoated ELISA kit, and mouse IL-10 precoated ELISA kit (Dakewe). All experiments were repeated three times.
Complement-dependent cytotoxicity assay Complement-dependent cytotoxicity (CDC) was assayed at a serum dilution of 1:50 with MCF-7 cells cultured for 12 h at 37 8C in 96well cell culture plates with the density of 5000 cells per well. Next, pre- and postimmunization sera were diluted 25-fold and added to the plate (50 mL per well). After incubation for 30 min at 37 8C, rabbit complement (1:2 diluted in culture medium, AbD seroTec, 50 mL per well) was added to the plate and incubated for 8 h, at 37 8C. Next, MTT solution (0.5 % MTT in PBS solution) was added to the plate (20 mL per well). The culture medium was removed after incubation for 4 h, formazan, violet crystal, was dissolved in DMSO (150 mL), and the optical density was measured at the wavelength of 490 nm. Cells incubated in culture medium were assayed as control. All assays were performed in triplicate. The cytotoxicity was calculated according to the formula: cytotoxicity [%] = [1 (experimental OD/control OD)] 100.
Acknowledgements We thank Prof. Oren Scherman and Prof. Horst Kunz for helpful discussions. We thank Prof. Xi Zhang for providing a Microcal VP-ITC apparatus and beneficial discussions. This work was supported by the Major State Basic Research Development Program of China (2013CB910700) and (2012CB821600), the National Natural Science Foundation of China (21332006, 91313301) and the Research Project of Chinese Ministry of Education (113005A).
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Communication Keywords: antitumor vaccines glycopeptides · host–guest systems
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cucurbit[8]uril
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Received: June 18, 2014 Published online on August 25, 2014
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