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Targeting Tumor Cells by Natural Anti-Carbohydrate Antibodies Using Rhamnose-Functionalized Liposomes Xuexia Li, Xiongjian Rao, Li Cai, Xuling Liu, Huixia Wang, Weinan Wu, Chenggang Zhu, Min Chen, Peng G. Wang, and Wen Yi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00173 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 17, 2016

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ACS Chemical Biology

Targeting Tumor Cells by Natural Anti-Carbohydrate Antibodies Using Rhamnose-Functionalized Liposomes

Xuexia Li†,&,‡, Xiongjian Rao†,&,‡, Li Cai#,‡, Xuling Liu†, Huixia Wang†, Weinan Wu†, Chenggang Zhu†, Min Chen∆, Peng G. Wang∆,§ and Wen Yi†,&,*

†

Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou

310058, China.

&

Collaborative Innovation Center for Diagnosis and Treatment of

Infectious Diseases, Hangzhou 310003, China. #Division of Mathematics and Science, University

of

South

Carolina

Salkehatchie,

Walterboro,

SC

29488

United States. ∆School of Life Science and the State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. §Department of Chemistry, Georgia State University, Atlanta, GA 30303 United States. ‡These authors contributed equally to this work.

* To whom correspondence should be addressed: [email protected] (W.Y.)

ACS Paragon Plus Environment


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ABSTRACT

Recruitment of antibodies in human immune systems for targeted destruction of tumor cells has emerged as an exciting area of research due to its low side effect, high efficacy and high specificity. The presence of large amounts of anti-carbohydrate natural antibodies in human sera has prompted the research efforts to utilize carbohydrate epitopes for immune recruitment. Here we have developed a general strategy for specific targeted destruction of tumor cells based on rhamnose-functionalized liposomes. Tumor cells artificially decorated with rhamnose epitopes were subjected to complementmediated cytotoxicity in vitro and showed delayed tumor growth in vivo. This study highlights the therapeutic potential for activation of endogenous immune response through cell-surface glycan engineering.


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Harnessing the immune system to target and eliminate tumor cells (also termed cancer immunotherapy) is a promising therapeutic strategy with exceptional specificity and efficacy.1-3 One emerging approach is to recruit pre-existing natural antibodies to the surface of target cells, thereby activating antibody-mediated immune responses, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), for cell destruction.1,4-6 To direct antibodies to the target tumor cells, cell surfaces need to be decorated with antibody-recognition molecules. The small molecule 2,4-dinitrophenyl (DNP) is among the most widely used defined antigens for antibody recruitment experiments.7,8 However, its intrinsic molecular property and the low abundance of naturally occurring anti-DNP antibodies in human sera largely limit its therapeutic application. Recently, high-throughput assays to probe carbohydrate-protein interactions in human sera have identified a number of high-titer pre-existing natural antibodies with specificity toward distinct carbohydrate epitopes.9 These carbohydrate epitopes include galactose-α-(1,3)galactose (α-Gal), mono-L-rhamnose (Rha), and various blood group antigens. Among them, Rha is a simple deoxy monosaccharide not expressed in human cells but prevalent in microbes. Studies have shown that anti-Rha antibodies are present in higher abundance in human sera than other carbohydrate epitopes, and that Rha could form relative stable complexes with anti-Rha antibodies6, indicating that Rha might be an attractive reagent for activation of endogenous immune response for targeted immunotherapy of cancer. Recently, two Rha conjugates have been synthesized to evaluate their ability to induce cell cytotoxicity.4,6 These Rha conjugates, in which Rha is appended to a phospholipid or



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N-hydroxysuccinimide (NHS), tend to incorporate Rha epitopes onto the surface of all cell types, thus imparting no targeting specificity. Moreover, the ability of Rha conjugates to induce destruction of tumor cells has not been evaluated in mouse models. To address these issues and systemically evaluate Rha-based immunotherapy, we have developed a targeting strategies using ligand-incorporated Rha-functionalized liposomes to deliver Rha-lipid conjugates onto target cells in a cell-surface receptor dependent manner (Figure 1). Tumor cells selectively targeted by this approach were subjected to significant cell cytotoxicity in vitro and showed delayed tumor growth in vivo. We chose to use liposomes as targeting vehicles due to their compatibility, synthetic facileness, in vivo stability, and broad clinical applications. In this strategy, liposomes containing ligands specific for cell-surface receptors and Rha-lipid conjugates were generated. Cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) was also used as a lipid component to facilitate membrane fusion and surface presentation of Rha conjugates rather than intracellular uptake.10,11 Decoration of tumor cell surface with Rha epitopes promotes recruitment of anti-Rha antibodies, thereby activating immune responses for complement-mediated cell killing. To demonstrate our strategy, we chose to target folate receptor (FR)-expressing tumor cells with folic acid-incorporated liposomes. FR is one of the most widely used cell surface receptors for active tumor targeting and validated for clinical use.12 Its expression is restricted in normal tissues but highly expressed in tumor tissues including breast, ovarian, colon and lung.13 Folic acid-lipid conjugates (DSPE-PEG2k-FA) and Rha-lipid conjugates (DSPE-PEG2k-Rha) were synthesized according to the literature14 or based on the synthetic scheme detailed in the Supporting Information. Targeted


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liposomes were composed of DOPE, DSPE-PEG2k-FA, DSPE-PEG2k-Rha (molar ratio, 1000:1:X, X ranges from 100 to 3000), and DOTAP (0.02% w/w with respect to DOPE). To characterize the biophysical properties of the generated liposomes, we use dynamic light scattering (DLS) and zeta potential measurements. The liposomes exhibited good qualities with average diameters ranging between 159.5 and 189.7 nm (Table S1). The liposomes were stable at 4°C up to one month without change of particle size distribution and noticeable aggregation. We first optimized the incubation time for liposomal targeting of FRoverexpressing HeLa cells (FR+ HeLa) using fluorescence liposomes composed of DOPE, DSPE-PEG2k-FA, DSPE-PEG2k-FITC and DOTAP. In the control liposomes, DSPE-PEG2k-FA was replaced with DSPE-PEG2k-OMe. Membrane targeting and fusion experiments were carried out at different time points and imaged using fluorescence microscopy (Figure S1). Notably, distinct fluorescence signals were observed on the surfaces of cells treated with targeted liposomes after 2 h incubation and up to 4 h, whereas only minimal background fluorescence was observed with control liposomes. Longer incubation (> 6 h), on the other hand, led to increased fluorescence signals by treatment with control liposomes, suggesting intracellular uptake of both targeted and control liposomes. Therefore, in the following targeted experiments, 4 h incubation time was chosen for the optimal targeted effect. Having determined the optimal incubation time, we next examined whether the Rha-lipid conjugates in the targeted liposomes could be successfully targeted and presented onto the surface of cells. Targeted liposomes containing DSPE-PEG2k-Rha and DSPE-PEG2k-FA (molar ratio 1000:1) were incubated with FR+ HeLa cells for 4 h,



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and immunostained with an anti-Rha antibody. Importantly, strong immunostaining for Rha was observed on the surfaces of cells with targeted liposomes, as compared to control liposomes in which either FA or Rha was omitted, indicating efficient presentation of the Rha epitopes (Figure 2A). To further confirm the incorporation of the Rha conjugates onto the cell surfaces, co-immunostaining with the anti-Rha antibody and the anti-FR antibody was carried out, and imaged using confocal fluorescence microscopy. Notably, immunostaining signals showed significant colocalization on the cell surfaces, thus confirming the surface presentation of the Rha conjugates (Figure 2B). To demonstrate that the targeted strategy is dependent on the presence of FR expression, we performed the targeting experiment using a lung cancer cell line A549, which is known to be deficient in FR expression. Minimal fluorescence signals were detected, thus further validating our targeting strategy (Figure 2A). To investigate the membrane half-life of the exogenously presented Rha conjugates, we treated the cell surface Rha conjugates with the anti-Rha antibody, followed by incubation with FITC-conjugated secondary antibodies, and assayed using flow cytometry (Figure S2). Fluorescence signals were relatively stable for the first 6 h and started to decrease. The signal decreased to half of the original level after 12 h. After 18 h, only weak fluorescence signals were detected, indicating that Rha-lipid conjugates had been internalized into the cells. We further demonstrated that our strategy could be used to target other FRexpressing cell lines, such as 4T1, a murine breast cancer cell line. Similarly, strong fluorescence on the cell surface was observed with targeted liposomes. Treating cells


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with control liposomes without FA or Rha resulted in low or minimal fluorescence (Figure S3). We next sought to investigate whether the surface presentation of Rha could result in complement-dependent lysis of targeted tumor cells. We treated FR+ HeLa cells with targeted liposomes containing different FA/Rha ratios ranging from 1:100 to 1:3000, and incubated treated cells with human serum, a source of both anti-Rha antibodies and complement. Cell cytotoxicity was assayed using commercially available CellTiterGlo reagents, a well-validated method for measuring CDC.4 Results showed that cells treated with targeted liposomes exhibited significant cell cytotoxicity, and the degree of cytotoxicity has shown dependence on the FA/Rha ratio (Figure 3A). To further probe the mechanism of the observed cytotoxicity, we treated cells with liposomes without FA or Rha. These cells showed minimal cytotoxicity, suggesting that cell lysis effect was dependent on FA targeting and the presence of Rha (Figure 3B). To determine whether the cell death was complement-mediated, targeted cells were exposed to heat-inactivated serum, in which critical complement proteins were destroyed while anti-Rha binding was not affected. No significant cell lysis was observed in this case. Tumor cells are known to express membrane proteins to inhibit complement activation.15 Treatment of cells with antibodies that target CD55 and CD59, two known complement inhibitors, has further augmented the cytotoxicity effect (Figure 3B). As another confirmation, targeted treatment resulted in significant cell cytotoxicity in 4T1 cells, but minimal effect in A549 cells (Figure S4). Taken together, our results suggested that targeted destruction of tumor cells by Rha presentation was complement-mediated.



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To further confirm the treatment efficacy of the liposomal approach, we performed a comparative experiment in which FR+ HeLa cells were subjected to two different treatments for cell cytotoxicity analysis. In one experiment, the cells were treated with targeted liposomes with FA:Rha ratio of 1:1000. In another parallel experiment, the cells were treated with Rha-lipid conjugates only (DSPE-PEG2k-Rha, 100 µM, equivalent to the amount of Rha in the liposomes). Cell cytotoxicity analysis showed that both treatments resulted in a comparable degree of cell lysis (Figure S5), thus further confirming the surface presentation of Rha and the treatment efficacy using liposomes. Finally, we explored whether our liposomal targeting strategy could be of any therapeutic benefits in a preclinical mouse model to inhibit tumor growth. Notably, recent studies have demonstrated that standard wild-type laboratory mouse strains possess low basal levels of natural anti-Rha antibodies, and that immunization with Rha immunogens could result in production of high titers anti-Rha antibodies, which is comparable to those found in human sera.5,16 Thus, standard mouse strains could be a perfect model for evaluation of Rha-based immunotherapy. To confirm that high titers of anti-Rha antibodies could be induced in standard wild-type mice, we immunized a group of 5 Balb/c female mice with Rha-OVA conjugates. After three immunization periods, ELISA assays showed significantly high titers of anti-Rha antibodies, as compared to the control group treated with PBS, consistent with the previous reports (Figure S6).5,16 Next we investigated the effect of our liposomal targeting strategy on tumor growth in the mouse model. Balb/c mice were firstly immunized subcutaneously with Rha-OVA conjugates three times at two weeks interval to induce the production of anti-Rha antibodies. One


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week after the third immunization, mice were challenged with 4T1 breast cancer cells. Groups of mice (n = 5) were then treated with targeted liposomes (FA:Rha 1:2500), control liposomes without FA, control liposomes without Rha, or PBS. Treatments were given daily for two weeks. Tumors were measured twice a week using calipers. Results showed that treatment with targeted liposomes significantly delayed tumor growth, whereas in mice treated with either control liposomes tumors grew at a similar rate compared to those treated with PBS alone (Figure 4). Notably, there was no significant weight loss in mice treated with liposomes, indicating the minimal toxicity of the treatment (Figure S7). This is important because even though FR is generally absent in most normal tissues, low levels of expression have been found in choroid plexus, thyroid, and kidney.12 The lack of toxicity of our liposomal treatment may also stem from the low-affinity and multivalent interactions between carbohydrates (Rha) and antibodies (anti-Rha).1 Such interactions are particularly useful in distinguishing normal and diseased cells in physiological systems.17 Taken together, these results demonstrated the therapeutic benefits of our liposomal targeting strategy in the inhibition of tumor growth in vivo. In conclusion, we have developed an immuno-targeing strategy for specific destruction of tumor cells based on Rha-functionalized liposomes. Artificial display of Rha epitopes onto the target tumor cells promotes recruitment of anti-Rha antibodies, thereby activating immune responses for cell destruction with high specificity and high efficacy both in vitro and in vivo. The liposome-based strategy is a synthetically modular and general approach for cell targeting. The facileness to incorporate various cell surface receptor-specific ligands onto liposomes vastly expands the power of this strategy to



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target different cell types, including but not limited to FR-expressing cells. In addition, given the variety of naturally occurring anti-carbohydrate antibodies in human sera, incorporation of different carbohydrate epitopes onto the targeted liposomes might further augment the cytotoxicity effect and therapeutic benefits. Thus, our study represents a significant advancement in carbohydrate-mediated immunotherapy, and highlights the therapeutic potential to harness immune systems for active targeting and treatment of cancers through cell-surface engineering.

References: (1) Carlson, C. B., Mowery, P., Owen, R. M., Dykhuizen, E. C., Kiessling, L. L. (2007) Selective tumor cell targeting using low-affinity, multivalent interactions. ACS Chem. Biol. 2, 119-27. (2) Galili, U. (2005) The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunol. Cell Biol. 83, 674-686. (3) McEnaney, P. J., Parker, C. G., Zhang, A. X., Spiegel, D. A. (2012) Antibody-Recruiting Molecules: An Emerging Paradigm for Engaging Immune Function in Treating Human Disease. ACS Chem. Biol. 7, 1139-1151. (4) Jakobsche, C. E., Parker, C. G., Tao, R. N., Kolesnikova, M. D., Douglass, E. F., Spiegel, D. A. (2013) Exploring Binding and Effector Functions of Natural Human Antibodies Using Synthetic Immunomodulators. ACS Chem. Biol. 8, 2404-2411. (5) Chen, W. L., Gu, L., Zhang, W. P., Motari, E., Cai, L., Styslinger, T. J., Wang, P. G. (2011) L-Rhamnose Antigen: A Promising Alternative to alpha-Gal for Cancer Immunotherapies. ACS Chem. Biol. 6, 185-191.


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(6) Sheridan, R. T. C., Hudon, J., Hank, J. A., Sondel, P. M., Kiessling, L. L. (2014) Rhamnose Glycoconjugates for the Recruitment of Endogenous Anti-Carbohydrate Antibodies to Tumor Cells. Chembiochem 15, 1393-1398. (7) Farah, F. S. (1973) Natural Antibodies Specific to 2,4-Dinitrophenyl Group. Immunology 25, 217-226. (8) Ortega, E., Kostovetzky, M., Larralde, C. (1984) Natural Dnp-Binding Immunoglobulins and Antibody Multispecificity. Mol. Immunol. 21, 883-888. (9) Oyelaran, O., McShane, L. M., Dodd, L., Gildersleeve, J. C. (2009) Profiling Human Serum Antibodies with a Carbohydrate Antigen Microarray. J Proteome Res. 8, 4301-4310. (10) Pulsipher, A., Griffin, M. E., Stone, S. E., Brown, J. M., Hsieh-Wilson, L. C. (2014) Directing Neuronal Signaling through Cell-Surface Glycan Engineering. J. Am. Chem. Soc. 136, 6794-6797; (11) Dutta, D., Pulsipher, A., Luo, W., Yousaf, M. N. (2011) Synthetic Chemoselective Rewiring of Cell Surfaces: Generation of Three-Dimensional Tissue Structures. J. Am. Chem. Soc. 133, 8704-8713. (12) Sudimack, J., Lee, R. J. (2000) Targeted drug delivery via the folate receptor. Adv. Drug Deliver Rev. 41, 147-162. (13) Weitman, S. D., Lark, R. H., Coney, L. R., Fort, D. W., Frasca, V., Zurawski, V. R., Kamen, B. A. (1992) Distribution of the Folate Receptor Gp38 in Normal and Malignant-Cell Lines and Tissues. Cancer Res. 52, 3396-3401. (14) Gabizon, A., Horowitz, A. T., Goren, D., Tzemach, D., Mandelbaum-Shavit, F., Qazen, M. M., Zalipsky, S. (1999) Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: In vitro studies. Bioconj. Chem. 10, 289-298. (15) Mikesch, J. H., Buerger, H., Simon, R., Brandt, B. (2006) Decay-accelerating factor (CD55): A versatile acting molecule in human malignancies. BBA-Rev Cancer, 1766, 42-52.



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(16) Sarkar, S., Lombardo, S. A., Herner, D. N., Talan, R. S., Wall, K. A., Sucheck, S. J. (2010) Synthesis of a Single-Molecule L-Rhamnose-Containing Three-Component Vaccine and Evaluation of Antigenicity in the Presence of Anti-L-Rhamnose Antibodies. J. Am. Chem. Soc. 132, 17236-17246. (17) Kiessling, L. L., Gestwicki, J. E., and Strong, L. E. (2000) Synthetic multivalent ligands in the exploration of cell-surface interactions. Curr. Opin. Chem. Biol. 4, 696-703.

Acknowledgements: This work was supported by the National Science Foundation of China (NSFC, grant nos. 31270865, 31322019 and 31570804), the Natural Science Foundation of Zhejiang Province (LR15C050001), the Thousand-Young-Talents Recruitment Program, and the Fundamental Research Funds for the Central Universities. W.Y. also acknowledged the support from the Development Fund for Collaborative Innovation Center of Glycoscience of Shandong University. L.C. also acknowledged the support by an ASPIRE grant from the Office of the Vice President for Research at the University of South Carolina.

Competing financial interests: The authors declare no competing financial interests.

Additional information: Supporting information is available in the online version of the paper.


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Figure legends Figure 1. Strategy of targeting tumor cells using ligand-incorporated rhamnosefunctionalized liposomes.

Figure 2. Efficient targeting and cell-surface presentation of Rha conjugates. (A) Confocal fluorescence microscopy images of FR+ HeLa cells and A549 cells treated with targeted liposomes containing FA and Rha, or control liposomes containing either FA or Rha. (B) Confocal fluorescence microscopy images of FR+ HeLa cells treated with targeted liposomes and immunostained with anti-Rha or anti-FR antibodies. Scale bar: 20 µm.

Figure 3. Cell cytotoxicity induced by cell-surface presentation of Rha. (A) Correlation of cytotoxicity with FA:Rha ratio. (B) Control experiments for the complement-mediated cytotoxicity assays. Error bars denote the standard deviation of the mean (± SD) from three independent experiments. Statistical analysis was performed by Student’s t-test (* P < 0.05).

Figure 4. Comparison of tumor growth rate in mice treated with targeted and control liposomes. Statistical analysis was performed by Student’s t-test (* P < 0.05).



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