Enhanced Spacer Length between Mannose Mimicking Shikimoyl and

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Brief Article

Enhanced Spacer Length between Mannose Mimicking Shikimoyl- and Quinoyl- Head Groups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell based DNA Vac-cination: A Structure-Activity Investigation Chandrashekhar Voshavar, Rakeshchandra Reddy Meka, Sanjoy Samanta, Srujan Kumar Marepally, and Arabinda Chaudhuri J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01556 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017

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Journal of Medicinal Chemistry

Enhanced Spacer Length between Mannose Mimicking Shikimoyland Quinoyl- Head Groups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell based DNA Vaccination: A Structure-Activity Investigation Chandrashekhar Voshavar† *, Rakesh C. R. Meka† , Sanjoy Samanta† , Srujan Marepally† , and Arabinda Chaudhuri † § * †

Biomaterials group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India

§

Academy of Scientific and Innovative Research (AcSIR), Taramani, Chennai 600 113, India

KEYWORDS. Liposomal DNA vaccine carriers; cationic transfection lipids; dendritic cells; DNA vaccination; mannose-mimicking ligands. ABSTRACT: In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl head-group are promising DNA vaccine carriers for DC transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking head groups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the antimelanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57/BL6J mice under prophylactic settings.

Introduction DNA immunizations in animal models have been used to elicit protective immunity against various infectious pathogens and malignancies.1, 2 Generation of cellular immune response against complex diseases in humans indicated the possibility for clinical application of this immunization technique.3 The stability, ease of production, strong induction of immunity and long lasting cytotoxic T lymphocyte (CTL) responses make DNA vaccines as an attractive strategy compared to traditional vaccines.4 Dendritic cells (DCs) are known to be professional antigen presenting cells (APCs) that play a critical role in the induction and regulation of immune responses.5, 6 Dendritic cell based genetic immunization (DNA vaccination) involves administration of tumor antigen encoded plasmid DNA which primes the immune system.3, 4, 7 Such DC-based genetic immunizations are being increasingly used in cancer immunotherapy.8, 9 Several different DNA vaccine carriers, both viral and non-viral, have been developed for ex vivo loading of DCs.10, 11 Over expression of endocytic mannose receptors (MRs) on DC cell surfaces has been elegantly exploited in the past for developing efficient DNA vaccine carriers in DC-based immunotherapy.12-14 Previously, we demonstrated that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl- and quinoyl- head-groups are promising DNA vaccine carriers for DC-transfection.15 Subsequently we showed that introduction of a lysine or a guanidinylated-

lysine moiety between the shikimoyl- & quinoyl- head groups and hydrophobic region of cationic amphiphiles possess significantly enhanced DC-transfection efficacies.16, 17 However, systematic structure-activity studies aimed at probing the role of varying methylene spacers between quaternary nitrogen center and glycomimicking head groups in modulating DC transfection efficacies have not yet been undertaken. To this end, herein we report on the design, synthesis and bio-activity evaluation of a novel series of cationic amphiphiles (1-10, Fig. 1A) in which the spacer lengths between the mannosemimicking shikimoyl- or quinoyl- head groups and the hydrophobic tails have been varied using amino acids containing 1-5 methylene units. We show that ex vivo DC-transfection efficiencies can be modulated by increasing the number of methylene groups. Further, we show that ex vivo immunization with melanoma antigen (MART1) encoded DNA vaccine with liposomes of the most efficient DC-transfecting lipids containing five methylene units in the spacer arm induces long lasting anti-melanoma immune responses in C57BL/6J mice. Results and Discussion Chemistry. Synthetic routes adopted for preparing the mannose receptor specific glycomimicking cationic amphiphiles 1-10 and control mannosyl analog 11 are shown schematically in Fig. 1A & Fig. S2, respectively. Lipids 110 were synthesized by coupling O-acetylated

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B. Figure 1A. Synthesis of mannose receptor targeting cationic amphiphiles 1-10.

Figure 1B. Structures of control mannosyl analog (Lipid 11) and co-lipid (Lipid 12).

shikimic acid and quinic acids to the 1o amine group of the hydrophobic derivatives of glycine, β-alanine, γ-amino butyric acid, 5-amino valeric acid and 6-amino caproic acid (Intermediates IIA, Fig. 1A) followed by deacetylation of the resulting product (Fig. 1A). Briefly, N-BOC protected amino acids (IA, Fig. 1A) upon peptide coupling with N-2-aminoethyl-N,N-di-n-hexadecylamine followed by acid mediated N-BOC deprotection afforded primary amine intermediates IIA (Fig. 1A). The intermediates IIA upon coupling with O-acetyl protected shikimic and quinic acids15 provided intermediates IIIA (Fig. 1A). Quaternization of intermediate IIIA with excess methyl iodide, O-acetyl deprotection of the resulting quaternized iodides with potassium carbonate & methanol, and finally chloride ion exchange chromatography of the deacetylated products over amberlite IRA 400 Cl- resin afforded the title/target mannose mimicking cationic amphiphiles 1-10 (Fig. 1A). The control mannosyl analog (Lipid 11, Fig. 1B) was synthesized from D-Mannose using the synthetic scheme shown in Fig. S2, supporting information. The details of the synthetic procedures, spectral characterizations and reverse phase HPLC profiles of all the target compounds (Lipids 1-11) are provided in the supporting information. Physico-chemical characterizations. First, we examined the DNA binding properties of the liposomes of lipids 1-10 (100-350 nm size) prepared with two conventio-

Figure 2. Flow cytometric GFP expression profiles in mbmDCs transfected with lipoplexes of lipids 1-12 & pα5GFP (A). Reduced GFP expression in mbmDCs pre-incubated with mannan for lipoplexes of lipids 5, 10 & 11 & pα5GFP (B). Filled traces are for untreated control mbmDCs.

-nally used co-lipids namely, cholesterol and DOPE (1,2dioleoyl-sn-glycero-3-phosphoethanola-mine). However, we did not observe significant lipid:DNA binding properties (representative DNA binding data with Cholesterol as co-lipid are shown in Fig. S3). Subsequently, instead of cholesterol or DOPE, we used lipid 12 as co-lipid which previously showed high DNA binding, membrane fusogenicity and stability profiles.18 Lipids 1-11 showed high DNA binding properties when lipid 12 was used as colipid (Fig. S4A). DNAse I sensitivities were evaluated for the lipoplexes of transfection efficient lipids 5, 10 & 11 along with lipid 12 which further confirmed the strong DNA binding properties of these lipids across the lipid:DNA charge ratios 8:1-1:1 (Fig. S4B). Interestingly, the sizes of the liposomes of lipids 1-12 were found to be large in general (240-980 nm) and surface potentials varied across the range 3-40 mV (Table S1). Similarly, the sizes of lipoplexes were found to be large (300-900 nm) across the lipid:DNA charge ratio 8:1-2:1 (Table S2A) and the global surface potentials of all the lipoplexes within the charge ratios 8:1-2:1 were found to be positive within the range of 4-16 mV (Table S2B). It is important to note that the dynamic light scattering experiments provide hydrodynamic radii of the liposomal aggregates.


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Figure 3. Cellular (A) and Humoral (B) immune responses in C57BL/6J mice (n = 4) s.c. immunized with mbmDCs pretransfected with lipoplexes of lipids 5, 10-12 and pCMV-MART1. Splenocytes were isolated and used directly without any in vitro re-stimulation for measuring secreted IFN- γ (A, *P< 0.001) and IL-4 (B, *P< 0.005). (C) Target cell lysis by CD8+ T cells from immunized mice. Untreated mbmDCs were used as controls. Experimental details are as described in supporting information.

Since the quinoyl head-groups of lipids 6-10 possess four hydroxyl groups compared to three hydroxyl group in lipids 1-5 with shikimoyl- head groups, the liposomes of lipids 6-10 could be more hydrated than liposomes of lipids 1-5. Thus, the observed higher size range for the liposomes of lipid 6-10 than those for lipids 1-5 may partly originate from more water of hydration in the head-group region. Similarly, the co-lipid 12 used in the present study, in addition to having one hydroxyl headgroup, contains two amide linkers near its polar headgroup region which may bring additional water of hydration compared to liposomes prepared using cholesterol as co-lipid. The observed higher size range of liposomes prepared with lipid 12 as co-lipid than those for liposomes prepared with cholesterol as co-lipid (contains only one hydroxyl head-group) may also owe its origin to higher degree of head-group hydration. In vitro DC Transfection Biology. Isolated dendritic cells from mouse bone marrow (femur & tibia) were first subjected to phenotypic analysis using cell surface markers unique to DCs by flow cytometry. After confirming the expressions of the distinguishing DC-surface markers (including Mannose receptor, MHC-II, F4/80, H2kB, CD86, CD11c, CD45R and CD40) in the isolated mbmDCs (Fig. S5), we measured the DC-transfection efficiencies of the lipids 1-12. The cultured mbmDCs were transfected with lipoplexes of lipids 1-12 and pα5GFP plasmid (plasmid DNA encoding green fluorescent protein) and the transfection efficiencies were measured by flow cytometry. Lipids 5, 10 & 11 were found to be the most efficient (up to 6-8% DCtransfection efficiencies) (Fig. 2A). Importantly, the liposomes of only lipid 12 (used as co-lipid in preparing liposomes of lipids 1-11) were found to be essentially transfection incompetent (Fig. 2A). Notably, the DCtransfection efficiencies of lipids 5, 10 & 11 were found to be significantly reduced (by >50%) in DCs pre-treated with mannan, a high affinity natural ligand of mannose receptor (Fig. 2B). This finding is consistent with mannose receptor selective uptake of the lipoplexes of lipids 5, 10 & 11 in DCs. Thus the findings summarized in Fig. 2A-B clearly show that the DC-transfection efficiencies of cationic amphiphiles with mannose mimicking shikimoyl- & quinoyl- head-groups increase with increasing

spacer unit length between the quaternary nitrogen atom and DC-targeting ligand. Toward gaining some insights into whether the varying transfection efficiencies of the lipoplexes of lipids 1-12 with different spacer lengths (in between the polar head-groups and hydrophobic tails) owe their mechanistic origin to varying cellular uptake efficiencies, we initially performed both transfection and cellular uptake experiments (using RhPE labeled liposomes) in mannose-receptor positive mouse macrophage cells (RAW 264.7 cells, a model antigen presenting cells). The degrees of cellular uptake of the lipoplexes of Rh-PE labeled liposomes of lipids 1-12 were obtained from the average fluorescence intensities of the transfected RAW264.7 cells using ImageJ software. Representative epifluorescence images from which quantitative cellular uptake data were computed using ImageJ software are shown in Fig. S6 for the lipoplexes of lipids 5, 10-12. Interestingly, for the lipid series 1-5 containing shikimoyl- head-groups, the most efficient liposomes of lipid 5 also showed best cellular uptake properties (now included in Fig. S7A-B). However, the degree of cellular uptake for the most efficient liposomes of lipid 10 in the quinoyl- head-group containing lipid series 6-10 was not found to be the highest (Fig. S7A-B). Thus, cellular uptake is unlikely to play the most important role in modulating the in vitro APC transfection efficiencies of the presently described lipids 1-10. Presumably, subsequent intracellular events (such as cytosolic release of plasmid DNA from endosomes, endosomal/lysomal degradation of plasmid DNA, etc.) also play some role in influencing the transfection properties of these lipids. Clearly, further studies need to be carried out in future toward demonstrating the mechanistic origin of the varying efficacies of lipids 1-10 in transfecting antigen presenting cells under in vitro settings. Immune response assays. After confirming the DCtransfection efficiencies of lipids 5, 10 & 11, we evaluated their efficacies in mounting cellular immune response in mouse model using lipid 12 as control. Initially, cellular immune responses (anti β-gal response) in mice were measured after subcutaneous administration of DCs pre-transfected with lipoplexes of lipids 5 & 10-12 with pCMV-SPORT-β-gal as a model DNA vaccine. ELISA


Journal of Medicinal Chemistry assay with serum samples of immunized mice revealed significant anti-β-gal antibody humoral immune response for lipoplexes of lipids 5, 10 & 11 compared to that in mice immunized with lipoplexes of lipid 12 (Fig. S8). Next, IL-4 and IFN-γ (two well known signature cytokines for humoral and cellular immune responses, respectively)19 were evaluated with splenocytes collected from mice subcutaneously immunized with DCs pretransfected with lipoplexes of lipids 5 & 10-12 using pCMV-MART-1 DNA (encodes Human MelanA/MART1 antigen of human melanoma tumor where 68.6% of amino acid sequence matches with its murine equivalent).20, 21 The results showed that lipids 5 & 10 are capable of inducing significant cellular immune response against MART-1 antigen as indicated by the elevated IL4 and IFN- γ secretion (Fig. 3A & B) than control lipids 11 & 12. Furthermore, cytotoxic T lymphocytes (CTL) assay was carried using splenocytes isolated from the mice immunized with pre-transfected DCs (using splenocytes as effector cells and B16F10 cells as target cells). Data from CTL assay revealed significant MART-1 antigen specific T-cell responses for lipids 5 & 10 than lipids 11 & 12 (Fig. 3C).

in inducing both humoral and cellular immune responses. Finally, the efficiencies of the cationic liposomes of the most promising lipids 5, 10 along with that of control mannosyl analog 11 and co-lipid 12 in mounting long lasting immune response against B16 melanoma by DCbased genetic immunization were evaluated in C57BL/6J mice under prophylactic settings. MART1 antigen is known to induce immune response in mouse against the aggressive murine B16 melanoma.15, 16, 22, 23 C57BL/6J mice were immunized (twice with a seven day interval) by subcutaneous administration of mbmDCs pretransfected with the lipoplexes of lipids 5, 10-12 and plasmid DNA encoding MART-1 antigen using untransfected DCs and naked plasmid as negative controls.

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Figure 4. (A) Protection against melanoma challenge in mice (n = 5) immunized with mbmDCs pre-transfected with lipoplexes of lipids 5, 10-12 and pCMV-MART1. Mice groups immunized with naked pCMV-MART1 and with untransfected DCs were used as control groups. Two 5 weeks after, mice were challenged with B16F10 cells (1 x 10 cells in 100µL HBSS). Results represent the means +/- SD (*P < 0.05 for lipids 5, 10-12 & naked MART-1 treated group compared with control group. (B) Survival study of immunized mice subsequently challenged (s.c.) with B16F10 cells. Experimental details are in supporting information.

Results from immune response & CTL assays indicate that lipids 5 & 10 hold more potential than lipids 11 & 12

Figure 5. Protection against spontaneous growth of i.v. administered pLuc-B16F10 cells in mice (n = 5) immunized with mbmDCs ex vivo pre-transfected with indicated lipoplexes. Mice immunized with untransfected DCs was used as control group. A. Luciferase activities in lung extract prepared from one sacrificed mice from each group on day 30 post tumor challenge. B. Representative images of excised lungs. C. Long term survival study of the remaining mice (n = 4) challenged with pLuc-B16F10 cells. Experimental details are in supporting information.

Two weeks after the last immunization, in one set of experiments mice were challenged (s.c) with lethal dose of B16F10 cells. In another set of experiments, following a widely used secondary lung metastasis model,24 mice were i.v. challenged with lethal dose of B16F10 cells stably transformed with luciferase gene (pLuc-B16F10) which spontaneously accumulated in lung. Mice s.c. immunized with DCs pre-transfected with lipoplexes of lipids 5 & 10 & MART1 encoded plasmid DNA showed significant protection against melanoma (subcutaneous tumor challenge). No tumor was detected for 29 days in mice s.c. immunized with lipoplexes of lipids 5 & 10 (Fig. 4A). Importantly, lipoplexes of lipid 5 was found to be the most efficient in mounting long-lasting (80%


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of s.c. immunized mice survived for 120 days post tumor challenge) protection against melanoma (Fig. 4B). The survivability for other groups were significantly less (Fig. 4B). Lipoplexes of lipid 5, 10 & 11 were found to be highly efficient in protecting mice from i.v. administered Luc-B16F10 cells growing on mouse lung during the first 30 days post luc-B16F10 challenge (Fig. 5A-B). However, long-lasting immune response against melanoma (150 days post luc-B16F10 challenge) was observed in 75% of mice s.c. immunized with DCs pre-transfected with the lipoplexes of pCMV-MART1 and lipids 5 and while the survival rate for the other groups were found to be significantly less (Fig. 5C). In conclusion, our present structure-activity findings demonstrate that mannose-receptor selective cationic amphiphile containing 5 methylene units in the spacer arm between the hydrophobic tail and mannosemimicking shikimoyl- & quinoyl- head groups (lipids 5 & 10) hold promise for use in ex vivo DC-transfection based DNA vaccination under prophylactic settings. Importantly, use of lipoplexes of pCMV-MART1 and lipid 5 in ex vivo DC-transfection were found to be the most efficient in mounting long-lasting anti-melanoma immune response in mice challenged with B16F10 cells using two different challenge modes. Experimental Section

tional Centre for Cell Sciences (NCCS), Pune, India. B16F10, pLuc-B16F10 Cells were grown at 37° C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS in a humidified atmosphere containing 5%CO2/95% air. 6-8 weeks old C57BL/6J mice (male & female, each weighing 20-22 g) were purchased from National Institute of Nutrition, Hyderabad, India. All the in vivo experiments were performed in accordance with the Institutional Bio-Safety and Ethical Committee Guidelines using an approved animal protocol. Statistical Analysis. Experimental data presented is the mean ± SD of triplicate experiments. Data were compared among the different groups for individual experiment using the Student t test and p < 0.05 was considered as significant.

ASSOCIATED CONTENT Supporting Information Detailed information on the syntheses and purifications, 1H NMR & ESI-Mass spectra and the reverse phase analytical HPLC profiles of the purified lipids 1-11; details for DCtransfection, isolation and culture of mbmDCs, DC-based immunization protocols, ELISA assays, and FACS protocol. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION

General procedures and reagents. ESI-Mass spectra for all the lipids and their intermediates were acquired by micromass Quatro LC triple quadrapole mass spectrometer or LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA). 1H NMR spectra were recorded on a Varian AV 300 MHz NMR Spectrometer. Cell culture media, fetal bovine serum, Amberlite Cl- ion exchange resin, TMS-triflate, EDCI, HOBt and cholesterol were purchased from Sigma-Aldrich (St. Louis, USA). Antibiotics and agarose were procured from Himedia, India. TFA, trichloro acetonitrile and methyl iodide were purchased from Spectrochem, India. DMannose was procured from SD Fine, India. Column chromatography was performed with silica gel (Acme Synthetic Chemicals, India, 60-120 mesh). Unless otherwise stated all reagents were purchased from local commercial suppliers and were used without further purification. Mouse GM-CSF, IL-4, IFN-γ, ELISA and CTL assay kits were purchased from Thermo Scientific and Promega USA. Mouse FITC conjugated CD11c, CD206, CD40, CD86, CD45R, H2kB, F4/80, anti-MHC II antibodies were purchased from Chemicon, USA. FACS data was analyzed and processed using FCS Express 4 Research Edition (USA) software. Structures of all the synthetic intermediates were confirmed by 1H NMR and ESI-MS. The purity of all the target compounds was confirmed by reverse phase analytical HPLC and found to be >95%. Cell lines and Animal models. B16F10 & pLuc-B16F10 (murine melanoma cells) were procured from the Na-

Corresponding Authors * E-mail: [email protected], Tel: +91-9542816932 [email protected], Tel: +91-9542816932

Present Addresses CV is at BioSatva Technologies, Golnaka, Hyderabad 500013, India; RCRM is at Department of Microbiology & Immunology, School of Medicine, University of Maryland, Baltimore, MD 21201 USA; SS is at Department of Cancer Biology, UMASS Medical School, Worcester, MA 01605, USA; SM is at Centre for Stem Cell Research, CMC Campus, Bagayam, Vellore 632002, India

Funding Sources This work was supported by the Department of Biotechnology and Council of Scientific and Industrial Research, Government of India, New Delhi (NWP-0036, CSC0123 & CSC0302).

Notes Conflict of Interest: The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dr. Van den Eynde from Ludwig Institute for Cancer Research, Brussels, Belgium, Dr. N. M. Rao & Dr. G. Pande from Centre for Cellular and Molecular Biology, Hyderabad, India, for providing us with pCMV-MART1, pCMV-SPORT-β-Gal, pCMV-Luc and pCMV-α5-GFP plasmids, respectively.



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ABBREVIATIONS

logue as DNA Vaccine Carrier in Dendritic Cell Based Genetic Immunization. J. Med. Chem. 2010, 53, 1387-1391.

APC: Antigen presenting cells; DC: Dendritic cell; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; GMCSF: Granulocyte Macrophage Colony Stimulating Factor; GFP: Green fluorescent protein. HBSS: Hank’s Balanced Salt Solution; IFN-γ: Interferon gamma; IL-4: Interleukin-4; mbmDC: Mouse bone marrow derived dendritic cells; MHC: Major Histocompatibility Complex.

16. Srinivas, R.; Garu, A.; Moku, G.; Agawane, S. B.; Chaudhuri, A. A Long-Lasting Dendritic Cell DNA Vaccination System Using Lysinylated Amphiphiles with Mannose-Mimicking Head-Groups. Biomaterials 2012, 33, 6220-6229.

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Protection against s.c. Melanoma challenge in mice

mbmDCs ex vivo pre-transfected with lipoplexes of pCMV-MART1 & Lipids 5 & 10

s.c.Immunization with mbmDCs

Tumor Challenge post immunization

2500

Tumor Volume (mm3)

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Control Lipid 5 + MART-1

2000


1500

Lipid 12 + MART-1 Naked MART-1

1000 500 0 0

18

20

22

25



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Enhanced Spacer Length between Mannose Mimicking Shikimoyl and

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