l-Rhamnose Enhances the Immunogenicity of Melanoma-Associated

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L-Rhamnose Enhances the Immunogenicity of Melanoma Associated Antigen A3 for Stimulating Anti-Tumor Immune Responses Huajie Zhang, Bin Wang, Zhongrui Ma, Mohui Wei, Jun Liu, Dong Li, Houcheng Zhang, Peng George Wang, and Min Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00081 • Publication Date (Web): 15 Mar 2016 Downloaded from http://pubs.acs.org on March 21, 2016

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Bioconjugate Chemistry

T cell-mediated antitumor immunity induced by Rha-tMAGE-A3 in the presence of anti-Rha antibodies. In details, Rha-tMAGE-A3 was taken up by APCs via the interaction between anti-Rha antibodies and Fcγ receptors (FcγR). The Rha-tMAGE-A3 was processed and displayed to activate T lymphocytes to generate tMAGE-A3 specific CTLs, which showed ability to lyse tumor cells expressed MAGE-A3. 677x409mm (300 x 300 DPI)

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L-Rhamnose Enhances the Immunogenicity of Melanoma-Associated Antigen A3 for Stimulating Anti-Tumor Immune Responses Huajie Zhang,† Bin Wang,† Zhongrui Ma,† Mohui Wei,‡ Jun Liu,† Dong Li,§ Houcheng Zhang,† Peng George Wang,*,†,‡ and Min Chen*,†

†

National Glycoengineering Research Center, the State Key Laboratory of Microbial

Technology and School of Life Science, Shandong University, Jinan, Shandong 250100, China ‡

Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, United States

§

Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, Shandong 250012,

China *

Co-corresponding authors: E-mail: [email protected] (Min Chen); [email protected]

(Peng George Wang).

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ABSTRACT Vaccines based on melanoma-associated antigens (MAGEs) present a promising strategy for tumor immunotherapy, whereas with weak immunogenicity. In this study, the xenoantigen L-rhamnose (Rha) was chemically conjugated with truncated MAGE-A3 (tMAGE-A3) to generate Rha-tMAGE-A3. The product showed good antigenicity with anti-Rha antibodies purified from human serum. FITC-labeled Rha-tMAGE-A3 was detected in THP-1 human macrophage cells via anti-Rha antibody-dependent antigen uptake process. Furthermore, peripheral blood mononuclear cells (PBMCs) stimulated with Rha-tMAGE-A3 in the presence of anti-Rha antibodies showed better cytotoxicity towards A375 human melanoma cells surfaced by MAGE-A3 antigen compared to PBMCs stimulated with tMAGE-A3. All data reveal that linking of Rha epitopes to MAGE enhances the immunogenicity of MAGE by harnessing the immune effector functions of human naturally existing anti-Rha antibodies. Rha epitopes could become immunogenicity enhancer of tumor associated antigens in the development of tumor immunotherapies.

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INTRODUCTION Melanoma-associated antigens (MAGEs) are a group of well-characterized cancer-testis antigens (CTAs) that are highly expressed in different types of tumor cells.1-3 MAGE-A3 antigen, one of these MAGEs, is strictly tumor specific and expressed in a large proportion of tumor types such as melanoma, myeloma, bladder carcinoma and lung carcinoma.4-6 Previous studies have shown that MAGE-A3 played a definite role in inducing tumor-killing effect though its antigenic epitopes recognition by specific cytotoxic T cells (CTLs).7-14 MAGE-A3 has drawn particular interest for tumor immunotherapy and already been the target of extensive clinical research, especially for melanoma and non-small cell lung cancer (NSCLC).15-18 However, tumor regression responses were observed in less than 30% of melanoma patients vaccinated with MAGE-A3 peptide or MAGE-A3 recombinant protein.3,15 In addition, the clinical study of MAGE-A3 associated NSCLC vaccine was recently suspended due to relatively weak patient responses. Thus, there is an urgent need to increase the immunogenicity of MAGE-A3 associated tumor vaccines. One avenue for boosting the immune responses to tumor antigens of interest involves linking of xenoantigens to them. The xenoantigen binds to relevant xenoantibodies and facilitates the uptake of the tumor antigens by antigen presenting cells (APCs). Anti-α-Gal antibodies are one such xenoantibody in human serum.19,20 Past studies have shown that installation of α-Gal as an xenoantigen on model vaccines HIV gp120 and flu increased the immunogenicity of vaccines 100-fold.19,21 Further, α-Gal epitopes would enhance the immune responses towards tumor cells that can express α-Gal epitopes on their surface.22,23 Compared

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with α-Gal, recent human serum studies have revealed that even more abundant human natural xenoantibodies are presented against monosaccharide L-rhamnose (Rha)24-27, which was an xenoantigen only found in some microbes and plants.28-32 Because of its natural abundance and structural simplicity, Rha has become a promising alternative in the development of tumor immunotherapies.33,34 In this work, we hypothesized that the immunogenicity of MAGE-A3 could be increased via conjugation with Rha epitopes, which could stimulate human serum Rha antibody-mediated antigen uptake process. To explore this hypothesis, the NHS-activated rhamnosyl donor was chemically synthesized followed by conjugation to truncated MAGE-A3 (tMAGE-A3) containing many immunodominant epitopes recognized by CTLs,35 to obtain a glycoprotein Rha-tMAGE-A3. Furthermore, the immune responses of Rha-tMAGE-A3 were investigated in the presence of human serum anti-Rha antibodies in vitro.

RESULTS AND DISCUSSION Purification of human serum anti-Rha antibodies. High anti-Rha titers pre-existing in human serum have shown potential in clinical applications. To facilitate its use in further in vitro research, we purified anti-Rha antibodies from human serum. To achieve this goal, we performed Rha-coupled affinity chromatography in which we purified 4 mg anti-Rha total antibodies (named as RGM) from 100 ml of human serum (Figure 1a). Further, we purified anti-Rha IgG (named as RG) from RGM via Protein G Mag Sepharose, with one heavy (~55

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KDa) and one light chain (~25 KDa) (Figure 1a). In addition, we performed ELISA experiments to evaluate the titers of our purified RGM and RG, respectively. As a result, expected high levels of anti-Rha IgG (1:12800) and IgM (1:800) were confirmed in RGM (Figure 1b). Meanwhile, high levels of anti-Rha IgG (1:12800) were detected in RG with almost no anti-Rha IgM (Figure 1c). In addition to these verifications, we did free monosaccharides competitive ELISA to investigate the specificity of RGM. In this experiment, only free Rha pulled down IgG (Figure 1d) and IgM (Figure 1e) from RGM whereas the other common monosaccharides did not. This indicated the antibodies including IgG and IgM were specific to Rha in RGM. All of these results suggested that our purified RGM and RG had high affinity and specificity to Rha epitopes, which could be used in in vitro immunological evaluations.

Figure 1. Evaluation of human serum anti-Rha antibodies. (a) Reducing SDS-PAGE analysis

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of purified human serum anti-Rha antibodies; (b) ELISA assays of anti-Rha IgG and IgM antibodies in RGM; (c) ELISA assays of anti-Rha IgG and IgM antibodies in RG; (d) Competitive ELISA assays of 7 common monosaccharides performed with IgG of RGM; (e) Competitive ELISA assays of 7 common monosaccharides performed with IgM of RGM. Rha conjugated BSA was used as immobilizing antigen, and free D-glucose (Glc), D-galactose (Gal), D-GalNAc, D-fucose (Fuc), D-mannose (Man), L-arabinose (Ara), and L-rhamnose (Rha) were used as competing antigens (2-fold dilutions from 100 to 6.25 mM). RGM: solutions containing anti-Rha total antibodies; RG: solutions containing anti-Rha IgG.

Synthesis of Rha-tMAGE-A3. After successful purification of human serum anti-Rha antibodies, we accordingly designed and synthesized L-rhamnosyl MAGE-A3 for in vitro anti-tumor immunological evaluation. Previous screening of immunodominant epitopes derived from MAGE-A3 showed a epitope peptide from aa 232 to aa 246 that contained Th epitopes, CTL epitopes and B epitopes.35 Based on these findings, the protein used in this study were truncated from aa 195 to aa 314 of MAGE-A3 (named as tMAGE-A3). The tMAGE-A3 has strong immunogenicity to elicit immune system and is more suitable for prokaryotic expression compared to the intact MAGE-A3. For the expression of rhamnosyl acceptor tMAGE-A3, we transformed E. coli BL21 (DE3) with pET15b-tMAGE-A3. Cultures were grown and induced as required followed by 6×-His-tagged tMAGE-A3 that were purified via Ni-NTA agarose and then characterized by anti-MAGE-A3 western blot (Figure 2a). To prepare Rha-tMAGE-A3, activated rhamnosyl donor 8 was required (Scheme

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1). Thiorhamnoside 3 was synthesized from L-Rha 1 via peracetylation and SnCl2 mediated glycosylation with PhSH. Then glycosylation of thiorhamnoside 3 and azido linker 4 provided intermediate 5, which was subsequently deacetylated and reduced to give amine 6. The reaction between 6 and succinic anhydride yielded regioselective amination product 7 with a terminal carboxylic acid group, which was then activated by TSTU to generate the N-hydroxysuccinimide (NHS) activated rhamnosyl donor 8, determined by mass spectrometry (MS) (Supplementary Figure S1). The spacers bearing a NHS ester could help Rha moieties be readily conjugated with multiple lysine residues on carrier proteins under mild physiological conditions.36 Following the synthesis of NHS-activated rhamnosyl donor 8 and acceptor tMAGE-A3, their ligations were carried out in 3×PBS for 1 h and quenched with ultrafiltration to remove the excess linkers. The conjugation results were also characterized by anti-MAGE-A3 western blot and anti-Rha western blot displaying a shift towards a higher mass of glycoprotein compared to unconjugated tMAGE-A3 (Figure 2a and b). The corresponding Rha moiety had a loading ratio of 9 by MALDI-TOF mass spectrometry analysis (Figure 2c). Considering the number of free amino groups in tMAGE-A3 was 12, the Rha loading efficiency on the tMAGE-A3 was about 75%. Scheme 1. Synthesis of NHS-activated rhamnosyl donor.

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Ac2O/Py

O

HO HO

AcO

OH

PhSH

O

AcO

SnCl2

OAc

O

AcO AcO

OAc

HO

O

N3

NIS/TfOH

AcO

O

HO HO

NH2

succinic anhydride

O

HO

6

2) Pd/C, NaBH 4

5

O CO2H

TSTU

O

HO

OH

N H

1) NaOMe/MeOH

OAc

O O

N3

O

AcO

3

2

1

4

SPh

OAc

OH

O

O

O

HO HO

OH

O O

N H

7

N

O

OH 8

Figure 2. Characterization of tMAGE-A3 and Rha-tMAGE-A3 by (a) anti-MAGE-A3 western blot, (b) anti-Rha western blot and (c) MALDI-TOF analysis.

Human

serum

anti-Rha

antibody

mediated

phagocytosis

of

FITC-labeled

Rha-tMAGEA3 by THP-1 cells. With the synthetic Rha-tMAGE-A3 and human serum

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anti-Rha antibodies ready, our first goal was to verify human serum anti-Rha antibody-mediated phagocytosis of Rha-tMAGE-A3 by macrophages. After co-incubation of FITC-labeled Rha-tMAGE-A3 (Supplementary Figure S2) and macrophage cell line THP-1 in the presence of RGM, the FITC-stained THP-1 cells (Figure 3a) increased more than that incubated with FITC-labeled tMAGE-A3 (Figure 3b). Meanwhile, anti-Rha antibodies were necessary to enhance the phagocytosis of Rha-tMAGE-A3 by THP-1 cells (Figure 3a and c). This might be because adding FITC-labeled Rha-tMAGE-A3 could result in in situ complexes of anti-Rha/Rha to effectively target FITC-labeled Rha-tMAGE-A3 to THP-1 cells through the interaction between the Fc portion of the anti-Rha antibodies and Fcγ receptors (FcγR) on THP-1 cells. All results suggested that Rha epitopes facilitated the uptake of Rha-tMAGE-A3 by APCs in the presence of human serum anti-Rha antibodies.

Figure 3. FITC-stained THP-1 cells detected by flow cytometry. THP-1 cells were incubated with (a) FITC-labeled Rha-tMAGE-A3 in the presence of RGM, (b) FITC-labeled tMAGE-A3 in the presence of RGM, (c) FITC-labeled Rha-tMAGE-A3 in the absence of RGM or (d) PBS control in the absence of RGM.

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In vitro killing of A375 tumor cells by stimulated PBMCs. Following the human serum anti-Rha antibody-mediated phagocytosis of Rha-tMAGE-A3 by macrophages, we hypothesized that these tMAGE-A3 specific antigen epitopes might be presented to T cells to generate tMAGE-A3 specific CTLs that could specifically kill tumor cells with MAGE-A3 antigens. To verify our hypothesis, the stimulated PBMCs and human tumor cell line A375 (surfaced by MAGE-A3 antigen) was co-cultured and then developed by CCK8. This facilitated a sensitive colorimetric assay for the determination of cell viability. The specific killing activity of the PBMCs stimulated with Rha-tMAGE-A3 against the A375 was much more obvious than that stimulated with tMAGE-A3 and PBS at effector:target ratios from 5:1 to 20:1 (Figure 4). These results showed that Rha epitopes of Rha-tMAGE-A3 helped to lyse tumor cells that expressed the MAGE-A3 antigen.

Figure 4. Cytotoxicity of stimulated PBMCs. PBMCs were stimulated with Rha-tMAGE-A3, tMAGE-A3 or none of them, such stimulated PBMCs were used to kill A375 cells. The ratios

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between effector PBMCs and target A375 were 5:1, 10:1, 20:1. Results were expressed as the arithmetic mean ± SD indicated by error bars. One-way ANOVA was used for analyses when three groups were compared. Differences were indicated with symbols (*: P

l-Rhamnose Enhances the Immunogenicity of Melanoma-Associated

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