以化學酵素法合成Globo H衍生物作為癌症疫苗用之研究

國立臺灣大學理學院化學所 博士論文

Department of Chemistry College of Science

National Taiwan University Doctoral Dissertation

以化學酵素法合成 Globo-H 衍生物 作為癌症疫苗用之研究

Chemoenzymatic synthesis of Globo H Analogues as haptens for cancer vaccine design

李信佑 Hsin-Yu Lee

指導教授：翁啟惠 博士、吳宗益 博士 Advisor: Chi-Huey Wong, Ph.D.

Chung-Yi Wu, Ph.D.

中華民國 104 年 2 月

February 2015

I

論文摘要

大部分癌症細胞表面會表現多量的特定醣抗原，Globo H (Fucα1→2Galβ1→

3GalNAcβ1→ 3Galα1→4Galβ1→ 4Glc)六碳醣是其中一種，以 Globo H 為基礎的 治療疫苗已經在應用在 乳癌、前列腺癌、卵巢癌、胰腺癌、大腸直腸癌及肺癌。

在這篇研究中，我們合成一系列 Globo H 衍生物以提升癌症疫苗的免疫性。Globo H 衍生物是由酵素法合成而來並和載體蛋白(CRM) 197 (DT)接合，並搭配 C34 醣脂類為佐劑(adjuvant)。在注射完 Balb/c 老鼠實驗，免疫的反應由醣晶片來分 析，結果顯示在 Globo H 還原端六號位上的 glucose 修飾氟化（F），疊氮基(N₃)，

苯基（phenyl)和在 Globo H 非還原端六號位上的 fucose 修飾疊氮基(N3)亦有強烈 的 IgG 免疫球蛋白反應辨認 Globo H 和 Gb5 和 SSEA4。上述的疫苗可以辨認 Globo H 表現的癌細胞(MCF-7)和具有補體依賴的細胞毒殺能力。由上述資料顯 示這樣的化學修飾癌症醣抗原可以促進癌症疫苗的發展。

II

Abstract

Globo H-based therapeutic cancer vaccines have been tested in clinical trials for the treatment of late stage breast, ovarian and prostate cancers. In this study, we explored Globo H analogue antigens with an attempt to enhance the antigenic properties in vaccine design. The Globo H analogues with modification at the reducing or non-reducing end were synthesized using chemoenzymatic methods, and these modified Globo H antigens were then conjugated with the carrier protein diphtheria toxoid cross-reactive material (CRM) 197 (DT), and combined with a glycolipid C34 as an adjuvant designed to induce a class switch of antibody to form the vaccine candidates. The immune response was studied by a glycan array and the results showed that modification at the C-6 position of reducing end glucose of Globo H with the fluoro, azido or phenyl group elicited robust IgG antibody response to specifically recognize Globo H (GH) and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4). However, only the modification of Globo H with the azido group at the C-6 position of the non-reducing end fucose could elicit strong IgG immune response.

Moreover, the antibodies induced by theses vaccines were shown to recognize GH expressing tumor cells (MCF-7) and mediate the complement-dependent cell cytotoxicity against tumor cells. Our data suggest a new potential approach to cancer vaccine development.

III

Abbreviations

Ac Acetyl

AcOH Acetic acid Ac2O Acetic anhydride

ADCC Antibody dependent cellular cytotoxicity Boc Tert-Butoxycarbonyl

Bn Benzyl

br broad(spectra)

BSA Bovine serum albumin

Bz Benzoyl

Cbz Benzyloxycarbonyl

CDC Complement dependent cytotoxicity CRM cross-reactive material

CSA Camphorsulfonic acid

CTL Cytotoxic T lymphocytes, CD8+ T cells

δ Chemical shift in ppm

DAST Diethylaminosulfur trifluoride

DC Dendritic cell

DCM Dichloromethane

DIPEA N,N-Diisopropylethylamine DMAP 4-dimethylamino pyridine DMF N,N-dimethylformamide

DT Diphtheria toxoid cross-reactive material (CRM) 197

EDCI 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

Equiv equivalent

Et Ethyl

Et3N Triethyl amine EtOAc Ethyl acetate

FACS Fluorescence-activated cell sorting Fmoc Fluorenylmethyloxycarbonyl

Fuc Fucose

g gram

Gal Galactose

GalNAc N-Acetylgalactosamine

GH Globo H

IV

Glu Glucose

GluNAc Acetylglucosamine

h hour

HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HRMS High resolution mass spectrometry

Hz Hertz

J Coupling constant

KH-1 Adenocarcinoma antigen KLH Keyhole limpet hemocyanin

Lac Lactose

Le Lewis

Lev Levulinate

Man Mannose

MALDI Matrix-assisted laser desorption/ionization

MBS m-maleimidobenzoyl-N-hydroxy succinimde ester

Me methyl

MeOH Methanol

MHC Major histocompatibility complex

MHz Mega hertz

mL milli litter

mol mole

mmol milli mole

MS Molecular sieves

NaOMe Sodium methoxide Neu5Ac 5-N-Acetylneuraminic acid NHS N-hydroxysuccinimide NIS N-iodosuccinimide

NMR Nuclear magnetic resonance spectroscopy

OVA Ovalbumin

PBS Phosphate buffered saline

Ph Phenyl

ppm parts per million

PSA Polysialic acid

Py Pyridine

rt room temperature

RFU relative fluorescence units

s singlet(spectra)

V SSEA Stage-specific embryonic antigen

sTn sialyl 2-6-α-N-acetylgalactosaminyl TACA Tumor associated carbohydrate antigen TBAF Tetra-n-butylammonium fluoride TBABr Tetrabutylamino bromide

TBAI Tetrabutylamino iodide

TBDPSCl Tert-Butylchlorodiphenylsilane

TCR T cell receptor

TF Thomsen-Friendenreich TFA Trifluoroacetic acid

TfOH Trifluoromethanesulfonic acid Th T helper or CD4+ T

THF Tetrahydrofuran

TLC Thin layer chromatography Tol p-methyl-phenyl

Troc 2,2,2-trichloroethoxyl carbonyl

TT Tetanus toxoid

VI CONTENTS

論文摘要... I

Abstract ... II Abbreviations ... III CONTENTS... VI CONTENT OF FIGURES ... VIII CONTENT OF TABLES ... XI

Chapter I General Introduction ... 1

1.1 Introduction ... 1

1.2. Carbohydrate-based vaccine ... 1

1.3. Immune response to carbohydrates ... 3

1.4. Tumor-associated carbohydrate antigens (TACAs) ... 5

1.5. Challenges of carbohydrate-based cancer vaccine development ... 8

1.6. Carbohydrate-based cancer vaccine ... 8

1.1.1 Classical approach ... 10

1.1.2. Fully synthetic carbohydrate-based cancer vaccines ... 14

1.7. Modification of carbohydrate antigens structures (MCAS) and cross-reactivity based immunotherapies ... 18

1.8. Synthesis of Globo H hexasaccharide... 20

1.9. Glycolipids as adjuvants ... 27

1.10. Glycan arrays are tools for passive immunization ... 30

Chapter II Immunogenicity study of Globo H analogues with modification at the reducing or non-reducing end of the tumor antigen ... 32

2.1. Introduction ... 32

VII

2.2. Results and discussion ... 36

2.2.1. Chemical synthesis of Globo H and analogues ... 36

2.2.1.1. Synthesis of lactose building block (18) ... 36

2.2.1.2. Synthesis of galactose building block (17) ... 38

2.2.1.3. Synthesis of GalNAc building block (16) ... 39

2.2.1.4. Synthesis of galactose building block (15) ... 40

2.2.1.5. Synthesis of fucose building block (12) ... 41

2.2.1.6. Synthesis of disaccharide (13) and trisaccharide (14) building blocks42 2.2.1.7. One-pot synthesis and Deprotection of Globo H (11) ... 43

2.2.2. Chemoenzymatic synthesis of Globo H and analogues ... 46

2.2.2.1. Synthesis of lactose building blocks (62 - 66) ... 46

2.2.2.2. Chemoenzymatic synthesis of Globo H-Lac analogues ... 49

2.2.2.3. Chemoenzymatic synthesis of Globo H-Fuc analogues ... 51

2.2.3. Synthesis of GH-Lac and GH-Fuc DT-conjugates ... 53

2.2.4. Glycan array analysis of the immunogenicity of the GH-Lac and GH-Fuc DT-conjugates ... 54

2.2.5. Binding studies of the mouse antisera induced by GH-Lac and GH-Fuc DT-conjugates to GH–expressing cancer cells ... 71

2.2.6. Complement-dependent cytoxicity (CDC) of the Mouse Antisera induced by GH-DT conjugates to MCF7 breast cancer cell ... 73

Chapter III Experiment section ... 75

References: ... 123

APPENDIX (1H & 13C NMR spectrums)………133

VIII

CONTENT OF FIGURES

Figure 1.1. A possible pathway of immune response to carbohydrate by

glycoconjugate. ... 4

Figure 1.2. Tumor associated carbohydrate antigens. ... 6

Figure 1.3. Globo H-KLH and Globo H-DT vaccine. ... 111

Figure 1.4. Tn-KLH trimeric clustered vaccine. ... 12

Figure 1.5. The unimolecular vaccine-1 displaying TF, Le^y and Tn. ... 13

Figure 1.6. The unimolecular vaccine-2 displaying globo H, Le^y and Tn. ... 14

Figure 1.7. The unimolecular pentavalent vaccine containing Globo H, GM2, STn, Le^y and Tn. ... 14

Figure 1.8. Structure of the dimeric Tn-Pam3Cys vaccine. ... 15

Figure 1.9. A two-component vaccine containing a T helper epitope and the MUC-1 peptde with STn. ... 166

Figure 1.10. Multiple antigenic glycopeptide trimeric Tn-PV. ... 177

Figure 1.11. Three-component vaccine was designed by Boons group. ... 188

Figure 1.12. a) S-linked GM2 and GM3 vaccine; b) C-linked STn vaccine; c) structures of fluorinated STn analogs vaccines ... 20

Figure 1.13. Chemoenzymatic synthesis of Globo H and SSEA4 with sugar nucleotide regeneration... 288

Figure 1.14. Structure of agelasphins (glycolipid) from the sponge ... 288

Figure 1.15. α-GalCer analogs. ... 30 Figure 1.16. Fabrication of carbohydrate microarrays and their applications for

IX biological and biomedical research. ... 31 Figure 1.17. Glycan-binding specificity profiling for the diagnosis of disease state or antibody validation... 31 Figure 2.1. Serum titers for C57BL/6 mice immunized with PBS (A), WT mTNF-α (B), pNO2Phe86mTNF-α(C), or Phe86 mTNF-α (D). ... 33 Figure 2.2. Structure of the GHN-BSA and N-BSA-GH. ... 34 Figure 2.3. Structure of the GH-Lac and GH-Fuc modified vaccines (1 DT-10 DT). . 35 Figure 2.4. The IgM antibody (200-fold dilution) elicited by GH-analogues DT

conjugates against (A) GH, (B) Gb5 and (C) SSEA4. ... 60 Figure 2.5. The IgG antibody elicited by GH-analogues DT conjugates against (a) GH, (b) Gb5 and (c) SSEA4. ... 59 Figure 2.6. The IgG subclasses distribution of GH-analogue DT-conjugates induced antibody against GH. ... 65 Figure 2.7. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-DT (1-DT). ... 66 Figure 2.8. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-F-DT (2-DT). ... 66 Figure 2.9. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-N3-DT (3-DT). ... 67 Figure 2.10. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-phenyl-DT (4-DT). ... 67 Figure 2.11. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-phenylNO2-DT (5-DT)... 68

X Figure 2.12. The glycan binding profiles of IgG (2000-fold dilution) collected from GH-NO2-DT (6-DT). ... 68 Figure 2.13. The glycan binding profiles of IgG (2000-fold dilution) collected from OH-GH-DT (7-DT). ... 69 Figure 2.14. The glycan binding profiles of IgG (2000-fold dilution) collected from N3-GH-DT (8-DT). ... 69 Figure 2.15. The glycan binding profiles of IgG (2000-fold dilution) collected from F-GH-DT (9-DT). ... 70 Figure 2.16. The glycan binding profiles of IgG (2000-fold dilution) collected from acetylenyl-GH-DT (10-DT). ... 70 Figure 2.17. GH-analogues DT conjugates-induced mouse antibodies recognize GH expressing tumor cell (MCF-7). ... 71 Figure 2.18. Staining of anti-SSEA3 (Gb5), anti-SSEA4 and anti-Globo H antibodies (red) against GH-negative A375 melanoma cells. ... 72 Figure 2.19. Antibodies elicited by GH analogues mediate complement-dependent cytotoxicity (CDC) to eliminate GH-expressing tumor cells……….….73 Figure 3.1. Serum IgG antibodies response against GH in immunized mice with GH-analogues DT/C34. ... 117 Figure 3.2. Serum IgG antibodies response against Gb5 in immunized mice with GH-analogues DT/C34. ... 118 Figure 3.3. Serum IgG antibodies response against SSEA4 in immunized mice with GH-analogues DT/C34. ... 119

XI

CONTENT OF TABLES

Table 1.1. Existing polysaccharide and conjugate vaccines on the market. ... 3 Table 1.2. Summary of common patterns of tumor associated carbohydrate antigens on malignant tissues. ... 7 Table 1.3. Examples of carbohydrate-based cancer vaccines ... 9 Table 2.1. MALDI-TOF analysis of average carbohydrate incorporation. ... 54 Table 2.2. 96 kinds of other home-made oligosaccharides and functional linkers. ... 555 Table 3.1. Mice serum of IgG and IgM antibody titers. ... 120

1

CHAPTER 1 General Introduction 1.1 Introduction

Carbohydrates are the most abundant and constitute one of the major classes of biomolecules, together with proteins and nucleic acids. They are energy sources in cells and structure elements in the cell wall of bacteria and plants. Carbohydrates also play important roles in many areas of biological sciences, including cell-cell recognition and communication, bacterial and viral infection, toxin interaction, tumor metastasis, inflammatory and immune reactions.¹ In most cases, complex carbohydrates are covalently attached to a protein or a lipid to form a glycoconjugate.

1.2. Carbohydrate-based vaccine

Carbohydrates have been demonstrated as potential therapeutic agents. For example, Oseltamivir (known as trade name Tamiflu), a sialic acid-mimic prodrug, is an anti-infective agent to prevent bacterial binding to host cells.² Heparin, a highly sulfated glycosaminoglycan, is an anticoagulant, preventing the formation of clots within the blood.³ The unusual carbohydrate antigens on the surface of pathogen are used as potential vaccine.⁴ The polysaccharides were first used as vaccine in the late 1940s against a number of bacteria pathogen.⁵ These pathogens have cell surface capsular polysaccharides or lipopolysaccharides cell, which helps to infect host cells.

These polysaccharides mask the cell surface of pathogen that cannot be attacked by the host immune system and phagocytosis. Thus, antibodies specifically bind to the polysaccharides on the surface of bacteria could be a way to improve elimination of the pathogens.

In 1983, the first commercial polysaccharide vaccine, PneumoVax, is extracted

2 from Streptococcus pneumoni 14 seroypes and is consist of unconjugated capsular polysaccharide.⁶ Some of capsular polysaccharides vaccine have been discovered and approved against Neisseria meningitides A, C, W135 and Y and Salmonella typhi.⁷ The poor immunogenicity to polysaccharides is the major obstacle of the carbohydrate-based vaccine. To overcome the T-cell independent properties of polysaccharides, capsular polysaccharides are conjugated to a strong immunogenic carrier protein, such as diphtheria toxoid (DT), diphtheria toxoid cross-reactive material (CRM197), tetanus toxoid (TT), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).

Avery and Goebel first demonstrated that pneumococcal polysaccharides conjugate to an immunogenic carrier protein to improve the immunogenicity of carbohydrates.⁸ This vaccine can induce long-term protection of T-cell dependent immune response against pathogens. So far, four types of the glycoconjugates vaccines, such as Haemophilus influenzae type b, Neisseria meningitidis, Salmonella enterica, Serovar Typhi and Streptococcus pneumonia, have been used in patients preventing bacterial infection (Table 1.1).⁹ Moreover, many bacterial glyconconjugate vaccines are ongoing in clinical trails against Staphylococcus aureus, Shigella sonnei, Shigella flexneri and others. The success of this glycoconjugates approach has been widely used to develop other vaccine, such as antiviral and anticancer vaccines.¹⁰

3 Table 1.1. Existing polysaccharide and conjugate vaccines on the market.^a

aP: polysaccharide vaccine, C: conjugate vaccine; CPS: capsular polysaccharide;

OMPC: outer membrane protein complex (derived from Neisseria meningitides); DT:

diphtheria toxin; CRM197: diphtheria toxin mutant; and TT: tetanus toxid.

1.3. Immune response to carbohydrates

The possible mechanism of immune response to carbohydrate by glycoconjugate is showed in Figure 1.1.^11,12 When the glycoconjugate vaccine is injected into the human or mice, the glycoconjugate is captured by PRRs (pattern recognition receptors) on the surface of APCs (antigen presenting cell), such as DC (dendritic cells) and marcophages. After APC has phagocytosed glycoconjugate to form small glycopeptides, it migrates to the draining lymph nodes. In the lymph nodes, the small glycopteides is reacted to the TCR (T cell receptor) of T cell using MHC (major

4 histocompatibility complex) class I or II on the DCs. The Th cells (T helper or CD4+

T cells) are activated by MHC class II on the DCs and are essential in B cell antibody IgG class switching. The cytotoxic T lymphocytes (CTL or CD8+ T cells) are also activated by MHC class I on the DCs with the help of Th cells. The CTLs destroy target cells infected by pathogen such as bacteria or viruses. The activated-Th cells bring glycopeptide on the TCR and reacted with MHC II on the surface of B cells.

The interaction between Th cells and B cells are then stimulated with CD40L on the surface of Th cells, which bind CD40 on the B-cell leading cytokine production by Th cells. The binding combination and cytokine stimulation result in B cell proliferation, differentiation into memory B cells and plasma cells that secrete antibodies specifically against carbohydrate epitope. The secreted antibodies kill cancer cells through two major pathways: complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC).

Figure 1.1. A possible pathway of immune response to carbohydrate by glycoconjugate (Glycoconj J 2012, 29, 259-271).¹³

5

1.4. Tumor-associated carbohydrate antigens (TACAs)

The abnormal glycosylation has been shown to play a key role in tumor progression and metastasis, and is correlated with the survival rates of cancer patients.^14,15 Tumor-associated carbohydrate antigens are over-expressed and found on the surface of the tumor cells and include altered carbohydrate structures and expression on the glycoprotein and glycolipids (Table 1.2). The tumor-associated carbohydrate antigens (TACAs) were extracted from tumor cells and identified by NMR, mass spectrometry and monoclonal antibodies.^16-18 So far, quite a few TACAs have been found (Figure 1.2).

Tumor associated carbohydrate antigens can be classified into four groups that are globo series, lacto series, ganglio series and mucin core. The first group of TACAs is globo series that includes Globo H, Gb5, Gb3 and SSEA4. Globo H, known as the Mbr-1 antigen, isolated as a glycolipid from breast cancer MCF-7 cells.^19-21 Globo H has been found over expressed in many types of human cancer cells including breast, prostate, ovary, pancreas, brain, endometrium, gastric, colon and lung cancers.^22-24 The second group of TACAs is lewis blood type (or lacto series) antigens such as Le^y, sLe^X, Le^a, sLe^a and KH-1 (adenocarcinoma antigen). sLe^a (known as CA19-9) is expressed on glycolipids and glycoprotein and is a marker to monitor patients with pancreatic, colorectal and gastric cancer.^25,26 sLe^x is an adhesion molecule and relate to promote tumor metastasis.^27,28

6

O HO

AcHN OH HO

Mucin-core

OSer/Thr

O HO

AcHN O HO

OSer/Thr

O HO

AcHN OH O

OSer/Thr O

COOH

HO AcHN

OH OH HO

O HO

OH OH HO

Tn sTn TF

O O O

HO

HO HO OH

OH OH

O HO

HO OH

O O HO

NHAc OH

O O

HO

HO O OH

O

O OH

HOOH

O

O O

HO

HO HO OH

OH OH

O OH

HO OH

O O HO

NHAc O

O O

HO

O OH OH

O O

COOH

HO AcHN

OH OH HO

O COOH

AcHN OH HOHO HO

Globo-series

Globo H DSGG

O NHAc OH O HO

O OH OH

O O

COOH

AcHN OH HOHO HO

O O

HOOH HO

sLe^a

O NHAc OH O HO

HO OH OH

OO O

HOOH HO

Le^a

O NHAc OH

OO

sLe^x

O HO

O OH OH

O COOH

AcHN OH HOHO

HO O OH

HOOH O

NHAc OH

OO

Le^y

O HO

HO O OH

O OH

HOOH

O OH

HOOH

O O O

O

HO HO OH

OH OH

O

Fucosyl GM1

O HO OH O HO

HO O OH

O

O OH

HOOH

AcHN

O COOH

OH AcHN HOHO

HO

O O O

O

HO HO OH

OH OH

O

GM2

O HO OH

HO AcHN

O HOOC

OH AcHN HOHO

HO

O

O O

O

HO HO OH

OH OH

O

GD2

O HO OH HO

AcHN

O HOOC

OH AcHN OHO

HO O HOOC

AcHN HO HOOH HO

O

O O

O

HO HO OH

OH OH

OH

GD3

O HOOC

OH AcHN OHO

HO O HOOC

AcHN HO HO OH HO

Lacto-series

Ganglio-series

O COOH

HO AcHN

OH OH HO

O COOH

HO AcHN

OH O HO

O COOH

HO AcHN

OH O HO

n=10 ~50

PSA

Figure 1.2. Tumor associated carbohydrate antigens. The carbohydrate antigens mentioned above could be classified into four groups that are globo series, lacto series, ganglio series and mucin core.

The third group of TACAs is ganglio series, such as GM2, GD2, GD3 and fucosyl GM1, isolated as glycolipids from human melanomas and neuroblastoma but is also detectable on normal cells. GD2 is overexpressed on the surface of neuroblastoma, melanoma, and small cell lung cancer and brain tumors. GD2 antibody (Dinutuximab) has finished phase III trail.²⁹ It is ongoing review in the EU as a treatment for neuroblastoma. The last group of TACAs is glycan on the mucins,

7 which are a family of high molecular weight and densely glycosylated protein produced by epithelial tissue. The abnormal glycans Tn, STn and Thomsen-Friedenrich (TF) are linked to peptide backbone of mucins.³⁰ They are found on breast, colon, prostate, lung or stomach cancer. STn (known as CA72-) is used to monitor gastric carcinoma.³¹ Mucins also can be diagnosis markers for clinical patients. For example, MUC-1 (known as CA15-3) is a transmembrane mucin overexpressed and found in more than 90% patients with breast cancer.³² MUC-1 is used to check prognosis and recurrence of patients with breast cancer.

Table 1.2. Summary of common patterns of tumor associated carbohydrate antigens on malignant tissues.

Globo H, globo hexaosylceramide; Le, Lewis; PSA, polysialic acid; s, sialyl; TF, Thomsen–Friedenreich; Tn, 2-6-α-N-acetylgalactosaminyl.

8

1.5. Challenges of carbohydrate-based cancer vaccine development

Although glycoconjugates vaccines have been achieved, there are some problems need to be addressed.^4,33 The first problem is the source of carbohydrates. Initially, the carbohydrates of interest are usually extracted from the pathogens or tumor cells and are hard to get desired quantities to do further experiment. Next, the heterogeneity of the carbohydrates from extraction is difficult to get the pure form carbohydrate. Thus, the scientist relies on the chemical synthesis method to get desired carbohydrate antigens. The chemists can synthesize carbohydrate antigen in large scale (milligrams to kilograms) with high purity. A lot of carbohydrate antigens with functional group in the reducing end are synthesized and can linked to carrier proteins, glycan array or others uses.

The second problem is the poor immunogenicity of carbohydrates that are T-cell independent antigens. These carbohydrates induce the production of short-living IgM antibodies without the production of high affinity IgG antibodies. Moreover, The TACAs is observed as self-antigen by immune system because they are present trace quantities on the surface of normal cells. Therefore, the robust IgG immune response against TACAs has been demonstrated more challenging than the robust IgG immune response against viral and bacterial carbohydrate antigens. Many scientists have discovered several approaches for improvement of the immunogenicity of TACAs.

After a brief introduction of the immune response to carbohydrates and TACAs, some carbohydrate-based cancer vaccine will be described later.

1.6. Carbohydrate-based cancer vaccine

The classical treatment methods for cancers are surgery, radiotherapy and chemotherapy. Recently, cancer immunotherapy has the chance to become the next

9 generation of cancer therapy. The development of cancer vaccines is initially from taking tumor cells from patients, inactivating tumor cells and re-injecting in the patients with an attempt to elicit the immune response against cancer cells.³⁴ However, most of these experiments were failed because of the poor knowledge of cancer cell.

In the following years, it is found that the unique carbohydrate antigens located on the surface of cancer cells play important role for the development of cancer vaccine (Table 1.3). Moreover, a better understanding of the immune system could help us discover carbohydrate-based cancer vaccine.³⁵

Table 1.3. Examples of carbohydrate-based cancer vaccines

10

1.1.1 Classical approach

TACAs on the surface of cancers provide potential targets for the development of carbohydrate-based cancer vaccine. Based on the successful study of bacterial carbohydrate antigens,⁹ classical carbohydrate-based cancer vaccines are also including the conjugation of a TACA to a T-cell-dependent carrier protein, such as diphtheria toxoid (DT), diphtheria toxoid cross-reactive material (CRM197), tetanus toxoid (TT), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).³⁶ Moreover, these vaccines often combine with an adjuvant for enhancement the immunogenicity.

Several adjuvants are found, such as Alum, BCG, CpG, MPLA, QS-21 and so on.³⁷ Gangliosides-protein conjugate cacncer vaccines are the pilot study.³⁸ The Helling group found that the GD3 linked to KLH with administration the adjuvant QS-21 elicit IgM and IgG immune response that could mediate complement dependent cytoxicity to GD3-expressing human melanoma cells.³⁹ Some of ganglioside-KLH conjugate have shown potential in clinical trials, especially GM2-KLH (known as GMK).⁴⁰ GM2-KLH is tested in Phase III clinical trials to prevent relapse of malignant melanoma in high-risk patients, but it is failed to show effectiveness of relapse-free survival over observation.⁴¹

Several keyhole limpet hemocyanin (KLH) monovalent vaccines are synthesized and studied, such as Globo H-KLH,^42,43 PSA-KLH,⁴⁴ Le^y-KLH,^45,46 Le^x-KLH,^47,48 Le^b-KLH, Tn-KLH,⁴⁹ STn-KLH^50,51 and TF-KLH⁵². One of the most promising glycoconjugates is Globo H-KLH. It is found that Globo H-KLH adjuvanted with QS-21 induced high-titer IgM and weak IgG antibodies response to Globo H (Figure 1.3.a). Antisera induced by this vaccine could bind to GH-expressing MCF7 human breast cancer cell lines and mediate the complement-dependent cell cytotoxicity

11 against tumor cells.⁴³ With improvement in synthesis,^53,54 it is now in phase III clinical trial in Taiwan and phase II clinical trial in USA, Korea, Hong-Kong and India for late stage breast cancer patients and in phase II clinical trial for ovarian cancer patients in Taiwan. However, these early stage clinical results showed that the induced IgM antibodies were still much higher than IgG antibodies.^43,55,56 Recently, our group developed a better vaccine using diphtheria toxoid cross-reactive material (CRM) 197 (DT) as carrier and a glycolipid C34 as adjuvant to induce a class switch with robust IgG antibody response against GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4), all found on breast cancer cells and the cancer stem cells only (Figure 1.3.b).²³

O OH HO OH O

O

O O

O OH OH

HO

OH HO OH O

HO

HO OH

O O

O HO OH

NHAc O

HOOH OH

O N

H HN

O

N O

O S KLH

Globo H-KLH

(a)

O OH HO OH O

O

O O

O OH OH

HO

OH HO OH

O HO

HO OH

O O

O HO OH

NHAc O

HOOH OH

Globo H-DT

DT 4

O N

H

O O

N 4 H

Figure 1.3. (a)Globo H-KLH and Globo H-DT vaccine. (b)Structures of the globo

12 series glycosphingolipisd Globo H, Gb5 (SSEA3) and SSEA4 (R = ceramide)

The linkage between the glycan antigen and the carrier protein is a key factor for the carbohydrate-based cancer vaccines. Most of classical monoepitope glycoconjugates are linked through an allyl linker that after ozonolysis to get an aldehyde group, which direct coupled with a carrier protein by reductive amination.

Another conjugation approach is to use carbohydrate antigen with thiol linker that can be coupled to a maleimide modified carrier protein. So far, some linkers between the carrier protein and the carbohydrate antigen are recognized by the immune system and suppres the antibodies response against the TACAs.^57,58 Thus, Linkers that are not immunogenic could be used for next generation of carbohydrate-based cancer vaccines.

For enhancement of immunogenicity against cancer, monomeric clustered vaccines are discovered. These trimeric clustered vaccines include same TACA unit linked to a peptide backbone, then conjugated to a carrier protein, such as Tn(c)-KLH,⁵⁹ TF(c)-KLH,⁵² STn(c)-KLH⁶⁰ and Le^y(c)-KLH⁶¹. All conjugate are conjugated to KLH via the heterobifunctional linker m-maleimidobenzoyl-N-hydroxy succinimde ester (MBS). So far, Tn(c)-KLH and TF(c)-KLH with adjuvant QS-21 treat for prostate cancer patients in phase I clinical trials and induced high titer IgM and IgG antibody response (Figure 1.4).^52,59

Figure 1.4. Tn-KLH trimeric clustered vaccine.⁵⁹

13 The other idea is the combination of several monomeric vaccines to induce a broader immune response. The pool monomeric-KLH vaccines (GM2, Globo H, Ley, TF(c), Tn(c), STn(c) and glycosylated MUC1) with adjuvant QS-21 treat epithelial cancer patients and induce low concentration of IgM and IgG antibody response.^62,63 The antibody titers for all antigens are significantly reduced in comparison to the corresponding monomeric vaccine. The result can be explained due to the large amount of carrier protein (KLH) in the study. It is found that high immunogenicity of the protein suppress immune response against the TACAs.

One approach to improve the immune response to TACAs is to use a unimolecular polyvalent vaccine to minimize the amount of protein. The first unimolecular vaccine-1 containing Tn, Le^y and TF, α-O-linked to serine sidues was synthesized and conjugated to KLH through MBS linker (Figure 1.5).⁶⁴ Another unimolecular vaccine-2 containing Tn, Le^y and Globo H, α-O-linked to hydroxynorleucine residues was synthesized and conjugated to KLH through MBS linker (Figure 1.6).⁶⁵ The immune result showed unimolecular vaccine-2 induced higher IgM and IgG antibodies than unimolecular vaccine-1. The antisera induced by both vaccines reacted with tumor cells known to express each TACAs selectively.

Figure 1.5. The unimolecular vaccine-1 displaying TF, Le^y and Tn.⁶⁴

14 Figure 1.6. The unimolecular vaccine-2 displaying globo H, Le^y and Tn.⁶⁵

Then, an elegant pentavalent unimolecular vaccine containing five TACAs was synthesized to study immune response. This vaccine containing Globo H, Le^y, Tn, TF and STn antigen, α-O-linked to hydroxynorleucine residues was synthesized and conjugated to KLH through MBS linker (Figure 1.7).⁶⁶ This vaccine adjuvanted with QS-21 induced IgM and IgG antibodies against each of the five TACAs and react to cancer cells overexpressing these antigens.⁶⁷

Figure 1.7. The unimolecular pentavalent vaccine containing Globo H, GM2, STn, Le^y and Tn.⁶⁷

1.1.2. Fully synthetic carbohydrate-based cancer vaccines

The approach of smaller fully synthetic carbohydrate vaccines is a viable way to enhance the immune response against TACAs. These vaccines contain TACAs

15 conjugated to T cell epitope or TLR ligand, which activates APCs to elicit the immune response against carbohydrate antigen. The first example reported by Toyokuni group is a two-component vaccine that contains a dimeric Tn antigen and Pam3Cys adjuvant (Figure 1.8).⁶⁸ The antisera induced by this vaccine induce high titer IgM antibodies against Tn antigen by ELISA assay. Professor Danishefsky is also interested in fully synthetic two-component vaccines. Some vaccine containg monomeric Le^y, trimeric Le^y61 or trimeric Tn⁶⁹ linked to Pam3Cys adjuvant. After immunization with adjuvant QS-21 in mice, the immune result showed only IgM antibodies but no IgG antibodies are induced these vaccines. Based on result above, the IgM immune response is induced by these vaccines without the presence of a carrier protein. For the purpose to induce a class switch to IgG antibodies, some Th epitopes are used to some carbohydrate-based vaccine.

Figure 1.8. Structure of the dimeric Tn-Pam3Cys vaccine.⁶⁸

Some vaccines include TACAs and Th epitope to enhance the immune response against carbohydrate antigen. The two-component vaccine designed by Kunz group containing the STn antigen on a MUC-1 peptide linked to Th epitope (OVA323-339) induced high titer IgG antibodies to specifically recognize glycosylated MUC-1 peptide (Figure 1.9).⁷⁰ Interestingly, it was found that STn-MUC-1 peptide conjugated

16 to a carrier protein Tetanus Toxoid (TT) elicited higher titer immune response than two-component vaccine described above.⁷¹

Figure 1.9. A two-component vaccine containing a T helper epitope and the MUC-1 peptde with STn.⁷⁰

A similar vaccine contains a multiple antigenic glycopeptide (MAG) that including several copies of TACAs and Th epitope was synthesized to enhance the immune response against carbohydrate antigen. For example, this MAG vaccine contains Tn antigen linked to Th epitope PV (derived from polio virus) through a non-immunogenic polylysine backbone (Figure 1.10).⁷² This vaccine with adjuvant QS-21 was tested in mice and nonhuman primates and induced high IgG titiers against Tn and improve the survival rate of xenograft mice.⁷³

Professor Dumy discovered a regioselectively addressable functionalized template (RAFT) that is a decapeptide containing of proline, glycine and lysine amino acids to linked Tn antigens through an oxime linkage on the side chain of lysine.⁷⁴ This vaccine induced high IgG titers against Tn antigen on tumor cells but no peptide backbone and oxime linkage.

17 Figure 1.10. Multiple antigenic glycopeptide trimeric Tn-PV.⁷²

After the great advance of the two-component vaccine, the three-component vaccine was designed and synthesized by Boons group in 2005.⁷⁵ The three-component vaccine contained a B cell epitope Tn, a Th epitope YAF derived from an outer-membrane protein of Neisseria meningitides and a built-in adjuvant Pam3Cys (Figure 1.11). This vaccine was formed liposome to study the immunogenicity in the mice. The result that weak IgG antibodies were induced by this vaccine with or without adjuvant QS-21. Next, the optimized three-component vaccine contained a glycosylated MUC-1 peptide with Tn antigen as B cell epitope, a Th cell epitope derived from polo virus and a built-in adjuvant Pam2CysK4 or Pam₃CysK₄. These two vaccine were formed as liposome to study the immunogenicity in the mice.⁷⁶ The result showed that both two vaccines induced robust IgG antibodies to recognize MUC-1 peptide with Tn antigen. The vaccine containing the Pam3CysK4 unit could induce stronger immune response than the other vaccine containing the Pam₂CysK₄ unit. The further study showed that the vaccine

18 containing the Pam₃CysK₄ unit elicited CTLs and ADCC.⁷⁷

Figure 1.11. Three-component vaccine was designed by Boons group.⁷⁶

In 2008, professor Dumy reported that the four-component vaccine containing Tn linked on RAFTs, a Th cell epitope PADRE, a CTL epitope OVA257-264 and palmitic acid was synthesized.⁷⁸ This water soluble vaccine was studied in mice and induced strong Th and CTL immune response and resulted in the reduced of the tumor sized on the mice inoculated with MO5 cells.

1.7. Modification of carbohydrate antigens structures (MCAS) and cross-reactivity based immunotherapies

Previous studies showed that modification of carbohydrate antigen structures (MCAS) could effectively elicit a higher level of immune response.^13,79 For example, in the modification study of the capsular polysaccharide of group B meningococci, the N-acetyl groups of α-(2,8)-linked polysialic acid (PSA) was replaced with the N-propinoyl group and was then conjugated into tetanus toxoid (TT).^44,80 This modification elicited a high antibody response to recognize not only the N-propinoyl PSA, but also the native N-acetyl PSA and bactericidal immune response in mice.⁸¹

Another case GD3 lactone was synthesized and then conjugate to KLH to overcome the weak immunogenicity of GD3. The clinical results showed that GD3

19 lactone-KLH induce higher IgG and IgM antibody response than the native form GD3-KLH. The serum induced by GD3 lactone-KLH could lyse GD3-expressing SK-Mel-28 cells.⁸² Similar approach was applied to GD2 and GD2 lactone-KLH also has good result in patients with melanoma.⁸³

The modification of glycosidic bonds between glycans is the way to improve immunogenicity. The O-linked of oligosaccharides was replaced with the S-linked of oligosaccharides and was then conjugated into carrier protein. S-linked GM3,⁸⁴ GM2,⁸⁴ trimannose⁸⁵ and tetra mannose⁸⁶ were synthesized by Bundle and co-workers and incorporated into TT (Figure 1.12.a). The result showed that no significant immune response between the modified form and native antigen. C-linked STn is synthesized by Linhardt lab and the sera induce by these modified antigen have moderate immune response to the native STn antigen (Figure 1.12.b).^87,88

Professor Ye is also interested in the modification of carbohydrate antigens structures and immunotherapies. Quite a few structurally modified STn were synthesized and these antigens were screened using competitive ELISA assay.⁸⁹ Some fluoro-containing analogues on the STn can significantly increase immune response to recognize the native STn antigen and the antisera can bind to the STn-expressing cancer cells (Figure 1.12.c).

20

O O

COOH

HO HO

AcHN HO

HO

OHOH

AcHN OH

HN a)

O O O

X

HO HO OH

OH OH

RO

O HOOC

OH AcHN HOHO

HO

O

OH HN

O H

N

X = O or S R = H or D-GalNAc

b)

O O O

COOH

HO HO

R₂ O HO

HO

OHOH

R₁

n c)

R₁=AcNH, R₂=CH₂FCONH R₁=AcNH, R₂=CHF₂CONH R₁=R₂=CH₂FCONH

KLH KLH

n TT

n

Figure 1.12. a) S-linked GM2 and GM3 vaccine are prepared by Bundle group⁸⁴; b) C-linked STn vaccine is synthesized by the Linhardt group^87,88; c) structures of fluorinated STn analogs vaccines that induced high immune responses are synthesized by the Ye group⁸⁹.

In the modification study of MUC-1 glycopeptide containing TF antigen, The C-6 and C-6’ position hydroxyl group of TF antigen was replaced with fluorine group and was conjugated to tetanus toxoid (TT).⁹⁰ These vaccines elicit IgG immune response and recognized the native MUC-1 antigen present on MCF7 breast cancer cell. Moreover, the Schultz group reported that incorporation of a p-nitrophenylalanine into the tumor necrosis factor-α (TNF-α) could break immune tolerance and induces more antibody response to TNF-α.^91,92

1.8. Synthesis of Globo H hexasaccharide

Globo H (GH; Fucα1→2Galβ1→3GalNAcβ1→3Galα1→4Galβ1→4Glc) is a member of the globo series glycosphingolipids. It was first found and characterized in

21 human teratocarcinoma cells and breast cancer MCF-7 cells in 1983,^19,20,22 and was subsequently found overexpressed in many types of human cancer cells including breast, prostate, ovary, pancreas, brain, endometrium, gastric, colon and lung cancers.^21,23,24 Because of its biological importance as a tumor marker and potential anticancer vaccine, Globo H has been target for scientists. Since the difficulty of isolating Globo H from natural sources, total synthetic approach was the viable way to get homogenous Globo H. It was first synthesized in 1995 by Danishesky and co-workers using glycal assembly strategy,^93,94 Schmidt et al. reported the synthesis of Globo H using trichloroacetimidate methodology.⁹⁵ A two directional glycosylation strategy was published by Boons and co-workers.⁹⁶ The synthesis of Globo H was prepared via a one-pot strategy by our group using computer program OptiMer.^53,97 Many other chemical methods have also been explored, such as linear synthesis,⁹⁸ automated solid phase synthesis⁹⁹ by Seeberger and co-workers, multi-component one-pot synthesis and chemoenzymatic synthesis approach.¹⁰⁰

In 1995, Danishesky and co-workers discovered the first total synthesis of globo H using glycal assembly approach (Scheme 1.1).⁹⁴ The glycal assembly approach uses the glycan building blocks, possessing three hydroxyl groups and an olefinic handle, that server to build complex carbohydrates. A retrosynthetic divided into A3 and A9.

A3 was synthesized from acceptor A2 and donor A1 using Mukaiyama Nicolaou condition to get 54 % yiels of A3. A9 was synthesized from acceptor A7 and donor A5 with methyl triflate. It was feasible to synthesize from A9 to A10 via iodosulfonimidation and function group rearrangement. The [3+3] glycosylation strategy was perform with methyl triflate to get 10: 1 mixture of α;β. Treatment of A11 with 3,3-dimethyldioxirane followed by coupling of the epoxide with lipid A12 under zinc chloride, after acetylation, to get A13. After general deprotection, the

22 Globo H ceramide was get. In the following years, they disclosure a second-generation synthetic strategy, which gives easily access to significant quantities of materials.¹⁰¹

O HO BnO

OBn

OBn

O O

OBn O

BnO PMBO

OBn

OBn F

BnO O

O BnO

OBn

OBn

O O

OBn

BnO O

BnO HO

OBn

BnO

O OTIPS O O O

O OTIPS HO HO

O OTIPS O O

O O

OTIPS HO O OH

O OBn BnOOBn

F

O OTIPS O O

O O

OTIPS HO O O O OBn BnOOBn

O OTIPS O O

O O

OTIPS HO O O O OBn BnOOBn

NHSO2Ph SEt

A1 A2 A3

A5

A6

A7

A8

A9

A10 d

b c

a

e

n-Bu3SnO (CH2)12Me OBn

N3

O O

BnO OBn

OBn

O O

OBn

BnO O

BnO O

OBn

BnO O

OTIPS O O

O O

OTIPS HO O O O OBn BnOOBn

Ph2OSHN

A11

O O

BnO OBn

OBn

O O

OBn

BnO O

BnO O

OBn

BnO O

OTIPS O O

O O

OTIPS HO O O O OBn BnOOBn

Ph2OSHN

A13

O (CH2)12Me OBn

N3

OAc

g

Globo H ceramide f

A3

A12

Scheme 1.1. Synthesis of Globo H using glycal assembly.⁹⁴ (a) (i) SnCl₂, AgClO₄, DTBP, Et2O, 54% (α:β = 3: 1); (ii) DDQ, CH2Cl2, H2O, 84%; (b) (i) DMDO, CH2Cl2; (ii) ZnCl2, THF, 87%; (c) SnCl2, AgClO4, di-tert-butylpyridine, Et2O, 47%; (d) (i) I(coll)2ClO4, PhSO2NH2, THF, 47%; (ii) EtSH, LiHMDS, DMF, 75%; (e) MeOTf, 70%～85% (α:β = 1 : 10); (f) (i) DMDO, CH2Cl2; (ii) Zn(OTf)2, THF, 20%; (iii) Ac₂O, Et₃N, DMAP, CH₂Cl₂, 95%; (iv) Lindlar’s catalyst, H₂, palmitic anhydride, EtOAc, 90%; (g) (i) TBAF, THF; (ii) NaOMe, MeOH, 94%; (iii) Na, NH₃, THF; (iv) Ac₂O, Et₃N, DMAP, CH₂Cl₂, 80%; (v) NaOMe, MeOH

23 Another effective synthesis of Globo H was reported by our group using programmable one-pot approach (Scheme 1.2).¹⁰² This approach uses a computer program called ‘‘OptiMer’’ to list the appropriate tolylthioglycoside building blocks from a database for the one-pot synthesis. With this strategy, oligosaccharides are rapidly assembled in minutes or hours without intermediate workup and purification.

To perform a significant reactivity difference between the glycosides and control of the stereoselectivity of each glycosylation, the approach for the synthesis of protected-Globo H A17 involved the use of two one-pot reactions.⁵³ The first one-pot reaction is to build the two β-linkages to form the building block A15. The other one-pot reaction is to build the two α-linkages using three building blocks A14 (RRV

= 7.2 x 104), A15 (RRV = 6) and A16 (RRV = 0) and the yield is 62 %. The reactivities are mainly regulated by electron-donating groups (benzyl ether and 2,2,2-trichloroethylcarbamate) and electron-withdrawing groups (benzoyl, p-nitrobenzoyl and o-chlorobenzyl ethers).

With further refinement, [1+2+3] one-pot strategy improve the yield of the synthesis globo H from 62% to 83% and the challenging Gal α(1→4) linkage is formed (Scheme 1.3).⁹⁷ Therefore, I followed this efficient one-pot synthetic approach with an attempt to synthesize the Globo H and its analogues described later in chapter II. The synthetic glycan analogues can be used to develop the carbohydrate based vaccine and glycan microarray.^23,103

24

O (ClBn)O (BzN)O

O

O O O

OPMB O OBn

BnO

BnO BnO OBn O

BnO BnO

OBn

O

O O

BzOOBz

TrocHN O OBn O OBn

STol

BnOOBn A14 (RRV = 7.2 x 10⁴)

A15 (RRV = 6)

O O O

OPMB OH

BnO

BnO BnO OBn A16 i

62 % ii

A17 O O

BnO BnO

OBn

OH O

O BzOOBz

TrocHN

O(NBz)

BnOOBn

OBn

OBn OBn

O (BzN)O O(NBz)

O(ClBn) STol

O O O

HO OPMB

HO HO OH

OH OH

O HO

HO OH

O O

HO

NHAc OH O O

HO HO

O OH

O

O OH HOOH

iiI

A18

Scheme 1.2. One-Pot synthesis of Globo H using (1+3+2) strategy.¹⁰² (i) NIS, TfOH, -40°C. (ii) NIS, TfOH, -30°C, 62% from 3. (iii) a) Zn, AcOH; b) Ac2O, Pyridine; c) NaOMe; d) H2, Pd/C, HCOOH, 45% over four steps.

25 Scheme 1.3. One-Pot synthesis of Globo H using (1 + 2 +3) strategy.⁹⁷ (i) NIS, TfOH, -40°C. (ii) NIS, TfOH, -30°C, 75% from 3. (iii) a) Zn, AcOH; b) Ac2O, Pyridine; c) NaOMe; d) H2, Pd-black, HCOOH, 70% over four steps.

In 2007, Seeberger and co-workers reported automated solid-phase assembly the protected TACAs Gb3 and Globo H (Scheme 1.4).⁹⁹ Six building blocks A23, A24, A25, A28, A30 and A31 are prepared for the Globo H. It is assembled using fluorenylmethoxycarbonyl (Fmoc) as a temporary protecting group that is stable under acidic glycosylation conditions and is easily removed by a weak base.

Installation of α-galactosidic linkage of protected Gb3 A27 was achieved by β-anomer A25 and A24 with better α-selectivity. Tetrasaccharide A29 wasd assembled by using building block A27 and A28. A30 and A31 were sequentially to assemble the Globo