A Dissertation Entitled Synthesis and Study of MUC1-Based Anti-Tumor

A Dissertation

entitled

Synthesis and Study of MUC1-Based Anti-tumor Vaccines

Partha Karmakar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Chemistry

______Dr. Steven J. Sucheck, Committee Chair

______Dr. Katherine A. Wall, Committee Member

______Dr. Jianglong Zhu, Committee Member

______Dr. Donald Ronning, Committee Member

______Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo

December 2015

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Synthesis and Study of MUC1-Based Anti-tumor Vaccines

Partha Karmakar

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in

The University of Toledo

Chemistry

It is important to obtain CD8+ T cell activation in the course of developing a potent anti-tumor vaccine. CD8+ T cell response to extracellular antigen is generated by processing of the extracellular antigen by antigen presenting cells (APCs). This step is followed by cross presentation of the corresponding epitope to the CD8+ T cell receptors via MHC class I molecules. Cross presentation can be facilitated by efficient antigen uptake via immune-complex-mediated maturation of the APCs. It is well known that vaccination with tumor associated cancer antigen (TACA)-containing MUC1 peptide with the variable number tandem repeat (VNTR) sequence can break self-tolerance in humanized MUC1 transgenic mice. We hypothesized that a MUC1 sequence

TSAPDT(GalNAc)RPAPGSTAPPAHGV that contains a CD8+ T cell epitope delivered on a targeted liposome surface could enhance antigen uptake. Anti-rhamnose antibodies are some of the most abundant naturally occurring antibodies found in humans. Our liposomes contain L-Rhamnose (Rha) epitopes displayed on their surface that can facilitate the natural antibody-dependent immune-complex formation and antigen uptake mechanism for better antigen presentation. To test this hypothesis, synthesis of a 20

iii

amino acid MUC1-Tn sequence, TSAPDT(GalNAc)RPAPGSTAPPAHGV was performed by solid phase peptide synthesis (SPPS) and a Toll-like receptor ligand

(TLRL) was covalently attached to it by Cu(I)-assisted click chemistry. The 20 amino acid MUC1 sequence contained B cell, CD4+ T cell, and CD8+ T cell epitopes. The

TLRL-MUC1-Tn vaccine was formulated onto liposomes that consisted of TEG- cholesterol or Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC) with a total lipid concentration of 30 mM. The vaccine was tested on groups of female C57BL/6 mice. Some of the groups of mice were immunized with Rha-Ficoll prior to vaccination, in order to generate anti-Rha antibodies in those mice. On vaccination, a 10 fold higher antibody production was observed against TLRL-MUC1-Tn by the anti-Rha expressing mice group that received the Rha-vaccine compared to the other groups of mice. The CD8+ T cell responses for the anti-Rha antibody expressing mice group that received the Rha-vaccine were higher compared to the others when they were evaluated by measuring CD8+ T cell proliferation, IFNγ production and cytotoxicity against a cancer cell line. These results indicate that antigen uptake was facilitated by the anti-Rha dependent immune complex formation that resulted efficient antigen uptake and the CD8+ T cell epitope was processed and presented by the APCs.

Acknowledgements

My whole graduate research work is the aftermath of great support, inspiration and generosity of many people. First, I would like to pay gratitude to my research advisor

Dr. Steven J. Sucheck for giving me an opportunity to work in his esteemed group and in very interesting projects. His motivation and mentoring inspired me through every challenge that came in my way throughout my graduate study. I deeply thank my co- advisor Dr. Katherine A. Wall for training and educating me in the fields of immunology that enabled me to perform all the immunological studies on the animal model. I highly appreciate the helpful suggestions and inspiration that I received from my committee members Dr. Jianglong Zhu and Dr. Donald Ronning.

I also acknowledge my past and present group members for the constructive discussions, and for keeping an extremely friendly environment in the work place. I am highly thankful to Dr. Yong Wah Kim for the training and help that I received from him in NMR and ESI-MS experiments.

I am extremely grateful to my family, especially to my mother and father, my younger brother and sister in law and my wife. I would like to mention my mother once more because of her inspiration during my graduate studies. I deeply acknowledge my friends for their constant encouragement and support throughout my study which converted the tedious graduate research into a joyful event of life. v

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of contents ...... vi

List of tables……………………………………………………………………………... ix

List of figures .……………………………………………………………………..…...... x

List of schemes ………………………………………………………………………… xii

List of abbreviations ...... xiii

1 Carbohydrate-based anti-cancer vaccine development:

background and significance...... 1

1.1 Introduction ...... 1

1.2 Immune system overview ………………………………………...2

1.3 Tumor-associated carbohydrate antigens ….……………………...8

1.4 Improved TACA-based vaccines ………………………………..13

1.5 MUC1-based cancer vaccine ...... 14

1.6 Multicomponent vaccines ...... 16

1.7 Immunocomplex mediated vaccine internalization; role of anti-

rhamnose antibodies …..…………...…………………………………………….17

1.8 References …………………………………………………….…18

2 Synthesis of a liposomal MUC1 glycopeptide-based immunotherapeutic

and evaluation of the effect of L-rhamnose on cellular immune response …..….24

2.1 Project summary …………...……………………………………25

2.2 Introduction………………………………………………….…..26

2.3 Results and discussions ………………………………………….31

2.4 Immunological results…………………………………………....38

2.5 Significance………………………………………………………46

2.6 Experimental procedure .....………………………………………47

2.7 References………………………………………………………..57

Supplementary data: Appendix A…………...…………………….…..…99

3 Mixed phase synthesis of gycopeptides: An extended use of N-peptidyl-

2,4-dinitrobenzene sulfonamide-thio acid ligation strategy in ...... 64

3.1 Project summary …...……………………...…………………….64

3.2 Introduction ...... 65

3.3 Results and discussion ...... 67

3.4 Significance...... 73

3.5 Experimental procedure …………………………………………73

3.6 References …………………………………………………...…..83

Supplementary data: Appendix B………………………………...….…106

4 Synthesis of L-rhamnosyl ceramide and evaluation of it’s binding to anti- rhamnose antibodies: Use of rhamnose ceramide as tumor marker……………..………87

4.1 Project summery ………………………………………………..87

4.2 Introduction …………………………………………………….89

vii

4.3 Results and discussion ………………………………….……….90

4.4 Significance ……………………………………………………..94

4.5 Experimental procedure ………………………………………...95

4.6 References ……………………………………………………...98

viii

List of Tables

1.1 Major classes of lymphocytes in Chapter 1…………………………...…………..5

2.1 Conditions for Cu(I)-assisted click reaction for synthesis of Pam3Cys-MUC1-Tn glycopeptide 4 in Chapter 2 ...... 37

2.2 Vaccination plan for the groups of mice in Chapter 2 ...... 40

3.1 N-Peptidyl-2,4-dinitrobenzenesulfonamides in Chapter 3..……………….……..69

List of Figures

1-1 Schematic representation of mechanism of antigen processing and presentation in

MHC II pathway ……………………………….…………………………………7

1-2 Schematic representation of mechanism of antigen processing and presentation in

MHC I pathway …………………………………………………………………..8

1-3 Glycoprotein associated Tn, TF and STn cancer

antigens…………………………………………………………………………....9

1-4 Examples of ganglioside glycolipid associated TACAs……………….……..….10

1-5 : Glycolipid-associated TACAs from Globo-serie ……...…………….………...11

1-6 TACAs of lacto-series or the Lewis antigens ………………………………...... 12

2-1 Anti-rhamnose antibody-mediated enhanced presentation of liposomal vaccine and generation of cellular immuneresponse

.…………………………………………………………………………..……………….24

2-2 Mechanism of anti-rha-mediated enhancement of cellular and humoral immuneresponse ...... 29

2-3 Design of the antigen: Structure of the TLR-2 agonist, linker, and MUC1 glycopeptide sequence. Known human and mouse CD8+ T cell epitopes ...... 33

2-4 Anti-MUC1 antibody titer for immunized and nonimmunized Mice ...... 39

2-5 Proliferation of CD8+ T cells from immunized mice…….……………….……...39 x

2-6 Anti-Rhamnose antibody titer for Groups B and D mice after the fourth boost with rha-ficoll ……………………………………………………………..…..…………41

2-7 Anti-MUC1 antibody titer for all groups of mice………………………………..42

2-8 CD8+ T cell proliferation of Group D at different peptide concentrations……………………………………………………………………………42

2-9 CD8+ T cell proliferation of all four groups at 25 μg/mL peptide………………43

2-10 CD8+ T cell specific IFNγ production of all four groups at 25 μg/mL petide……………………………………………………………………………………..44

2-11 Apoptosis of EL4 cells induced by CD8+ T cells from groups B and

D………………………………………………………………………….………………45

3-1 Strategic representation of mixed phase synthesis of glycopeptides by sulfonamide-thio acid ligation chemistry

……………………………………………………………………………………..…..…64

3-2 General reaction and mechanism of dNBS-thioacid ligation……………………66

4-1 Graphical representation of binding of anti-rhamnose antibodies to tumor cells displaying rhamnose epitope on its surface and detection of the binding by flow cytometry………………………………………………………………………….….….87

4-2 α-L-RhaCer 1 and the control ceramide 2……………………………………….89

4-3 Anti-rhamnose antibody titer of BALB/cJ mice immunized with rhamnose ovalbumin conjugate (after 2nd boost) ……………………………………………..……91

4-4 Fluorescence microscope images with liposomes under 60x magnification

……………………………………………………………………….…………………..92

4-5 Flow cytometry of EL4 cells ……………………………………………..……..93 xi

List of Schemes

2.1 Synthesis of Pam3Cys-MUC1-Tn 4 ...... 34

2.2 Synthesis of alkyne functionalized Pam3Cys 3 ...... 35

2.3 Synthesis of 8 amino acid CD8+ T cell epitope 5 ...... 38

3.1 General strategy for mixed phase synthesis of MUC1 peptide sequences………………………………………………………………………………...68

3.2 Coupling between Fmoc-Histidinyl thioacid and dNBS-glycopeptide 4 using thioacid-sulfonamide ligation chemistry………………………………….……………..70

3.3 Coupling between Fmoc-dipeptidyl thioacid and dNBS-glycopeptide 5 using thioacid-sulfonamide ligation chemistry ...... 72

xii

List of Abbreviations

AcOH……………acetic acid

Bn………………..Benzyl

Boc………………tert-butoxylcarbonyl

2-Cl-Z…………...2-chlorobenzyloxycarbonyl

Cbz……………....carbobenzyloxy

DMDO…………..dimethyldioxirane

DME…………….dimethoxyethane

DMF……………..N,N-dimethylformamide

DMSO…………...dimethylsulfoxide dNBS…………….2,4-dinitrobenzenesulfonyl chloride

EDCI…………….1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Fmoc…………….9-fluorenylmethoxycarbonyl

DCC.…………….1,3-dicyclohexylcarbodiimide

DIBAL-H………..diisobutylaluminum hydride

DIPEA…………. N,N-diisopropylethylamine

DMAP…………..4-(N,N-dimethylamino)pyridine

HATU…………..N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]-pyridin-1-ylmethylene]-

N-methylmethanaminium hexafluorophosphate N-oxide

xiii

HBTU…………..N-[(1H-benzotriazole-1-yl)(dimethylamino)methylene]-N- methylmethanaminium hexafluorophosphate N-oxide

HOBt…………...1-hydroxybenzotriazole

HOOBt…………3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

HONp…………..4-nitrophenol

HOSu…………...N- hydroxysuccinimide iPr………………isopropyl ivDde…………..1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl)

MBHA………….p-methylbenzhydrylamine resin mCPBA………...m-chloroperoxybenzoic acid

Me……………...methyl

MeOH………….methanol

MesNa…………2-mercaptoethane sulfonic acid, sodium salt

MUC1………….mucin 1

NMP……………N-methylpyrrolidone

NBS……………N-bromosuccinimide

PBS…………….phosphate buffered saline

PEG……………polyethylene glycol

Ph……………...phenyl

Py……………...pyridine

PyBOP………….(1H-benzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate tBu……………...tert-butyl

xiv

Rha……………..rhamnose

TACA………….tumor associated cancer antigen

TCEP…………..tris-carboxyethylphosphine

TES…………….triethylsilane

TFA…………….trifluoroacetic acid

TFE…………….2,2,2-trifluoroethanol

Tf2O……………triflic anhydride

THF……………tetrahydrofuran

TIPS ………. ….triisopropylsilane

TLC……………thin layer chromatography

TMP…………....2,4,6-trimethylpyridine

TMSOTf……….trimethylsilyl trifluoromethanesulfonate = trimethylsilyl triflate

Tn……………...2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-galactopyranosyl

Trt……………...trityl

TrtSH…………..trityl mercaptan

UV……………...ultraviolet

VNTR…………..variable number tandem repeat

Chapter 1

Carbohydrate-based anti-cancer vaccine development: background and significance

1.1 Introduction.

A malignant neoplasm, commonly known as cancer, is an unregulated and uncontrollable growth of abnormal cells in the body. Over two hundred different forms of cancers have been identified in humans, affecting more than sixty organs and claiming millions of lives worldwide. According to GLOBOCAN reports, 14.1 million new cancer cases and 8.2 million cancer deaths occurred in 2012 worldwide.1 The American Cancer

Society has projected a total of 1,658,370 new cancer cases and 589,430 cancer deaths for the year 2015.2 Traditional methods for cancer treatment are chemotherapy, surgery and radiation therapy. These methods are often unselective, leading to severe side effects, and unsuccessful to prevent cancer recurrence and metastasis. Hence, development of new therapeutics is an urgent need in the fight against cancer.

The first example of the use of immunotherapy in cancer treatment is dated in

1893 when Coley injected bacterial toxin into a malignant tumor that generated a strong immune response.3 Coley’s method has been modified in numerous ways in the last one hundred years, including the use of the patient’s own inactivated tumor cells as a vaccine construct.4 Though this method was considered effective due to its tumor specific 1

immune-response, the high cost and complexity in implementation have limited the use of this strategy.5 One very well-known approach of cancer immunotherapy is generation of monoclonal antibody (mAbs)-mediated passive immunity against existing tumors. A number of US FDA approved mAbs targeting malignant cancer cells in humans are presently in use.6 Though this passive immune therapy approach is popular, it only provides a temporary protection against a malignant tumor and can extend the patient’s life only for some months. Therefore, development of a vaccination strategy for a permanent solution to this life threatening disease is very important.

1.2 Immune system overview.

The main physiological function of the immune system is to defend our body from any infection, mainly against microbes. The immune system is broadly divided into two major parts, (i) innate immune system and (ii) adaptive immune system (Table 1).

The defense provided by these two parts of the immune systems are known as innate immunity and adaptive immunity, respectively.

1.2.1 Major classes of immune system: The major components of innate immunity are (a) physical and chemical barriers, e.g. skin and other epithelial tissues, (b) phagocytic cells (macrophages, neutrophils) and natural killer (NK) cells, (c) blood proteins and (d) cytokines that regulate and coordinate different activities of the cells of innate immunity.7 Innate immunity provides the first line of defense against any microbial infection.

Adaptive immunity is more complex and plays the major part in host defense. The term “adaptive” is specifically used due to its “ability to remember” and respond better to 2

repeated exposure to an infection. This unique nature of adaptive immunity paved the way for developing immunotherapy to treat an infectious disease. The major components of adaptive immunity are different lymphocyte cells, e.g. B lymphocytes and T lymphocytes and their products, e.g. antibodies. The foreign substances that induce the specific immune response are called antigens.7

1.2.2 Sub-classes of adaptive immunity: Adaptive immunity is subdivided into two major classes, (1) humoral immunity and (2) cell-mediated immunity. Humoral immunity is mainly regulated by the antibodies that are generated by the B lymphocytes in response to an external antigen. Humoral immunity provides the primary defense against the extracellular microbes in the adaptive immunity because the antibodies can bind to the microbial antigens and assist in their elimination.7 Cell-mediated immunity or cellular immunity, on the other hand, works on the intracellular microbes, e.g. virus and some bacteria that proliferate inside the host cell where they are inaccessible to the circulating antibodies.

1.2.3 Fundamental characteristics of adaptive immunity: Both the humoral and cellular immune response against a foreign antigen have the following fundamental characteristics:

1. An adaptive immune response is specific to different portions of a particular

antigen. These specific portions of the antigen that are specifically recognized

by the immune system are called the determinants or epitopes.7

2. For repeatinFollowing repeated exposure to a specific antigen, the ability of

the immune system to respond is higher, which is known as immunogenic

memory. For each exposure to an antigen, the number of clones of the

lymphocytes specific to the antigen increases as well as the number of

memory cells increases. The memory B lymphocytes generate antibodies that

bind to the antigen with higher affinity for a repeated exposure.

3. After antigen stimulation, the immune response specific to the antigen

gradually returns to its resting basal state known as homeostasis and stays in

that state until further stimulation has occurred.

4. The immune system is highly capable of differentiating between foreign and

the self-antigens and does not produce an immune response against the self-

antigen generally. This phenomenon is known as self-tolerance. Although

abnormality in maintaining self-tolerance causes auto-immune diseases.

1.2.4 Cellular composition of adaptive immune system: The main cellular components of the adaptive immune system are the lymphocytes, effector cells and antigen presenting cells. The lymphocytes are divided into different subclasses (Table 1).

The B lymphocytes are the cells that produce the antibodies against the foreign antigens and thus are one of the most important parts of the humoral immune system. The T lymphocytes, on the other hand are mainly the components of the cellular immune system. The major class of the T lymphocytes is the helper T cells and cytotoxic T lymphocytes. The third type of lymphocytes is the Natural Killer (NK) cells. Although the NK cells are an important subclass of the lymphocytes, they are more active in the innate immune system.7

Table 1: Major classes of lymphocytes.

Selected Antigen Lymphocytes Function Phenotype Receptor markers

T lymphocytes

Stimuli for B cells to CD4+ (Helper growth and differentiation. CD3+, CD4+, T αβ heterodimers Cytokine secretion to CD8- lymphocytes) activate macrophages

CD8+ Killing of tumor cells, virus + - CD3 , CD4 , (Cytolytic T infected cells and rejection αβ heterodimers + CD8 lymphocytes) of allografts

Fc receptors,

B MHC class II Production of antibodies Immunoglobulin lymphocytes molecules, CD19,

CD21

Antibody dependent

Natural killer cellular toxicity, killing of Killer cell Ig- Fc receptor for

cells tumor cells and virus like receptor IgG (CD16)

infected cells

There are immunological cells, e.g. mononuclear phagocytes, different T lymphocytes and leukocytes that often take part in various mechanisms of elimination of antigen. These are called the effector cells.

The antigen presenting cells (APCs) are one of the very important classes of cells in the adaptive immune system. The T lymphocytes of the cellular immune system can only recognize specific peptidyl epitopes of an antigen.7 The antigen presenting cells internalize the antigen, process and present the right epitope to the T lymphocytes via major histocompatibility complex (MHC) molecules on their surface (Figure 1). The most effective antigen presenting cells to induce a T cell response are the dendritic cells.8

The MHC molecules are mainly two types, MHC II and MHC I. In case of foreign microbial infection, the protein antigen that is endocytosed into the vesicle of the APCs

(like dendritic cells), are processed inside the vesicle and bind to the class II MHC molecules. The MHC II molecules then present the peptidyl antigen epitope to naïve

CD4+ T cells and effector T cells that have been generated in previous antigen stimulation (Figure 1).7

Figure 1: Schematic representation of mechanism of antigen processing and presentation in MHC II pathway.

On the other hand, in the case of the MHC I pathway, the foreign protein antigen that in the cytosol is processed by the proteasome and carried into the endoplasmic reticulum

(ER) by the transporter associated with antigen processing (TAP) in the dendritic cells.

The processed antigen epitopes in the ER bind to the MHC I molecules and are presented to the naïve CD8+ T cells. Viral protein antigens and mutated tumor antigens are the most common antigens that are present in the cytosol. This is why it is highly important that the immune system recognize the cytosolic proteins. If the virus infected cell or the tumor cell is captured by the APCs, sometimesthe external viral or tumor antigen is processed and presented on MHC I to naïve CD8+ T cells and activates them. This mechanism is called cross presentation since one cell type (APC) captures antigen from another cell 7

type (virus infected cell or tumor cell) and presents the antigen to a different cell type

(CD8+ T cells) to prime or activate them specific to the antigen (Figure 2).7

Figure 2: Schematic representation of mechanism of antigen processing and presentation in MHC I pathway.

1.3 Tumor-associated carbohydrate antigens.

The mammalian cell surface contains a number of different glycoproteins and glycolipids that are associated with different biological processes. Tumor cells are characterized by over expressed aberrant short oligosaccharide sequences and show unusual glycosylation of cell-surface glycolipids and O- and N-linked glycoproteins that play a key role in tumor growth and metastasis.9 Recently, the identification of tumor associated carbohydrate antigens (TACAs) on tumor cell surfaces has created a broad 8

field of opportunities to develop new strategies for cancer immunotherapy.5, 10 Tumor cells can be distinguished from normal cells due to the presence of the TACAs on their surface.6a, 11 These TACAs have been widely studied to develop new cancer vaccines.

Several theories have been proposed to explain the change in environment and metabolism in tumor cells leading to aberrant gene expression that causes TACA formation.12

Figure 3: Glycoprotein-associated Tn, TF and STn cancer antigens.

Figure 4: Examples of ganglioside glycolipid-associated TACAs.

The TACAs are broadly classified into two types, glycoprotein antigens where the

TACAs are majorly glycosylated on serine or threonine residues of the peptide backbone and glycolipid antigens where the TACAs are hydrophobically bound to the lipid bilayer.

Widely studied glycoprotein-associated TACAs include Tn, TF and STn (Figure 3) and most common glycolipid associated TACAs include gangliosides (e.g. GD2, GD3, fucosyl GM1, GM2, GM3) (Figure 4), globo-series (e.g. Gb3, Gb4, Gb5 and Globo-H)

(Figure 5) and lacto-series or the Lewis antigens (e.g. SLea, SLex, SLex,Lex and Ley)

(Figure 6).5, 10

Figure 5: Glycolipid-associated TACAs from Globo-series.

TACAs are expressed in a wide range of epithelial cancer cell lines. For example,

Tn and Globo H are expressed on epithelial cancer cell lines such as prostate, breast and ovarian cancers.11a, 13 It has also been found that TACAs are highly expressed in cancer stem cells as well.14 Moreover, many TACA containing glycopeptides of cancerous origin are secreted into the serum, thus making the TACAs attractive targets for anti- tumor vaccine development.18-21

Figure 6: TACAs of lacto-series or the Lewis antigens.

1.4 Improved TACA-based vaccines.

Though TACAs appear as a very promising target, using TACAs alone as a vaccine candidate has some major drawbacks. TACAs are inherently very weakly immunogenic because of their T-cell independent character and since normal cells also express TACAs in low densities, TACAs appear as self-antigen to the immune system which develops immune-tolerance against the TACA-vaccine.5 Moreover, no memory cell or high affinity IgG antibody is generated against the TACAs and the immune response is primarily generated by the formation of low affinity IgM antibodies that are very short lived. In addition to this, it is difficult to isolate TACAs from natural sources in pure form and in significant amount. Though synthetic strategies like one-pot synthesis and automated oligosaccharide synthesis have been developed to prepare TACAs in the laboratories in large quantities, it is very laborious and expensive.15

An efficient vaccine should be able to successfully trigger the innate as well as the adaptive immune system against the corresponding antigen. On activation, antibodies against a particular antigen are produced by the B-cells of the immune system. Numerous antigens including carbohydrates are recognized by the membrane-bound Ig proteins of

B-cells.16 But for the activation of T-cells, the antigen must be taken into and processed by the antigen presenting cells (APCs) and then be presented to the T-cells through corresponding major histocompatibility complex (MHC) molecules.17 The most common

APCs are the dendritic cells (DCs).18 The most common antigens are the protein and peptide antigens that are well taken and processed into small peptidyl fragments by the

APCs and are displayed on the surface as a complex with the major histocompatibility complex (MHC) molecules.18 The antigen complexed on the MHC on the APCs interact 13

with the T cell receptors on the naïve T-cells leading to their activation that triggers the immune response.19 The activated CD4+ T-cells help in the production of high affinity

IgG antibodies and the activated CD8+ T-cells induce cytotoxic T-cell generation, which leads to programmed cell death called apoptosis. A considerable amount of memory cells are also generated by the activated helper T-cells, along with B-cell activation.18, 20 Due to the effectiveness of the protein and peptide containing antigens in activating the immune system, TACA-based vaccine constructs have been modified by conjugating different proteins and peptides to them.21

1.5 MUC1-based cancer vaccines.

Mucins are intensely glycosylated high molecular weight proteins that are found in tissues of epithelial origin and express TACAs heavily when these epithelial cells turn into cancerous cells.22 Mucin-1 (MUC1) is found in most of the breast, colon and ovarian carcinomas that express high density of Tn, STn, TF , Globo-H and Lewis antigens.23

MUC1 is also shed into the patient’s blood serum and hence has established itself as a marker for monitoring recurrence of epithelial cancer and is a promising target for cancer immunotherapy.22-24 It is well documented that the core-type TACA glycans like GalNAc and Gal-GalNAc remain bound to the peptide during antigen processing inside DCs and bind to the MHC II molecules as glycopeptides, thus generating the antigen specific response.25 A molecular modeling study by Apostolopoulos and co-workers with MUC1 octamer and nonamer variable number tandem repeat (VNTR) showed that GalNAc linked to the threonine residue in SAPDTRPA points into the C pocket and binds to the

MHC I molecules by van der Waal’s attraction and hydrogen bonding.26 14

Though MUC1 is a promising target, Finn and co-workers showed that the human

MUC1 from tumor ascites are poor immunogens.24, 27 The classical approach to improve the immunogenicity of the TACAs is to conjugate them to large carrier proteins, e.g. keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or bovine serum albumin

(BSA).28 The TACAs are picked up by the APCs along with the carrier protein and after processing are displayed on the surface as a complex with MHC II molecules, that activates the helper T-cells leading to the production of cytokines and activation of the B- cells.18 Administration of MUC1-KLH and MUC2-KLH along with QS-21 adjuvant in mice resulted in production of a considerable amount of both IgM and IgG antibodies.29

Follow up studies with several carbohydrate antigen KLH conjugates demonstrated similar results.30 Based on these successful preclinical results, phase II clinical trials were performed with a hexavalent monomeric KLH conjugate containing Ley, GM2, Globo-H, glycosylated MUC1-32 mer, TF, and Tn as well as with a heptavalent KLH conjugate consisting of Ley, GM2, Globo-H, Tn, STn, Tn and TF-MUC1 co-administered with QS-

21 as adjuvant.31

Though these vaccine constructs appear promising, one major drawback of using carrier protein conjugated to the cancer vaccine candidates is that the carrier proteins are themselves immunogenic and suppress the immune response against the antigen of interest.32 To overcome this problem Toll-like receptor ligands (TLRLs) have been conjugated to TACAs to trigger the release of cytokines that can activate the B-cells and the macrophages.33 The toll-like receptors (TLRs) on the DCs are capable to recognize the TLRLs on TACAs that helps better antigen internalization by the DCs. A TLR2 agonist Pam3Cys was covalently attached to several TACAs including dimeric and 15

trimeric Tn clusters.34 Immunization of mice showed the formation of IgM as the major antibodies against the respective antigens along with very low amount of the high affinity

IgG. Even with the co-administration of QS-21 adjuvant did not improve the IgG production.34 This result suggested the requirement of the presence of a helper T-epitope in the vaccine construct to trigger the production of the high affinity IgG antibodies.

1.6 Multicomponent cancer vaccines.

A two component vaccine was made by conjugating an STn antigen containing

MUC1 glycopeptide, derivative to a CD4+ helper T-cell epitope derived from ovalbumin.21f This conjugate was administered with complete Freund’s adjuvant (CFA) to ovalbumin T-epitope transgenic mice expressing T-cell receptors specific for that epitope, leading to generation of highly specific antibodies against the glycosylated

MUC1.21f A multi-antigenic glycopeptide (MAG) conjugated with a helper T-epitope derived from the polio virus or the PADRE and a trimeric Tn antigen was able to generate high amount of IgG antibodies against the Tn antigen when administered to mice with alum adjuvant.35

In search of a more robust synthetic vaccine, a three component vaccine was designed incorporating a carbohydrate B-cell epitope, a helper T-cell epitope and a TLR ligand that is a potent immune activator that renders self adjuvanting character.36 The vaccine was administered to mice as a liposomal formulation in mice with or without the external adjuvant QS-21.36 This first form of three component vaccine showed a moderate amount of IgG antibody formation against Tn.36 Based on this result, several conjugates with the TACA glycosylated MUC1 glycopeptide conjugated three 16

component vaccines with liposome have recently been synthesized that elicited strong humoral and cellular immunity along with cytokine induction when administered to

MUC1 transgenic mice.37

1.7 Immunocomplex-mediated vaccine internalization; role of anti-rhamnose antibodies:

One way to boost the immune response against a vaccine is by installing a xenoantigen onto the target antigen leading to triggering of in vivo immune complex formation resulting in enhanced antigen uptake by the APCs.38 The in vivo generated immune complex containing naturally occurring antibodies can interact with the Fc receptors on the APCs, thus facilitating the internalization of the vaccine.30 Since human sera are abundant in anti-α-Gal antibodies, α-Gal epitopes have been used in a number of vaccine models that showed promising immune responses.39 Recently, anti-rhamnose antibodies have been identified to be more abundant than anti-α-Gal antibodies in human sera.40 It has been shown that synthetic α-L-rhamnose epitopes can bind to anti-rhamnose antibodies and can be used as a tumor marker as well as a component on a vaccine construct that can induce immune-complex formation.40d, 41 Three component MUC1- based vaccines with α-L-rhamnose epitopes conjugated to the glycopeptide antigen or displayed on the surface of the liposome of the vaccine construct have recently been designed.41b, 42 The vaccines were tested on mice that were prepared to express anti- rhamnose antibodies that enhanced MHC II specific response.41b, 42 These results have inspired us to develop more robust vaccine candidates with α-L-rhamnose epitope conjugates to produce enhanced cellular immunogenicity against MUC1. 17

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Chapter 2

Synthesis of liposomal MUC1-based immunotherapeutic and evaluation of the effect of L- rhamnose on cellular immune response

Graphics 1: Anti-rhamnose antibody-mediated enhanced presentation of liposomal vaccine and generation of cellular immuneresponse

2.1 Project summary.

A CD8+ T cell response against extracellular antigen is generated by processing of the extracellular antigen by antigen presenting cells (APCs) and cross presentation to

CD8+ T cell receptors via MHC class I molecules. Cross presentation can be facilitated by efficient antigen uptake via immune-complex-mediated maturation of the APCs. It has been shown that vaccination with tumor associated cancer antigen (TACA)-containing

MUC1 variable number tandem repeat (VNTR) can break self-tolerance in humanized

MUC1 transgenic mice. For a potent anti-tumor vaccine, obtaining CD8+ T cell activation is very important. We hypothesize that delivering a MUC1-TACA VNTR containing a

CD8+ T cell epitope on a liposome surface could enhance the antigen uptake. Our liposome contains an L-Rhamnose (Rha) epitope displayed on its surface to facilitate the natural antibody-dependent immune-complex formation and antigen uptake mechanism for better antigen presentation. To justify this hypothesis, we synthesized a 20 amino acid

MUC1-Tn VNTR containing B cell, CD4+ T cell, and CD8+ T cell epitopes by SPPS. A

Toll-like receptor ligand (TLRL) was attached to it by Cu(I)-assisted click chemistry

(CuACC). The TLRL-MUC1-Tn vaccine was incorporated into liposomes, that consisted of TEG-cholesterol or Rha-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) with a total lipid concentration of 30 mM. The vaccine was tested on groups of female C57BL/6 mice, some of which were immunized with Rha-

Ficoll prior to vaccination in order to generate ant-Rha antibodies in those mice. On vaccination, it was observed that the anti-Rha containing mice that received the Rha- vaccine could produce up to 10-fold higher amount of antibodies against TLRL-MUC1- 25

Tn compared to the other groups of mice. The anti-Rha antibody containing mice showed a higher CD8+ T cell response compared to the others when they were evaluated by measuring CD8+ T cell proliferation, IFNγ production and cytotoxicity. These results suggest that the antigen uptake was facilitated by the anti-Rha dependent antigen uptake mechanism and the CD8+ T cell epitope was well processed and presented by the APCs.

Since anti-rhamnose antibodies are wide-spread and abundant in humans and this liposomal vaccine model contains Rha and antigen epitopes displayed separately on the liposome surface, this design could be used in a wide range of vaccine formulations using different the antigen epitopes.

2.2 Introduction.

It is always a big challenge to the immune system to detect tumors or viral infections since the virus or the disregulated oncogene hides inside the autologous cells, thus making the humoral immune mechanism inactive against them.1 This problem is resolved by the major histocompatibility complex class I (MHC I) antigen processing mechanism.1 In general, though MHC I molecules display autologous internal peptides that are ignored by the immune system. During viral infection or in the course of neoplast formation, the respective peptide displayed on MHC I is recognized by the receptors of

CD8+ T cells resulting in activation of the CD8+ T cells to produce an immune response.1,2 Hence, in order to make an effective antitumor vaccine, the antigen has to be optimally presented to CD8+ T cells by MHC I molecules.3 Unfortunately, such antigens are often too poorly presented by the MHC I molecules to trigger strong CD8+ T cell

responses. In order to overcome this problem, liposomes have successfully been used as carriers of a range of viral, bacterial and tumor antigens and their efficient delivery to the antigen presenting cells (APCs) by this method is also well known.4-9

Dendritic cells (DCs) are some of the most important APCs that have the ability to take up exogenously encountered antigens by phagocytosis or by receptor-mediated endocytosis, process, and present the antigens mainly via MHC II molecules to CD4+ T cells, but also via MHC I molecules to CD8+ T cells in a process known as antigen cross- presentation.3,10 The Fc receptors (FcRs) on the surface of the DCs play an important role in this antigen uptake and processing. For example, Fcγ receptors (FcγRs) facilitate immune-complex-mediated maturation of the DCs by binding to immune complexes, resulting in presentation of antigens to CD4+ helper T cells on MHC II, presentation of antigens to CD8+ cytotoxic T cells on MHC I followed by CD8+ T cell activation, and presentation of antigens to regulatory T cells.11 Herein, we attempted to develop a

MUC1-based tumor immunotherapeutic consisting of an effector CD8+ T cell epitope as well as CD4+ T cell and B cell epitopes. We incorporated these elements on a liposome surface that would trigger FcR-mediated antigen uptake as well as stimulate B cells to produce anti-MUC1 antibodies. The anti-MUC1 antibodies can also bind tumor cells and kill them through antibody dependent cell-mediated cytotoxicity (ADCC), that is mediated through FcγRs on myeloid cells.12

Mucins have recently become very attractive targets for development of potential antitumor immunotherapeutics because in various adenocarcinomas and in premalignant lesions leading to epithelial cancers, human mucin 1 (MUC1) reaches the cell surface aberrantly glycosylated. Cancer derived-MUC1 glycopeptide variable number tandem 27

repeats (VNTR) contain a GalNAc1-Ser/Thr modification (Tn antigen), and other truncated O-glycans due to a lack of core 1,3-galactosyltransferase.13 The changed glycosylation exposes the MUC1 as immunogenic peptide epitopes to the immune system. These peptide and glycopeptide antigens can be processed by the APCs and presented to the immune system to induce MUC1-specific cytotoxic T lymphocytes

(CTLs).14 Some examples of cancers that contain the MUC1 modification are breast, colon, lung, ovary, pancreatic and rectum adenocarcinomas.15-18 Moreover, MUC1 specific CTLs have been found in patients with breast 19, ovarian 20, and pancreatic 21 cancers and MUC1-specific antibodies are associated with a survival benefit. 22-24 As a result, a 2009 report from the National Cancer Institute (NCI) ranked MUC1 second out of 75 tumor-associated antigens on the basis of their therapeutic function, number of patients with antigen positive cancers, immunogenicity, specificity, oncogenicity, expression levels and % positive cells, stem cell expression, number of epitopes and cellular location of expression.25 These reports inspired numerous laboratories to explore innovative approaches to increasing the immunogenicity of MUC1 to develop an immunotherapeutic that could kill cancer cells as well as be used in the context of immunoprevention. 26-37 Goydos et al. demonstrated that a synthetic MUC1-based vaccine could safely be used in humans 38 and a phase I clinical study was reported with a

100 amino acid long MUC1-based vaccine against advanced pancreas and bile duct cancer.39,40 In addition to that, MUC1 peptide pulsed DCs have safely been used as a vaccine in humans with advanced metastatic breast and ovarian cancer. 41

Figure 1. Mechanism of Anti-Rha-mediated enhancement of cellular and humoral immuneresponse. 29

It is now well known that abundant and specific natural antibodies for a number of foreign antigens are widely distributed in the human population. One of these is the

Gal1-3Galß1-4GlcNAc-R (-Gal) epitope.42,43 Galili and co-workers have shown that in vivo interaction of -Gal epitopes (in model vaccines against HIV gp120 and flu virus) with naturally occurring anti--Gal antibodies in 1,3-galactosyltransferase knockout mice leads to FcγRs mediated anti--Gal antibody-dependent antigen uptake, that resulted in a greater than 100-fold increase in immunogenicity.44,45

Bovin, Gildersleeve and others identified additional naturally-occurring anti- carbohydrate antibodies in human sera.46-48 These studies made us interested in antibodies specific for the xenoantigen rhamnose (Rha) because Anti-Rha antibodies are among the most abundant in humans and can also be generated in readily available non-transgenic mice.9,49,50 In fact, Rha is now recognized as one of the most effective natural antibody eliciting molecules known 51 and Rha has been successfully conjugated to lipids to recruit anti-Rha antibodies to tumor cells 52,53.

Previously, it has been shown that the anti-Rha antibodies generated in mice can recognize a Rha moiety carried by a MUC1-based liposomal vaccine leading to better

MHC class II presentation to CD4+ T cells.9,49 In the present studies, we have asked whether bringing the anti-Rha antibodies into action via an anti-RhaFcγR mediated mechanism11 would increase the cross-presentation of MUC1 epitopes on MHC I by

DCs. In addition to IgM antibodies, natural anti-Rha antibodies are mostly IgG3 in the mouse9 or mostly IgG2 in humans.51 One good advantage is that these IgG isotypes generally bind poorly to inhibitory Fc receptors, thus enhancing the immune response.54

To explore this hypothesis we designed a vaccine in which the antigen is incorporated on a liposome surface with or without Rha epitopes.

In these studies we have shown that a glycosylated MUC1-derived glycopeptide anchored in a liposome decorated with Rha epitopes elicits both an improved humoral and cellular immune response in mice previously vaccinated with a Rha antigen (Figure

1). Antigen uptake and presentation was evaluated in groups of C57BL/6 mice by evaluating anti-MUC1 antibody production, CD8+ T cell proliferation, interferon gamma

(INFγ) production, and cytotoxic T cell-mediated apoptosis of cancer cells. We believe these studies are relevant to the development of many other types of vaccines for humans based on the high natural abundance of anti-Rha in the human population.

2.3 Results and Discussion.

2.3.1 Antigen Design. We sought an antigenic sequence that would contain a

CD8+ T cell epitope that could be presented on MHC I and would be a sequence that still contained B cell and CD4+ helper T cell epitopes. Ninkovic et al. have previously proposed that the monoglycosylated 21-mer GP3:

AHGVTSAPDT(GalNAc)RPAPGSTAPPA represents an ideal peptide for development as an anti-tumor vaccine candidate in humans.55 These claims are based on experiments showing that the GP3 is processed by immunoproteasomes or by cathepsin-L to produce a 10-mer glycopeptide SAPDT(GalNAc)RPAPG as a major product.

SAPDT(GalNAc)RPAPG was also shown to strongly bind to empty MHC class I HLA-

A*0201 molecules. The latter is the most frequent MHC class I allele found in

Caucasians. The same study also showed that the SAPDTRPAPG peptide and the 31

respective threonine-modified glycopeptide stimulated human cytotoxic T-cells in vitro.

Earlier studies defined the preferred processing sites for GP3:

AHGVTSAPDT(GalNAc)RPAPGSTAPPA (preferred cleavage sites of immunoproteasomes are underlined), showed the sequence to be efficiently processed, and determined that substrates glycosylated at the DTR motif were the best source of glycopeptides that would potentially bind to MHC I.56 We then considered the implications of stimulation of cytotoxic T-cells in C57BL/6 mice which contain the MHC class I H-2KbDb alleles. Apostolopoulos et al. have shown that the MUC1 8-mer

SAPDT(GalNAc)RPA binds more strongly to the MHC class I mouse allele H-2Kb compared with the corresponding unglycosylated peptide. Further, SAPDT(GalNAc)RPA binds to H-2Kb with high affinity and generates T cells both in vitro and in vivo, which recognize both SAPDTRPA and SAPDT(GalNAc)RPA peptides. 57 Findings made by

Finn and coworkers suggest that MUC1 variable number tandem repeats (VNTRs) containing TACAs were more potent at breaking self-tolerance in MUC1 transgenic mice than the unglycosylated VNTR, since they are more ‘foreign’-like epitopes in comparison to the unglycosylated MUC1. 58,59 Similar findings were made by Boons, who was able to demonstrate that a MUC1-based antigen having GalNAc, again at the DTR site, is more effective in MHC I stimulation of CD8+ T cell activation in MUC1-transgenic mice compared to the non-glycosylated MUC1 sequence 60. Based on these considerations we selected a 20-mer GalNAc modified glycopeptide related to GP3,

TSAPDT(GalNAc)RPAPGSTAPPAHGV (Figure 2), a sequence we hypothesized would contain an MHC-I epitope for both humans and potentially the C57BL/6 mice used in this study. However, we were uncertain at the onset of the antigen design phase as to whether 32

or not this specific glycopeptide would be processed to produce either CD8+

SAPDT(GalNAc)RPA or CD4+ helper T cell epitopes in C57BL/6 mice.

Figure 2. Design of the antigen: Structure of the TLR-2 agonist, linker, and

MUC1 glycopeptide sequence. Known human and mouse CD8+ T cell epitopes (boxes).

Small self-like glycopeptides are known to be not highly immunogenic.61 One approach to enhance immunogenicity has been to conjugate the peptides to synthetic

62-65 bacterial lipoproteins. Pam3CysSS is a synthetically prepared domain of the N- terminal region of Braun’s lipoprotein. 66 It has been shown to be able to stimulate virus- specific CTL responses against influenza virus-infected cells when conjugated with influenza nucleoprotein peptides 147-158 from influenza nucleoprotein63 and to elicit protective antibodies against foot-and-mouth disease when coupled to the antigenic

62 determinant of VP1 . We used the Pam3Cys triazole-linker moiety because we demonstrated in Sarkar et al. that it was sufficient to serve both as an anchor to the liposome and to act as an adjuvant9, 67 (Figure 2). 33

Scheme 1. Synthesis of Pam3Cys-MUC1-Tn 4.

2.3.2 Synthesis of an Azido-Functionalized MUC1-Tn Sequence. An azido functionality was appended to the N-terminus of the peptide as a chemical handle for

‘click’ conjugation with a propargylated Pam3Cys. We synthesized the 20 amino acid sequence, N3-linker-TSAPDT(GalNAc)RPAPGSTAPPAHGV (2) for our model vaccine,

Scheme 1. The protected azide-containing peptide was prepared by the Fmoc-strategy on an Omega 396 synthesizer (Advanced ChemTech, Louisville, KY) using solid phase peptide synthesis (SPPS) strategy. The peptide was analyzed by analytical reverse phase

HPLC and MALDI-TOF. The acetyl deprotection was achieved by treating the azidopeptide 1 with 6 mM sodium methoxide in methanol and the deprotected peptide 2 was purified by size exclusion chromatography using Bio-Gel (P-2, 45-90 μm) and water as solvent followed by lyophilization 34

2.3.3 Synthesis of Pam3Cys-MUC1-Tn 4. Propargyl Pam3Cys 3 was prepared by

Sarkar, S. as previously reported (Scheme 2).9 However, the copper (I)-assisted azide- alkyne Huisgen cycloaddition chemistry between the azidopeptide 2 with alkyne 3 was more challenging than previously reported. Previously, CuSO4 in H2O:THF:MeOH was used as catalyst and solvent, respectively 9. However, these conditions worked poorly with azido peptide 2. Moreover, the conditions also promoted methyl ester formation in the MUC1 peptide, observed by mass spectrometry. We explored various cycloaddition conditions (Table 1) and found CuI, TBTA, Na-ascorbate and DIEA in H2O:THF:DMF

(1:1:2) to be far more effective with substrate 2, Scheme 1, to afford Pam3Cys-MUC1-Tn

Scheme 2: Synthesis of alkyne functionalized Pam3Cys 3.

2.3.4 Synthesis of CD8+ T cell epitope 5. To overcome possible inefficient in vitro processing of the whole 20 amino acid Pam3Cys-MUC1-Tn 4 by the tumor in a

CTL assay, we prepared the putative 8 amino acid residue CD8+ T cell epitope

SAPDTnRPA (5) using solid phase peptide synthesis on Wang resin and purified through

Bio-Gel P-2 (45-90 μm) with water as eluent followed by lyophilization (Scheme 3).

2.3.5 Synthesis of Rha-TEG-Cholesterol. Rha-TEG-Cholesterol was prepared as previously described and used in formulation of the liposomes.9

2.3.6 Synthesis of a Rha-Ficoll Conjugate. To evaluate the effect of Rha in enhancing antigen uptake, we needed to generate anti-Rha antibodies in mice, since naïve mice do not have anti-Rhamnose antibodies.9 We achieved this by vaccinating mice using a Rha-Ficoll conjugate with an equal volume of Alum emulsion. We choose Ficoll as the carrier for Rha because it is well known for producing a T-independent antibody response. The Rha-Ficoll was prepared as described in the literature.9

2.3.7 Liposome Formulation. We prepared two different batches of liposomes for the four groups of mice. For the preparation of the normal liposomes 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) (80%) and cholesterol (20%) were used (Batch 2).

For the Rha-displaying liposomes, 10% Rha-TEG-Cholesterol was mixed with 10% cholesterol, keeping the other components the same (Batch 1). The liposomes were formulated by the extrusion method in a total lipid concentration of 30 mM. The concentration of Pam3Cys-MUC1-Tn 4 was 10 nanomolar in both kinds of liposomes.

Table 1. Conditions for Cu(I)-assisted click reaction for synthesis of Pam3Cys-MUC1-

Tn glycopeptide 4.

Reagents Conditions Solvent System Time Results

CuSO4.5H2O, H2O:THF:MeOH 42 ~50% Conversion with methyl ester hr. side product TBTA, Na-ascorbate (1:1:2)

CuSO4.5H2O, H2O:THF (1:1) 42 ~10% Conversion with high hr. amount of unidentified side product TBTA, Na-ascorbate formation

CuSO4.5H2O, H2O:THF:DMF 42 ~ 60% Conversion with high hr. amount of unidentified side product TBTA, Na-ascorbate (1:1:2) formation

CuOAc, H2O:THF:DMF 50 100% Conversion with high amount hr. of unidentified side product TBTA, Na-ascorbate (1:1:2) formation

CuI, TBTA, Na- H2O:THF:DMF 16 100% Conversion with negligible ascorbate, DIPEA hr. impurity (1:1:2)

Scheme 3. Synthesis of 8 amino acid CD8+ T cell epitope 5.

2.4 Immunological Results

2.4.1 Preliminary Study. To check the antigenicity of our vaccine, we first

b b started with two C57BL/6 mice (H-2K D ) and primed with Pam3Cys-MUC1-Tn incorporated on the Batch 2 liposomes. The mice were boosted twice with the vaccine at

14 day intervals. On the seventh day after the second boost, the mice and control non- immunized mice were bled and the sera were pooled to check the anti-MUC1 antibody titer. An ELISA was performed to demonstrate that the mice were producing anti-MUC1 antibody (Figure 3). The mice were then sacrificed on the same day and the CD8+ T cells were isolated. A T-cell proliferation assay was performed to ensure that the vaccine could stimulate the generation of CD8+ T cells. Isolated spleen CD8+ T cells were cultured with different concentrations of the liposomal Pam3Cys-MUC1-Tn 4 in the presence of

C57BL/6 DCs. A considerable amount of T-cell proliferation (Figure 4) demonstrated the presence of at least one CD8+ T-cell epitope in the vaccine.

Figure 3. Anti-MUC1 antibody titer for Immunized and Nonimmune Mice by

ELISA assay. Absorbance measured at 630 nm.

Figure 4. Proliferation of CD8+ T Cells from Immunized Mice. The radio active counts per minutes (CPM) goes higher with increasing peptide concentration.

2.4.2 Evaluation of Effect of Rha-TEG-Cholesterol-formulated Liposomal

Vaccines. Four groups of six female C57BL/6 mice were used for this study (Table 2).

Table 2. Vaccination plan for the groups of mice.

Group A Group B Group C Group D

Immunization - Rha-Ficoll - Rha-Ficoll

Vaccination Liposomal Liposomal

Pam3Cys-MUC1-Tn Pam3Cys-MUC1-Tn with

Rha-TEG-Cholesterol

2.4.3 Anti-Rha Antibody Generation in Mice. To test our hypothesis, we again generated anti-Rha antibodies in groups of mice. Mice of Groups B and D were injected subcutaneously (day 0) with 100 µL equivolume emulsion of Rha-Ficoll and Alum (100

µg of Rha-Ficoll per mouse). The mice were boosted with the same composition at 14 day intervals up to the fourth boost. On the seventh day after the fourth boost, blood was pooled from the mice and sera were isolated. An ELISA was performed to determine the anti-Rha antibody titer. The Rha-Ficoll immunized mice showed about 25-fold higher titer than non-immunized mice against microtiter plates coated with Rha-BSA (Figure 5).

Control wells were coated with BSA.

Figure 5. Anti-Rhamnose antibody titer for Groups B and D mice after the fourth boost with Rha-Ficoll by ELISA assay. Absorbance measured at 630 nm.

2.4.4 Tumor Antigen Vaccination. Once mice were producing anti-Rha, the second step was to vaccinate all the groups of mice with the corresponding liposome formulations containing the Pam3Cys-MUC1-Tn 4 tumor antigen according to the vaccination plan described in Table 2. One of the goals of our study was to evaluate the production of antibodies against the antigen and the stimulation of CD4+ helper T-cells.

The other major goal was to evaluate whether CD8+ T cells were being activated, resulting in cytokine production and anti-tumor cytotoxicity. Here we have evaluated the effect of the Rha ligand on antigen uptake and presentation using CD8+ T cell proliferation, cytotoxicity and interferon gamma (IFNγ) production.

2.4.5 Anti-MUC1 Antibody Production. The anti-MUC1 antibody titer of all four groups of mice was evaluated by ELISA. The study was performed on pooled sera isolated from two mice of each group after the 3rd boost of the vaccine. The ELISA

shows a considerable amount of anti-MUC1 antibody production by each group of mice, with over 10-fold higher titer in Group D mice (Figure 6). This demonstrated that CD4+

T cells were stimulated more effectively by targeting through Rha.

Figure 6. Anti-MUC1 antibody titer for all groups of mice.

Figure 7. CD8+ T cell proliferation of Group D at different peptide concentrations.

2.4.6 CD8+ T Cell Proliferation Study. We did a CD8+ T cell proliferation study to evaluate whether enhanced uptake of the Rha-TEG-Cholesterol-formulated liposomal vaccine resulted in more effective CD8+ T cell priming in vivo. We used the eight amino acid CD8+ T cell epitope 5 (Scheme 3) to restimulate the primed CD8+ T cells in vitro.

To determine the concentration of CD8+ T cell epitope 5 required to activate CD8+ T cells, we first performed a T cell proliferation assay with the CD8+ T cells from Group D in the presence of DCs pulsed with peptide concentrations of 2.5, 5, 10, and 20 µg/mL.

The assay indicates a continuous increase in proliferation with the increase of peptide concentration (Figure 7). In the proliferation assay with all four groups, peptide 5 was used at 25 μg/mL concentration. The comparative study indicates that Group D is the only group that shows enhanced peptide specific proliferation relative to the other groups

(Figure 8).

Figure 8. CD8+ T cell proliferation of all four groups at 25 μg/mL peptide.

Figure 9. CD8+ T cell specific IFNγ production of all four groups at 25 μg/mL peptide.

2.4.7 Interferon Gamma (IFNγ) Production. Following antigen stimulation, most CD8+ T cells release interferon gamma (IFNγ). The production of IFNγ is higher for primed CD8+ T cells. We performed an IFNγ-ELISA assay to evaluate if enhanced uptake of the Rha-TEG-Cholesterol-formulated liposomal vaccine resulted in more primed CD8+ T cells. Isolated CD8+ T cells from each group of mice were cultured with

DCs pulsed with CD8+ T cell epitope 5. After 24 hours incubation at 37 °C, the supernatant was collected for ELISA analysis. The assay indicated 4.6- to 10-fold higher

CD8+ T cell epitope 5 specific IFNγ production for Group D, compared to the other three groups (Figure 9).

Figure 10. Apoptosis of EL4 cells induced by CD8+ T cells from Groups B and

D. The ratio of EL4 cells to CD8+ T cells are 1:100 and 1:50.

2.4.8 JAM Assay for CTLs. One approach to generating a more effective antitumor immunotherapeutic vaccine may be to stimulate CD8+ T cells to specifically induce apoptosis of tumor cells. We assayed for cytotoxic CD8+ T cells from Group B and D mice, both of which contained anti-Rha antibodies. Radiolabeled EL4 tumor cells were pulsed with CD8+ T cell epitope 5, followed by incubation with the CD8+ T cells using EL4 to CD8+ T cell ratios of 1:100 and 1:50.68 Both groups showed killing of the

EL4 cells by apoptosis induction; however, Group D showed about twice the cytotoxicity (Figure 10).

2.5 Significance.

A new, fully synthetic, glycopeptide-based tumor antigen has been synthesized,

Pam3Cys-MUC1-Tn 4. The antigen was successfully formulated into liposomes along with a Rha-TEG-Cholesterol designed to bind anti-Rha antibodies that are found abundantly in human serum and can be generated in mice. In these studies anti-Rha antibodies were generated in C57BL/6 mice by Rha-Ficoll immunization to mimic human natural anti-Rha antibodies. The liposomal Pam3Cys-MUC1-Tn vaccine formulated with Rha-TEG-Cholesterol resulted in improved antibody and CD8+ T cell response against the Pam3Cys-MUC1-Tn antigen in anti-Rha expressing mice compared to mice lacking anti-Rha antibodies. This result suggests that the Rha epitope in the vaccine can form immune complexes with the anti-Rha antibodies in vivo followed by efficient antigen uptake in DCs, leading to cross-presentation on MHC class I molecules.

It is important to note that our CD8+ assays only measured T cells recognizing the CD8+ epitope contained in the added 8 residue peptide. Other T cells to additional cross- presented epitopes may also have been generated. The use of anti-Rha antibodies in immune-complex mediated maturation of antigen presenting cells to induce a CD8+ T cell response is a novel discovery. The Rha targeting allowed use of an antigen that was otherwise only weakly cross-presented and enhanced its ability to elicit CD8+ T cells while still generating a humoral response. The vaccine model contains separate Rha and antigen epitopes that gives the vaccine platform a wide range of applicability.

2.6 Experimental Procedures.

2.6.1 General Methods. The fine chemicals including copper salts, cholesterol,

L-Rhamnose, DIEA, DMF and other dry solvents were purchased from Acros Organics or Sigma Aldrich. All the solvents were purchased from Fischer Scientific. 1,2-

Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and Ficoll 400 were acquired from

Avanti Polar Lipids Inc. (Alabaster, AL). Silica gel (230−400 mesh) for flash column chromatography was purchased from Sorbent Technologies. Thin-layer chromatography

(TLC) pre-coated plates were obtained from EMD. TLC plates (silica gel 60, f254) were visualized under UV light or by charring (5% H2SO4−MeOH) or by staining with ninhydrin. Flash column chromatography was done with silica gel (230−400 mesh). Tris

[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA) was obtained from Anaspec

(San Jose, CA). Preloaded Fmoc-L-Ala-Wang resin, and all other Fmoc-L-amino acids were procured from Anaspec (San Jose, CA) or from Chem-Impex Inc. Imject Alum was purchased from Thermo Scientific. Goat anti-mouse IgG/IgM antibodies were obtained from Sigma. Dynabead kit for CD8+ T cell isolation was purchased from Invitrogen. All other secondary antibodies were obtained from Jackson Immunoresearch Laboratories

(West Grove, PA). Female C57BL/6 mice (6−8 weeks old) were obtained from the

Jackson Laboratory. All mice were maintained in the AAALAC-accredited animal facility at the University of Toledo Health Science Campus under a specific pathogen- free environment. All mouse experiments were performed according to NIH guidelines with approval of the UT Institutional Animal Care and Use Committee.

2.6.2 Synthesis of Glycopeptide Azide 1. Glycopeptide azide 1 was synthesized in Omega 396 synthesizer (Advanced ChemTech, Louisville, KY) by Fmoc-based SPPS strategy. The coupling of the amino acids, the acetyl protected Tn-Thr and the azide functionality was achieved by HOBt and DIC in the presence of NMP starting with a preloaded Fmoc-Ala-Wang resin. Fmoc deprotections were accomplished by treatment with 25% piperidine in dimethylformamide. A modified Reagent K cocktail consisting of

88% TFA, 3% thioanisole, 5% ethanedithiol, 2% water and 2% phenol was used for simultaneous resin cleavage and deprotection of the glycopeptide. The acetyl protections on the N-acetyl galactal survived the cleavage conditions. The cocktail mixture was filtered through a Quick-Snap after the cleavage and collected in a 20 mL ice-cold butane ether and the peptide was allowed to precipitate out for an hour at -20 °C. It was then centrifuged and washed twice with ice-cold methyl-t-butyl ether and the precipitate was dissolved in 20% acetonitrile in water and lyophilized affording peptide 2. The peptide was analyzed by analytical reverse-phase HPLC and MALDI-TOF (matrix assisted laser desorption ionization time of-flight) mass spectrometer, model 4800 from Applied

Biosystems. HR- MALDI-MS: [M+H] m/z calcd for C100H156N29O37, 2355.12; found,

2355.217.

2.6.3 Synthesis of Glycopeptide Azide 2. Peptide 1 (5 mg, 2.24 μmol) was taken in 2 mL dry methanol and 12 μL of freshly prepared 1 M sodium methoxide was added to the solution. The reaction was monitored by MALDI-TOF analysis. On completion, the reaction was neutralized with solid carbon dioxide. The solution was concentrated and purified by Bio-Gel (P-2, fine 45−90 μm, 12 g) size exclusion chromatography (column 48

bed length: 30 cm, diameter: 2.5 cm) using deionized water as eluent. Lyophilization of the pure fractions afforded 2 as a white powder (4.7 mg, 100%). HR-MALDI-MS:

[M+H] m/z calcd for C94H150N29O34, 2229.0895; found, 2229.336.

2.6.4 Synthesis of Pam3Cys-MUC1-Tn 4. CuI (134 μg, 0.54 μmol) and TBTA

(0.857 mg, 1.62 μmol) were dissolved in H2O-THF (1:1, 0.40 mL). Na-ascorbate (0.80 mg, 4.04 μmol) was added to the solution. The solution was stirred for 5 minutes.

Compound 3 (1.27 mg, 1.35 μmol) was taken in THF (0.40 mL) and added to the reaction mixture and stirred for 15 minutes followed by the addition of a solution of compound 2

(1 mg, 0.45 μmol) in H2O-DMF (1:3, 0.4 mL). The reaction mixture was stirred at 20 °C under N2 atmosphere for 16 h. The reaction mixture was concentrated, dissolved in

CHCl3, washed with 7.5 % aqueous citric acid solution, dried over sodium sulfate and the solvent was evaporated to afford compound 4 as a light yellow solid (1.9 mg, 100 %).

HR-MALDI-MS: [M+H] m/z calcd for C151H256N31O40S, 3175.86; found 3175.809.

2.6.5 Synthesis of CD8+ T-Cell Epitope 5.69 The CD8+ T-Cell epitope 5 was synthesized manually by assembling the amino acids on Fmoc-Ala-preloaded Wang resin by Fmoc-based SPPS strategy. The reactions were performed in a 20 mL syringe reactor cartridge and agitation was provided by a stream of dry nitrogen. The peptide synthesis was performed by coupling HOBt esters of Fmoc-protected amino acids in situ using

PyBOP as the coupling agent in presence of diisopropylethyl amine (DIPEA).

Deprotection of the N-α-Fmoc group was achieved by treatment with 25 % piperidine in dimethylformamide thrice, first for 5 min then a second and third time for 10 min each. 49

After the synthesis, the resin cleavage as well as global deprotection (except the acetyl protections on the carbohydrate moiety) was accomplished by treatment with TFA-H2O-

TIPS (95:2.5:2.5), for 4 h under N2 atmosphere. The mixture was filtered, washed with the cleavage cocktail (1-2 mL), followed by CH2Cl2. The filtrate was concentrated to dryness under vacuum. The dry peptide (5 mg, 2.24 μmol) was dissolved in 2 mL of dry methanol, and 12 μL of freshly prepared 1 M sodium methoxide was added and the reaction mixture was stirred at ambient temperature under N2 atmosphere for 4 h. The reaction was neutralized with solid carbon dioxide, concentrated and purified by Bio-Gel

(P-2, fine 45−90μm, 12 g) size exclusion chromatography (column bed length: 30 cm, diameter :2.5 cm) using deionized water as eluent. Lyophilization of the pure fractions afforded 5 as a white powder (4.7 mg, 100%). MALDI-MS: [M+H] m/z calcd for

C94H150N29O34, 1017.48; found, 1017.940.

2.6.6 Liposome Formulation. Different lipid stock solutions were prepared in chloroform and aliquots of the stock solutions were mixed in proportions to obtain a solution with a total lipid concentration of 30 mM in a total volume of 2 mL (Batch 1:

DPPC 80%, cholesterol 10%, Rha-TEG-Cholesterol 10%, and Pam3Cys-MUC1-Tn

0.69μM; Batch 2: DPPC 80%, cholesterol 20%, Pam3Cys-MUC1-Tn 0.69 μM). A constant stream of nitrogen was used to evaporate the chloroform and the resulting lipid films were dried under vacuum for 12 h. Two mL of HEPES buffer (pH = 7.4) was then added to hydrate the dry lipid films and the suspensions were incubated at 43 °C for 40 min. The suspensions were subjected to 10 freeze−thaw cycles (dry ice/acetone and water

at 40 °C). Final liposomes were prepared by extrusion (21 times) using a LipoFast Basic fitted with a 100 nm polycarbonate membrane to control the liposome size.

2.6.7 Immunization. Two female C57BL/6 mice (6−8 weeks old, The Jackson

Laboratory) were primed (day 0) and boosted three times (days 14, 28 and 42) with 100

μL intraperitoneal injections of Pam3Cys-MUC1-Tn conjugate 10 (10 nm per injection) incorporated on liposome (Batch 2) in PBS.

2.6.8 Anti-MUC1 Antibody ELISA. Ninety-six-well plate (Immulon 4 HBX) was coated with MUC1-Tn conjugate 2 (15 μg/mL) dissolved in 0.01 M phosphate buffered saline (PBS) and the plate was incubated over night at 4 °C. The plate was then washed 5 times with PBS containing 0.1% Tween-20 (wash buffer). Blocking was performed by incubating the plate for 1 h at 25 °C with BSA in PBS (1 mg/mL). The plate was then washed 5 times with the wash buffer and incubated for 1 h with serum dilutions in PBS/BSA. Unbound antibody in the serum was removed by washing and the plate followed by incubating for 1 h at 25 °C with Horseradish Peroxidase (HRP) goat antimouse IgG + IgM (Sigma) diluted 5000 times in PBS/BSA. The plate was washed with wash buffer and TMB (3,3′,5,5′-tetramethylbenzidine) one component HRP micro well substrate (Bio FX, Owings Mills, MD) was added to the plate and it was allowed to react for 10-20 min. Absorbance was recorded at 620 nm. The absorbance was plotted against log10[1/serum dilution].

2.6.9 CD8+ T-Cell Proliferation Assay. On day 49, selected mice were sacrificed and the spleens were removed and placed in 5 mL of freshly prepared spleen cell culture 51

medium (DMEM with 10% fetal calf serum). Single cell suspensions were prepared using modified sterile glass homogenizers. The cells were washed three times with culture medium and from the cell suspension, the CD8+ T cells were isolated using a

Dynabead FlowCompTM Mouse CD8 kit (Invitrogen). The cell concentration was brought to 5×106 cells/mL. 100μL aliquots of the spleen cell suspensions were added to 96 well plates (5×105 cells per well). The DC suspension9 cultured from the bone marrow of a non-immunized C57BL/6 mouse was pulsed with the antigen by incubating with the

Pam3Cys-MUC1-Tn liposomes at antigen concentrations from .02 to 2 µg/mL at 37 °C for 4 h. 100 μL aliquots of the pulsed DCs were added to the wells containing the CD8+ T cells (5×104 DCs per well). The plates were incubated at 37 °C for 4 days. On day 4, the cells were pulsed with radioactive [3H]-thymidine (40 μCi/mL, 25 μL per well) and incubated overnight at 37 °C in presence of 5 % CO2. The cells were harvested on glass- fiber filters and incorporation was determined by measurements on a Top Count scintillation counter (Packard, Downers Grove, IL). The radioactive [3H]-thymidine count per minute (CPM) is equivalent to the proliferation of the cells.

2.6.10 Immunization. Twenty-four female C57BL/6 mice were used in this study. They were divided into four groups as A, B, C and D, each group containing six mice. Groups B and D were injected subcutaneously (day 0) with 100 µL equivolume emulsion of Rhamnose-Ficoll (Rha-Ficoll) and Imject Alum (100 µg of Rha-Ficoll per mouse). The mice were boosted with the same composition on day 14, 28, 56 and 70. The mice were bled on day 77 and the sera were pooled to check for the presence of anti-Rha antibodies. 52

2.6.11 ELISA for Anti-Rha Antibody Titer.9 A ninety-six-well plate (Immulon

4 HBX) was coated with Rha-BSA conjugate9,49 (2 μg/mL) in PBS and incubated overnight at 4 °C. The ELISA was continued as described above.

2.6.12 Vaccination. Vaccination was started on day 82 after Rha-Ficoll immunization. Two different batches of liposomes were prepared, Batch 1 and Batch 2.

Group A and C were primed and boosted twice in 14 days intervals with 100 µL of Batch

2 liposomes per mouse (100 µL subcutaneous injection, 10 nm Pam3Cys-MUC1-Tn per mouse). Groups B and D were primed and boosted thrice in 14 days intervals with 100

µL of Batch 1 liposomes/mouse (100 µL subcutaneous injection, 10 nm Pam3Cys-

MUC1-Tn per mouse). After such preparation, the mice were kept in rest and two mice from each group were boosted with Rha-Ficoll (Groups B and D only, composition and amount as mentioned earlier) followed by liposomal vaccines (all groups, 12 days after the last Rha-Ficoll boost, composition and amount are same as stated above) prior to different assays.

2.6.12 Anti-MUC1 Antibody Titers ELISA. Mice from each group were bled after the third boost. The sera from individual mice in a group were pooled. The ELISAs were performed as described above.

2.6.13 CD8+ T Cell Proliferation Assay with All Groups of Mice. Two mice from each group were sacrificed on the seventh day after the fourth boost of vaccine and 53

cells were isolated from spleen and lymph nodes as described earlier. From the cell suspension, the CD8+ T cells were isolated using a Dynabead FlowCompTM Mouse CD8 kit (Invitrogen). The CD8+ T cell concentration was brought to 4×105 cells/mL and 100

μL aliquots were added to a 96 well plate (2×104 cells/well). One hundred μL of the DC suspension (2×103 cells/mL, 1×104 cells/well) cultured from the bone marrow of a non- immunized C57BL/6 mouse was also added to the wells containing CD8+ T cells, and also to the empty wells as control. Thus the DC to CD8+ T cell ratio was 1:10 and total volume was 200 μL per well. Combinations of CD8+ T cells with DCs were pulsed with

CD8+ T cell epitope 5 with effective concentration of 25 μg/mL and incubated for 6 days at 37 °C. On day 5, the cells were pulsed with radioactive [3H]thymidine (40 μCi/mL, 25

μL per well) and at the end of day 6, the cells were harvested on glass-fiber filters and incorporation was determined by measurements on a Top Count scintillation counter

(Packard, Downers Grove, IL). A preliminary assay to determine the optimal peptide concentration used cells from one group D mouse and a range of peptide concentrations from 2.5 to 20 µg/mL.

2.6.14 Interferon Gamma (IFNγ) ELISA. CD8+ T cells (5×105 cells/mL) isolated from two mice from each group were distributed on a 6 well plate (500 μL per well). The DC suspension (5×104 cells/mL, 2.5×104 cells/well) cultured from the bone marrow of a non-immunized C57BL/6 mouse was also added to the wells containing

CD8+ T cells. Thus the DC to CD8+ T cell ratio was 1:10 and total volume was 1 mL per well. The combination of CD8+ T-cells with DCs were pulsed with CD8+ T cell epitope 5 with effective concentration of 25 μL/mL and incubated for 24 hours at 37 °C. After 24 54

hour, the supernatant was collected and IFNγ production was measured by using Murine

IFNγ Mini ELISA Development Kit (Peprotech).

2.6.15 JAM Assay for Apoptosis.68 Two mice from each group were sacrificed on the seventh day after the fourth boost of vaccine and cells were isolated from spleen and lymph nodes as described earlier. The CD8+ T cells were isolated as before. C57BL/6

EL4 lymphoma cells (ATCC TIB 39) were freshly grown in DMEM medium and the concentration of the cell suspension was brought to 1×105 cells/mL. The cell suspension was pulsed with [3H]-thymidine (40 μCi/mL) for 3 hours. The cell suspension was then divided into two equal halves after washing and one half of the EL4 cells were pulsed

+ with CD8 T cell epitope 5 and incubated 12 hours at 37 °C in the presence of 5 % CO2.

Both preparations were then washed thrice and the EL4 cell concentrations were again brought to 1×105 cells/mL. One hundred μL aliquots of the EL4 cell suspensions were then added to 96 well plates (1×104 cells per well). Each group of CD8+ T cell suspensions was prepared with concentrations of 1×107 and 0.5×107 cells/mL. One hundred μL aliquots from each concentration were added to different wells containing

EL4 cells with or without peptide pulsing. One hundred μL of 2 μM staurosporin was added to some of the wells instead of CD8+ T cells as a positive control. The negative controls were EL4 cells alone and EL4 cells pulsed with CD8+ T cell epitope 5, without any CD8+ T cells. The plate was incubated for 6 hours at 37 °C in the presence of 5 %.

CO2. The cells were then harvested on glass-fiber filters and incorporation was determined by measurements on a Top Count scintillation counter (Packard, Downers

Grove, IL). Percent specific killing was calculated with respect to the negative control

EL4 cells pulsed with CD8+ T cell epitope 5, without any CD8+ T cells.

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Chapter 3

Mixed phase synthesis of glycopeptides: An extended use of N-peptidyl-2,4-dinitrobenzene sulfonamide-thio acid ligation strategy.

Barlos’s Peptide 1 Peptide 2 Resin

Wang P= Protecting group Resin

Peptide 2 Peptide 1

Graphics 1: Strategic representation of mixed phase synthesis of glycopeptides by

sulfonamide-thio acid ligation chemistry

3.1 Project Summary

A mixed phase peptide synthesis strategy has been developed using the solid phase peptide synthesis (SPPS) and coupling of N-peptidyl and N-glycopeptidyl 2,4- dinitrobenzene sulphonamides (dNBS) and C-terminal peptidyl-thioacids. Both the N- peptidyl dNBS and C-terminal peptidyl thioacid fragments were prepared by Fmoc-based

SPPS and the resulting peptides were coupled to generate longer peptides. The final dNBS-thio acid ligation reaction was completed within 15 to 20 minutes.

3.2 Introduction.

Nitrobenzenesulfonamides (NBS) were first used by Fukuyama and co-workers as protecting-activating groups for secondary amine synthesis.2,3 Latter, 2,4- dinitrobenzenesulfonamides (dNBS) were found by Tomkinson et al. to undergo condensation reactions with thioacids to form amides, thioamides, ureas and thioureas

(Figure 1).4,5 Since those early reports, only a few examples of the use of this chemistry for the chemoselective formation of amide bonds have been reported.

There is significant interest in developing chemoselective amide bond forming reactions for the preparation of peptides and glycopeptides.6 The most widely investigated techniques for this purpose are the native chemical ligation7 and its variants which include the use of auxiliaries to overcome the cysteine specific limitations.8,9 Other cysteine and auxiliary free ligations include the Staudinger ligation,10,11 reactions between α-ketoacids and N-hydroxylamines,12 isonitrile-mediated amide bond formation13 and iodinium-promoted nitroalkane-amine coupling.14 Direct aminolysis of thioesters has also been described towards the synthesis of cyclic peptides15 and glycopeptides16,17 but the reaction time varies from 48 to 96 hours for these cases.

Figure 1: General reaction and mechanism of dNBS-thioacid ligation

Examples of the use of thioacids for amide bond synthesis include silver ion mediated coupling18 with amines and the reaction of thioacids with electron deficient arenes and heteroarenes to form activated thio-esters.19 Thioacids have also been used in combination with isocyanates and isothiocyanates20 and N-terminal dNBS amino acid methyl esters21 to form glycosyl, peptidoglycosyl and peptidyl amide linkages. Coupling of thioacids and N-glycosyl-2,4-dinitrobenzenesulfonamides to afford N-glycosyl amides has been investigated.22 Crich et al. has demonstrated a solution phase peptide synthesis using a combination of electron deficient peptidyl benzenesulfonamides capable of reacting with peptidyl thioacids of differential reactivity.22 Seeking to further expand the application of peptidyl thioacids we have now completed an Fmoc-base SPPS of N-dNBS peptides in combination with the SPPS derived peptidyl thioacids to effect a mixed-phase synthesis of a MUC1 glycopeptide sequence.1 66

3.3 Results and Discussions.

3.3.1 Synthesis of d-NBS capped MUC1 sequence on Solid phase:

MUC1 is a heavily glycosylated transmembrane protein that is closely associated with cancers of epithelial origin and is a relevant target of glycopeptide-based anti-cancer therapeutics.24-26 The 20-amino acid-tandem repeat contains several possible glycosylation site at the threonine and serine residues found along the sequence NH2-

24,25 PDTRPAPGSTAPPA14H15G16VTSA-COOH. Ligation at the H15-G16 and A14-H15 sites were performed to determine the application of this chemistry as a potential coupling strategy for the mixed-phase synthesis of glycopeptides.1 The simultaneous presence of multiple unprotected functional groups, such as imidazole, carboxyl, and hydroxyl in the MUC1 fragment would also demonstrate the chemoselectivity of the methodology (Scheme 1).1

Solid phase peptide synthesis has been a convenient method for procuring medium sized peptide fragments (2-40 amino acid residues) and coupling of o- and p- nitrobenzenesulfonyl group to the N-terminus of a peptide bound to a rink amide MBHA resin is already reported.27 Thus it appears that dNBS groups could be introduced as well.

We performed a screening of a series of conditions potentially useful for introducing 2,4- dinitrobenzenesulfonyl chloride (dNBS-Cl) on solid phase. We used the comparatively more economical NH2-Ala-Wang resin for this study. The combination of dichloromethane and pyridine was found perfect for introducing the dNBS group onto 67

peptides in solution phase.21 But on solid phase, higher equivalents of dNBS-Cl (4 eq)

1 were required for completion of the reaction. Other conditions that included CH2Cl2-

DIEA, DMF-pyridine, DMF-DIEA, and CH2Cl2-pyridine-DMAP (0.1 eq), were unsatisfactory. It took 4 h to get a complete reaction and the reaction progress was monitored by Kaiser test.27,28 The dNBS-alanine was cleaved from the resin by TFA treatment to obtain the dNBS-alanine as the primary product. The dNBS group was stable to the acidic cleavage.

Scheme: 1 General strategy for mixed phase synthesis of MUC1 peptide sequences

Table: 1 N-Peptidyl-2,4-dinitrobenzenesulfonamides1*

*Compounds 1-4 was previously synthesized by Talan, R. S.,29 compound 3 and 4 were resynthesized in the present work.

Once the method was optimized, synthesis of longer 2,4-dinitrobenzenesulfonyl peptide and glycopeptides were prepared, starting from dipeptide to hexapeptide. The peptides were manually assembled on the Wang resin using Fmoc-based SPPS and coupling of the amino acids were accomplished using PyBOP, HOBt as coupling reagents and DIEA as base in DMF. The dNBS was installed after the last amino acid 69

coupling and removal of the Fmoc group (Scheme 1) using 4 equivalents of dNBS-Cl and 10 equivalents of pyridine in dichloromethane.

Scheme: 2 Coupling between Fmoc-Histidinyl thioacid and dNBS-glycopeptide 4 using thioacid-sulfonamide ligation chemistry.1

The successful solid phase coupling of dNBS on resin-bed at β-branched amino acids such as serine and valine and in the presence of protected glycan makes the strategy useful for ligation at hindered sites (Table 1)1 Cleavage of peptides from the resin is obtained normally by TFA in the presence of various nucleophilic additives to capture

12 12 highly reactive cationic species. The TFA-TIPS-H2O (95:2.5:2.5) cleavage mixture

worked as a cleavage mixture as well as acted as a global deprotecting system for these peptides and glycopeptides leaving the glycan residue protected. The dNBS remains attached to the peptide10,13 through cleavage from the resin, which until now was not been reported to survive TFA treatment.1 The peptides were purified by reverse phase HPLC using C-8 column and 254 nm cut off wavelength followed by lyophilization.

3.3.2 dNBS-Thioacid Ligation.

The solution phase dNBS-thioacid ligation was first successfully performed between dNBS-glycopeptide 4 and Fmoc-histidine thioacid 6 (Scheme 2).1 The Fmoc- histidine thioacid was prepared from Fmoc-N(trityl)-histidine, first converting it into the corresponding thiotrityl ester using HATU and tritylthiol in presence of DIEA in DMF followed by global deprotection with TFA-TIPS-CH2Cl2 (50:5:45). The final dNBS-thio acid ligation occurred in the presence of cesium carbonate at 0.5 M total concentration of the reactants in DMF and the reaction was completed in 15 minutes. The progress of the reaction was monitored by ESI-MS. A reverse phase HPLC purification followed by lyophilization afforded the hexapeptide 7 in 71% yield.

To validate this dNBS-Thioacid ligation strategy as a potential coupling procedure for longer chain peptide and glycopeptide synthesis, the reaction was also performed between the dNBS-glycopeptide 5 and the Fmoc-Pro-Ala-thioacid 8 (Scheme

3). The dipeptide was also prepared by Fmoc-based SPPS by manual assembling of the amino acids on Barlos’ resin. Cleavage from Barlos’ resin requires very mild condition.

Treatment with TFE-AcOH-DCM (2:2:6) followed by co-evaporation with toluene after 71

the cleavage afforded the dipeptide in considerably pure form. Recently, direct conversion of N-terminus protected peptidyl acid into thioacid has been reported using

30 CDI and Na2S in acetonitrile.

We used a combination of CDI and NaHS in dichloromethane to obtain the dipeptide thioacid, which was directly used in ligation with peptide 5 without extensive purification.1 The ligation reaction was completed in 15 minutes. The reaction was monitored by ESI-MS and on completion, purified by reverse phase HPLC followed by lyophilization to obtain the glycopeptide 9 with 67% yield.

Scheme 3: Coupling between Fmoc-dipeptidyl thioacid and dNBS-glycopeptide

5 using thioacid-sulfonamide ligation chemistry.

3.4 Significance.

The successful ligation at the hindered junctures of N-arylsulfonated MUC1 glycopeptide assembly with unprotected β-branches and O-linked protected glycans using the sulfonamide-thioacid ligation chemistry suggests that this methodology is an efficient and chemoselective ligation technique for peptide and glycopeptides synthesis. We believe it would facilitate the rapid adoption of this chemistry for mixed phase peptide synthesis.

3.5 Experimental Procedures:

3.5.1 General Information

Materials. L-amino acids were used in every peptide coupling. Fmoc Ala- preloaded Wang resin and H2N-Ala 2-chlorotrityl resin (Loading: varying from 0.6 to 0.8 mmol/g), amino acids and HOBt were obtained from Chem-Impex International. PyBOP was obtained from Acros Organics and Fmoc-Ac3-Tn-α-Thr-OH was obtained from

Sussex Research. All other fine chemicals were obtained from Acros Organics, Alfa

Aesar, Fisher Scientific and Sigma-Aldrich.

General Procedures. RP-HPLC analyses were performed on a Shimadzu LC-

20AT prominence liquid chromatograph equipped with DGU-20A3 prominence degasser and all the data was processed with Shimadzu LC solution software. Premier C8 column

(150 x 4.6 mm, 5 μm) with a flow rate of 1.0 mL/min, was used for analytical HPLC a

Restek UltraC8 column (150 x 10.0 mm, 5 μm) with a flow rate of 5.0 mL/min was used for semi-preperative HPLC. Eluents were used in a gradient of either water (0.1% TFA) and MeOH (0.1% TFA) or water (0.1% TFA) and acetonitrile. UV detection was at 254 73

nm. INOVA-600 (1H NMR 600 MHz; 13C NMR 150 MHz) or Varian VXR-400 (1H

NMR 400 MHz; 13C NMR 100 MHz) were used for proton and carbon nuclear magnetic resonance spectra (1H NMR and 13C NMR) recording.

3.5.2 Solid-Phase Peptide Synthesis for dNBS capped fragments. Peptides were manually assembled on Fmoc-Ala-preloaded Wang resin (100 mg to 150 mg) using

Fmoc-based SPPS strategy. The reactions were performed in a 20 mL syringe reactor cartridge with agitation with by a stream of dry nitrogen. The amino acids used are:

Fmoc-Ser(OtBu)-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-

His(Trt)-OH, Fmoc-Pro-OH and Fmoc-Ac3-Tn-α-Thr-OH. The synthesis involved the following steps: (a) Fmoc deprotection with 20% piperidine in DMF (3 mL) for 25 min;

(b) Kaiser test; (c) washing with DMF (3 x 3 mL, 5 min/wash); (d) coupling of Fmoc amino acid (2 eq) for 3-4 h with pre-activation (2 min) in PyBOP (2 eq), HOBt (2 eq),

DIEA (4 eq) and DMF; (e) Kaiser test; (f) washing with DMF (3 x 3 mL, 5 min/wash).

After full peptide coupling, the Fmoc group was removed and resin was washed with

DMF (3 x 3 mL, 5 minutes/wash), followed by CH2Cl2 (3 x 3 mL, 5 minutes/wash). The resin was treated with 2,4-dinitrobenzenesulfonyl chloride. The peptide was then detached from the resin by treating with TFA/H2O/TIPS (95:2.5:2.5), for 4 h under nitrogen atmosphere. The mixture was then filtered, washed with the cleavage cocktail

(1-2 mL) and CH2Cl2. The filtrate was concentrated to dryness in vacuo and the crude peptide was purified by reverse phase HPLC. Yields are calculated based on the resin loading provided by the manufacturer at the time of purchase.

3.5.3 General Procedure for N-Terminal Sulfonation. Pre-swollen peptidyl resin in CH2Cl2 was taken in a 20 mL syringe reactor cartridge and 2,4- dinitrobenzenesulfonyl chloride (0.096 g, 0.36 mmol), and pyridine (0.12 mL, 1.44 mmol) were added to the resin. Agitation was provided at room temperature with continuous dry nitrogen stream and CH2Cl2 (0.5 mL) was occasionally added whenever the reaction mixture tended to dryness. On completion, the mixture was washed with

CH2Cl2 (1 x 6 mL, 5 min/wash), DMF (3 x 3 mL, 5 min/wash) and CH2Cl2 (3 x 3 mL, 5 min/wash). The coupling procedure described above sometimes was required to be performed twice. The Kaiser test was used to monitor the completion of the reaction.

3.5.4 dNBS-Gly-Val-Thr-Ser-Ala-OH (3).28 Once the N-terminal capping with dNBS was done by the general procedure described earlier, a portion (20.3 mg) of the recovered resin (170 mg) was treated with the cleavage cocktail (0.2 mL) to give the crude peptide. The crude peptide was purified by semi-preparative reverse phase-HPLC

1 and lyophilized to afford a white solid (4.4 mg, 65%). H NMR (600 MHz, CD3CN) δ

8.65 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 2.4, 8.4 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.26 (d,

J = 6.6 Hz, 1H, NH), 7.21-7.14 (m, 3H, Val-NH, Thr-NH, and Ser-OH), 4.34-4.28 (m,

2H, Ser-α-CH, and Ala-α-CH), 4.26 (dd, J = 3.0, 7.2 Hz, 1H, Thr-α-CH), 4.18-4.14 (m,

1H, Thr-β-CH), 4.05 (t, J = 7.2 Hz, 1H, Val-α-CH), 3.96 (d, J = 17.4 Hz, 1H, Gly-α-CH),

3.92 (d, J = 17.4 Hz, 1H, Gly-α-CH), 3.80 (dd, J = 4.2, 12.0 Hz, 1H, Ser-β-CH), 3.67 (dd,

J = 4.2, 11.4 Hz, 1H, Ser-β-CH), 2.09-2.04 (m, 1H, Val-β-CH), 1.34 (d, J = 7.2 Hz, 3H,

Ala-CH3), 1.09 (d, J = 6.6 Hz, 3H, Thr-CH3), 0.88 (d, J = 3.6 Hz, 3H, Val-CH3), 0.87 (d,

+ J = 3.6 Hz, 3H, Val-CH3); mass spectrum (ESI-MS), m/z = 686.3 [M+Na]

(C23H33N7NaO14S requires 686.17).

28 3.5.5 dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (4). Once the N-terminal capping with dNBS was done by the general procedure described earlier, a portion (20.7 mg) of the recovered resin (205 mg) was treated with TFA/TIPS/H2O (95:2.5:2.5) (0.2 mL) for 3 h to give the crude glycosylpeptide. The crude material was purified by semi- preparative reverse phase-HPLC and lyophilized to afford a white fluffy solid (4.4 mg,

1 59%). H NMR (600 MHz, CD3CN) δ 8.66 (d, J = 2.4 Hz, 1H), 8.51 (dd, J = 2.4, 8.4 Hz,

1H), 8.28 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H, Ser-NH), 7.17-7.15 (m, 3H, Ala-

NH, Thr-NH, Val-NH), 6.82 (d, J = 9.0 Hz, 1H, NHAc), 5.33 (d, J = 1.8 Hz, 1H, H-4),

5.06 (dd, J = 3.6, 11.4 Hz, 1H, H-3), 4.99 (d, J = 3.0 Hz, 1H, H-1), 4.43 (dd, J = 2.4, 8.4

Hz, 1H, Thr-α-CH), 4.39-4.36 (m, 1H, Ser-α-CH), 4.33-4.27 (m, 3H, Ala-α-CH, H-2, and

H-5), 4.27-4.22 (m, 2H, Thr-β-CH and Ser-OH), 4.16 (dd, J = 6.6, 7.8 Hz, Val-α-CH),

4.06 (s, 1H, H-6’), 4.05 (d, J = 1.2 Hz, 1H, H-6), 3.97 (d, J =17.4 Hz, 1H, Gly-α-CH),

3.91 (d, J =17.4 Hz, 1H, Gly-α-CH), 3.74 (dd, J = 5.4, 11.4 Hz, Ser-β-CH), 3.69 (dd, J =

4.8, 11.4 Hz, Ser-β-CH), 2.09 (s, 3H, CH3CO), 2.08-2.04 (m, 1H, Val-β-CH), 1.97 (s,

3H, CH3CO), 1.90 (s, 3H, CH3CO), 1.87 (s, 3H, CH3CO), 1.36 (d, J = 7.2 Hz, 3H, Ala-

CH3), 1.18 (d, J = 6.6 Hz, 3H, Thr-CH3), 0.90 (d, J = 6.6 Hz, 3H, Val-CH3), 0.88 (d, J =

+ 7.2 Hz, 3H, Val-CH3); mass spectrum (ESI-MS), m/z = 1015.5 [M+Na]

(C37H52N8NaO22S requires 1015.28).

3.5.6 dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5). Once the N-terminal capping with dNBS was done by the general procedure described earlier, a portion (70 mg) of the recovered resin (150 mg) was treated with TFA/TIPS/H2O (95:2.5:2.5) (0.2 mL) for 3 h to give the crude glycosylpeptide. The crude material was purified by semi- preparative reverse phase-HPLC and lyophilized to afford a white fluffy solid (20 mg,

1 56%). H NMR (600 MHz, CD3CN) δ 8.98 (s, 1H, imidazole-NH), 8.62 (d, J = 1.8 Hz,

1H, aromatic), 8.53 (dd, J = 3.0, 8.4 Hz, 1H, aromatic), 8.41 (s, 1H, imidazole CH), 8.20

(d, J = 8.4 Hz, 1H aromatic), 8.05 (t, J = 6 Hz, 1H, Gly-NH), 7.88 (d, J = 8.4 Hz, 1H,

NH), 7.81 (d, J = 6.6 Hz, 1H, NH), 7.77-7.71 (m, 1H, NH), 7.61-7.58 (m, 1H, NH), 7.23

(s, 1H, imidazole CH), 7.12 (d, J = 12 Hz, 1H, AcNH), 7.01 (d, J = 9.6 Hz, 1H, NH),

5.30 (d, J = 3.0 Hz, 1H, H-4), 5.07 (dd, J = 3, 11.4 Hz, 1H, H-3), 5.00 (d, J = 3.6 Hz, 1H,

H-1), 4.54-4.50 (m, 1H, Thr-α-CH), 4.43-4.37 (m, 1H, Ser-α-CH), 4.29-4.22 (m, 3H,

Ala-α-CH, H-2 and H-5), 4.05 (d, J = 6.00, 1H, His-α-CH), 3.90-3.85 (m, 2H, Thr-β-CH,

Ser-OH), 3.78-3.76 (m, 1H, Val-α-CH), 3.71-3.70 (m, 2H, H-6), 3.26-3.23(m, 2H, Ser-β-

CH), 3.16-3.12 (m, 2H, His-β-CH), 2.10 (s, 3H, CH3CO), 1.97-1.94 (m, 7H, Val-β-CH, 2

CH3CO), 1.90 (s, 3H, CH3CO), 1.88 (d, J = 5.4, 3H, Ala-CH3), 1.34 (d, J = 7.2, 3H, Thr-

CH3), 1.22 (dd, J = 6.6, 15.6 Hz, 3H, Val-CH3), 0.89 (dd, J = 6.6, 22.2, 3H, Val-CH3);

+ mass spectrum (ESI-MS), m/z = 1130.5 [M+H] (C43H60N11O23S requires 1130.35).

3.5.7 N-α-N-Fmoc-N-im-Trityl-Protected L-Histidine Trityl Thioester. Fmoc protected N(trityl)-histidine (0.177 g, 0.29 mmol), HATU (0.166 g, 0.44mmol) and tritylmercaptan (0.087 g, 0.31 mmol) were dried in vacuum for 30 minutes and taken in dry DMF (1.0 mL) followed by the addition of DIEA (0.20 mL, 1.26 mmol). The 77

resulting solution was stirred for 2 hours at room temperature and completion of the reaction was confirmed by TLC. The reaction mixture was taken in ethyl acetate (20 mL), washed with water (2 x 10 mL) and brine (1 x 10 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure and the crude material was purified by flash column chromatography on SiO2 using stepwise gradient of acetone- ethyl acetate-hexane (5:5:90 and 10:10:80) to provide a colorless powder. Yield: 0.231 g

1 (92%); TLC Rf = 0.09 (acetone-ethyl acetate-hexane =7.5:7.5:85); H NMR (600 MHz,

CDCl3): δ 7.73 (d, J = 7.8 Hz, 2H, aromatic H), 7.26 (t, J = 9.0, 2H, aromatic H), 7.39 (s,

1H, imidazole H), 7.35 (t, J = 7.8 Hz, 2H, aromatic H), 7.31-7.25 (m, 8H, aromatic H),

7.22-7.08 (m, 24H, aromatic H), 7.02 (d, J = 7.8 Hz, 1H, NH), 6.61 (s, 1H, imidazole H),

4.58 (q, J = 7.8 Hz, 1H, α-CH), 4.45 (dd, J = 6.0, 9.0 Hz, 1H, Fmoc CH), 4.55-4.40 (m,

13 2H, Fmoc CH2), 2.95 (d, J = 5.4, 2H, β-CH); C NMR (150 MHz, CDCl3): δ 198.43

(SC=O), 156.23,144.17,144.03,143.88, 142.48, 141.38, 141.36, 138.96,136.19, 130.04,

129.96, 128.27, 128.25, 127.83, 127.77, 127.75, 127.24, 127.20, 127.10, 125.62, 125.46,

120.04, 119.59, 75.50, 70.11, 67.48, 61.25, 47.28, 29.88; mass spectrum (ESI-MS), m/z =

+ 878.0 [M+H] (C59H49N3O3S requires 878.3).

3.5.8 Fmoc-His-SH (6). The N-α-N-Fmoc-N-im-Trityl-Protected L-Histidine

Trityl Thioester (0.2 g) was treated with cleavage cocktail of TFA-TIPS-CH2Cl2

(50:5:45) for 15 minutes under nitrogen atmosphere and the mixture was concentrated to dryness under reduced pressure to obtain the crude material. The desired thioacid was detected by ESI-MS and the crude was directly taken to the next step of thioacid-dNBS ligation reaction without any more purification. 78

3.5.9 Solid Phase Synthesis of Fmoc-Pro-Ala-OH. The Fmoc-Pro-OH was manually assembled on a H2N-Ala preloaded 2-chlorotrityl resin (0.1 g) using Fmoc- based SPPS strategy. The reaction was performed in a 20 mL syringe reactor cartridge with agitation by a stream of dry nitrogen. The synthesis was performed as described in the general SPPS coupling strategy above. The resin was treated with cleavage cocktail of

TFE-AcOH-CH2Cl2 (2:2:6) (3 mL) for 4 h at room temperature under nitrogen atmosphere to cleave off the peptide. Evaporation of the solvents from the washings gave a thick oil which after azeotroping with toluene afforded the desired dipeptide as a white solid. The purity of the product, according to TLC, ESI-MS and 1HNMR, was good enough to proceed directly to the next step without further purification. Yield: 0.026 g

(91%). TLC Rf = 0.3 (methanol-dichloromethane = 1:9). This compound is mentioned in the literature. ‡

‡Basak A.; Bag S. S.; Basak A.; Bioorg.Med.Chem., 2005,

3.5.10 N-Fmoc-L-prolyl-L-alanine Trityl Thioester (9). The peptide (19.7 mg,

0.048 mmol) was treated with HATU (62.9 mg, 0.17 mmol), tritylmercaptan (32.6 mg,

0.12 mmol), DIPEA (0.035 mL, 0.21mmol) in DMF (0.75 mL) to obtain the thioester after 3 hours and then the General Procedure given above for amino acid thioesters was followed. The crude material was subjected to flash column chromatography on SiO2 (1.6 x 14 cm) using a gradient eluent MeOH-CH2Cl2 (0:100 and 0.5:99.5) to afford the thioester as a yellow film. Yield: 11.7 mg (36%); silica gel TLC Rf = 0.09 (MeOH-CHCl3

+ = 1:99); ESI-MS: m/z calcd for C42H38NaN2O4S 689.2, found: 689.1 [M+Na] . 79

3.5.11 Fmoc-Pro-Ala-SH (10). A solution of Fmoc-Pro-Ala-OH (0.013 g,

0.0318 mmol) in CH2Cl2 (1.5 mL) was added to a solution of CDI (0.025 g, 0.159 mmol) in CH2Cl2 (1 mL) and stirred for 30 min under N2 atmosphere. NaHS was then added to the mixture followed by stirring for another 3 h under N2 atmosphere. The completion of the reaction was confirmed by ESI-MS monitoring and on completion, the reaction mixture was diluted with CH2Cl2 (6 mL) and the pH was brought to 3 by adding ice cold

1(N) HCl. The organic layer was separated and dried over sodium sulfate. Evaporation of the solvent resulted the desired crude product as yellowish white semisolid (0.010 g,

74%) that was directly used in the thioacid-dNBS ligation reaction without further

+ purification. Mass spectrum (ESI-MS), m/z = 447.4 [M+Na] (C23H24N2O4SNa requires

447.5).

3.5.12 General Procedure for Thioacid-dNBS ligation: Peptidyl thioacid (1.2 eq) and Cs2CO3 (2 eq) was added to a DMF solution of the dNBS-peptide (1 eq) and the reaction mixture is stirred for 15 minutes under N2 atmosphere at room temperature. The completion of the reaction was monitored by ESI-MS and the DMF was azeotroped with toluene under reduced pressure to get the crude peptide as yellowish semisolid. The crude material was purified by semi-preparative reverse phase-HPLC and lyophilized to afford the pure product.

3.5.13 Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (7): According to the general procedure mentioned above, to a DMF(0.1 mL) solution of Fmoc-His-SH (0.84 80

mg, 0.0020 mmol) Cs2CO3 (1.1 mg, 0.0034 mmol) was added followed by DMF solution of the dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (1.7 mg, .0017 mmol) and the reaction mixture was stirred for 15 minutes under nitrogen atmosphere at room temperature. The completion of the reaction was monitored by ESI-MS and the DMF was azeotroped with toluene under reduced pressure to get the crude peptide as yellowish semisolid. The crude material was purified by semi-preparative RP-HPLC and freeze-

1 dried to afford the pure product. Yield: 1.4 mg (71%). H NMR (600 MHz, CD3OD ): δ

8.71 (s, 1H, imidazole H), 7.81 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 7.8 Hz, 2H), 7.40 (t, J =

7.2 Hz, 2H), 7.33-7.30 (m, 2H), 7.22 (s, 1H, imidazole H), 7.02 (d, J = 9.0 Hz,1H, NH),

6.65 (d, J = 8.4 Hz, 1H, NH), 5.34 (s, 1H, H-4), 5.14 (dd, J = 3.0, 11.4 Hz, 1H, H-3), 5.11

(d, J = 4.2 Hz, 1H,H-1), 4.63 (s, 1H, Thr-α-CH), 4.50 (t, J = 4.8 Hz, 1H, Ser-α-CH),

4.48-4.40 (m, 3H, His-α-CH & Fmoc CH2), 4.39-4.34 (m, 2H, H-2, Ala-α-CH), 4.30-4.28

(m, 3H, Thr-β-CH & 2 other protons), 4.20 (t, J = 6.0 Hz, 1H, Fmoc CH), 4.09-4.03 (m,

2H), 3.97 (d, J = 5.4, 2H, Gly-CH2), 3.82 (dd, J = 4.8, 11.4 Hz,1H, Ser-β-CH), 3.14-

3.09(m, 2H, His-β-CH), 2.19-2.17 (m, 1H, Val-β-CH), 2.14(s, 3H, CH3CO), 2.00( s, 3H,

CH3CO), 1.97 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO), 1.42 (d, J = 7.8 Hz, Ala-CH3), 1.28

(d, J = 6.6 Hz, Thr-CH3), 1.00 (d, J = 7.2 Hz, 3H, Val-CH3), 0.99 (d, J = 7.2 Hz, 2H, Val-

+ CH3); Mass spectrum (ESI-MS), m/z = 1122.3 [M+H] (C52H68N9O19 requires 1122.5).

3.5.14 Fmoc-Pro-Ala-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (11):

According to the general procedure mentioned above, to a DMF(0.1 mL) solution of

Fmoc-Pro-Ala-SH (1.53 mg, 0.0036 mmol) Cs2CO3 (1.95 mg, 0.006 mmol) was added followed by DMF solution of the dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (3.5 81

mg, .003 mmol). The reaction mixture was stirred for 15 minutes under N2 atmosphere at room temperature. The completion of the reaction was monitored by ESI-MS and the

DMF was co-evaporated with toluene under reduced pressure to get the crude peptide as yellowish semisolid. The crude material was purified by semi-preparative reverse phase-

HPLC and lyophilized to afford the pure product. Yield: 2.05 mg (67%). Mass spectrum

+ (ESI-MS), m/z = 1291.1 [M+H] (C60H80N11O21 requires 1290.55).

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Chapter 4

Synthesis of L-rhamnosyl ceramide and evaluation of its binding to anti-Rhamnose antibodies: Use of rhamnose ceramides as a tumor marker.

Graphics 1: Graphical representation of binding of anti-rhamnose antibodies to tumor

cells displaying rhamnose epitope on its surface and detection of the binding by flow

cytometry

4.1 Project Summary

Tumor cells contain a variety of antigens that can be used as tools in provoking an anti-tumor immune response. However, these tumor associated antigens are often poorly presented to the immune system. The antigen uptake by the antigen presenting cells can be enhanced if the tumor cells contain surface markers that can bind to existing

antibodies to form immune complexes. In this study, we showed that an α-L-rhamnosyl ceramide (1, α-L-RhaCer) can non-covalently label tumor cells and the resulting cells are strongly recognized by anti-rhamnose (anti-Rha) antibodies. In the course of α-L-RhaCer

1 synthesis, we have explored the use of an L-rhamnosyl thioglycoside and trichloroacetimidate as glycosyl donors and the acceptors desired for glycosylation, 3-O- benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D- xylose. Liposomes and EL4 cells were used as model systems for tumor cells to demonstrate the ability of 1 to insert into a lipid bilayer. The assembly of the liposomes or the EL4 cells with α-L-RhaCer 1 and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to demonstrate the ability of

α-L-RhaCer 1 to be displayed on the cell surface as well as to be recognized by anti-Rha antibodies. α-Rhamnose antibodies are highly abundant in human serum. Hence, the ability to non-covalently label tumor cells with a rhamnosyl ligand is postulated to serve as a surface marker for an autologous anti-tumor immunotherapy.

4.2 Introduction:

A protective anti-tumor immune response can be generated by tumor associated antigens (TAA) following anti-cancer vaccination via sufficient uptake of the TAA by antigen presenting cells (APC), which can be processed and displayed on MHC (major histocompatibility complex) molecules to generate cytotoxic T cells and helper T cells.

However, naturally occurring tumor cells often lack surface markers that facilitate the antigen uptake by APC. One approach for overcoming this problem is to decorate tumor cells with natural antibodies which can interact with Fcγ receptors (FcγR) on APC and 88

induce tumor cell uptake.1 For example, injection of heterogeneous glycolipids isolated from rabbit red blood cells containing terminal α-gal epitopes was injected into melanoma tumors implanted in mice that express a high level of anti-α-gal antibodies.1

The resulting tumor cells displayed the α-gal epitopes on their membranes and an anti- tumor response was observed.1

Potential drawbacks of this strategy are the need of isolation of α-gal glycolipids from rabbits and maintaining the heterogeneity of the isolates is not easy. Moreover, anti- rhamnose antibodies are more abundant naturally occurring antibodies than anti- α-gal.2

To overcome the problem of heterogeneity, we designed a homogeneous α-L-RhaCer 1 which we envisaged could be injected into melanoma or any epithelial tumor (Figure 1).

Model systems for generating and studying anti-α-gal antibodies require α-gal knock out mice. The advantage of studying the response to anti-Rha antibodies is that they can be generated in common laboratory mice.3

Figure 1: α-L-RhaCer 1 and the control ceramide 2.

Since the fatty acid tails are thermodynamically more stable when surrounded by other lipids as opposed to water, spontaneous insertion of α-L-RhaCer (1) into phospholipid-based cell membrane is expected to occur.1 This insertion causes the anti-

Rha epitopes to be displayed on the surface of the tumor cells that would allow binding of anti-Rha antibodies to the Rha epitopes. Thus, after forming the immune complex, the Fc portions of the anti-Rha antibodies would be expected to interact with the FcγR on APC analogous to what has been shown with α-gal glycolipids and anti-α-gal antibodies.1

These interactions allow the tumor cells to be accessible for adequate antigen uptake by the APC, resulting a substantial anti-TAA immune response. We previously showed that anti-Rha antibodies facilitate higher uptake and presentation of antigens that were delivered in Rha-bearing liposomes.3b In order to design a homogeneous glycolipid we looked into naturally occurring ceramides. Ceramides have varying N-acyl chain lengths, from 14 to 26 carbons (C14 to C26) in mammals and the most abundant are C16 (16:0) and C24 (24:0 and 24:1).4 With this rationale, a synthesis was designed to prepare an anomerically pure glycolipid, using L-rhamnose and ceramide (C16 or C24) to produce potential tumor marker for the development of an anti-cancer vaccine.D We chose rhamnosyl ceramide 1 as our model glycolipid and the ceramide 2 was used as a negative control (Figure 1)

4.3 Results and discussion:

Anomerically pure α-L-RhaCer 1 and its des-rhamnose analogue 2 were successfully synthesized by Long, D. E. using a high yielding synthetic plan. In order to access anti- rhamnose antibodies, two BALB/cJ mice were primed and boosted with 1 µg rhamnose- 90

ovalbumin conjugate (Rha-Ova)3b,c with Sigma Adjuvant System (1:1 ratio). The production of anti-Rha antibodies was confirmed by ELISA assay from the pooled blood sera of the two BALB/cJ (Figure 2). On day 38 following priming, blood was pooled from the two mice and the sera was isolated and antibodies were isolated by precipitation from the sera at 40% saturated ammonium sulfate solution (pH ~7) followed by dialysis against phosphate buffered saline (PBS). Control antibodies were isolated in a similar way from nonimmunized mice.

Figure 2. Anti-Rhamnose Antibody Titer of BALB/cJ Mice Immunized with

Rhamnose Ovalbumin Conjugate (after 2nd Boost) by ELISA assay. The absorbance was measured at 630 nm.

In order to demonstrate that synthetic α-L-RhaCer 1 can be recognized by anti- rhamnose antibodies, we first considered liposomes as our primary model for bi-lipid layered cell membrane. We prepared liposomes of approximately 100 nm in size that displayed α-L-RhaCer 1 or control ceramide 2 on their surface. The liposomes were formulated with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (80%) and 91

cholesterol (20%) in a total lipid concentration of 30 mM.3c The 100 nm size was obtained by extrusion method using 100nm porous polycarbonate membrane filter and a manual extruder. Three batches of liposomes were prepared to demonstrate the binding of the liposomal rha-ceramide with anti-rha antibodies, the first displaying the α-L-RhaCer

1 (10 nM), and the second displaying the ceramide 24 (10 nM). The third batch of liposomes did not contain any ceramide and was used as a background.

Figure 3. Fluorescence microscope images with liposomes under 60x magnification. (A) α-L-RhaCer liposomes with anti-rhamnose antibodies (B) α-L-RhaCer liposomes with non- rhamnose control antibodies (C) liposomes with anti-rhamnose antibodies. All with FITC-goat anti-mouse IgG/IgM

To test whether rhamnosyl-ceramide can interact with surface exposed anti-Rha antibodies, liposomes displaying the different ceramides on their surface were incubated with either anti-Rha antibodies collected from Rha-Ova immunized mice sera that contains high concentration of anti-Rha antibodies or antibodies collected from non- immunized mice sera. The resulting complexes were incubated with FITC-conjugated goat anti-mouse IgG/IgM secondary antibodies. The fluorescence imaging (Figure 3) 92

shows specific binding of rhamnosyl-ceramide displayed on the liposome surface to anti- rhamnose antibodies.

Once we were able verify that the α-L-RhaCer (1) displayed on liposome surfaces could successfully bind to anti-rhamnose antibodies, the next step was to see whether it could be recognized on real cancer cells. We used EL4 tumor cells as the model cancer cells. Our plan was to expose the EL4 cells to either α-L-RhaCer (1) or Cer (24) followed by anti-Rha or non-Rha antibodies. After a final incubation with FITC-conjugated goat anti-mouse IgG/IgM secondary antibody, a flow cytometry would be performed with the tumor cells in order to determine any binding. The EL4 cells are generally grown in

DMEM medium that contains 10% fetal calf serum.

Figure 4. Flow cytometry of EL4 cells. FL1-A = FITC (A) EL4 with α-L-RhaCer and anti-rhamnose antibodies (B) EL4 with α-L-RhaCer and non-rhamnose antibodies

(C) Black peak: EL4 cells with RhaCer and non-Rha antibodies; blue peak: EL4 cells with Cer and anti-Rha antibodies; red peak: EL4 cells with RhaCer and anti-Rha antibodies. All samples contained FITC-anti mouse IgG/IgM.

We realized that ceramides have strong affinity to the albumin that the fetal calf serum contains. Hence, after growing the EL4 cells in DMEM medium with 10% fetal calf serum, the cells were washed thoroughly with DMEM medium alone and finally, the incubation with α-L-RhaCer 1 or ceramide 2 were performed in DMEM medium alone.

After the incubation, the cells were taken in Hank’s balanced salt solution (HBSS) and then incubated with anti-Rha antibodies or non-Rha antibodies. The cells were washed and further incubated with FITC-conjugated goat anti-mouse IgG/IgM secondary antibody and taken in HBSS after washing off any unbound antibodies and flow cytometry was performed on an Accuri c6 flow cytometer. The flow cytometry data clearly shows that the synthetic α-L-RhaCer (1) is displayed on tumor cell surfaces and is recognized by anti-Rha antibodies (Figure 4).

4.4 Significance.

We have demonstrated that the ceramide 1 can be incorporated on the surface of on the surface of a liposomal phospholipid bilayer as well as on a tumor cell surface and can be recognized by anti-Rha antibodies of the α-Rha motif. These results suggest that 1 could be displayed on autologous tumor cells in a similar fashion. We hypothesize that the reinjection of these modified tumor cells into animals would be recognized by natural or induced anti-Rha antibodies to produce an improved immune-complex mediated anti- tumor response through FcγRs on APCs. We speculate that this new technique of using

α-L-RhaCer tumor cell markers to induce immune-complex formation that is recognized

by naturally occurring antibodies would create a platform to develop potent anti-tumor vaccines.

4.5 Experimental Procedure.

4.5.1 Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was purchased from Avanti Polar Lipids. Ovalbumin and horseradish peroxidase (HRP) goat anti-mouse IgG/ IgM were obtained from Sigma. FITC goat anti-mouse IgG/IgM was purchased from BD-Biosciences (San Jose, CA). Female, 6-8 week old BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in the

Animal Care Facility at the Health Science Campus, University of Toledo. All animal care procedures and experimental protocols was approved by the Institutional Animal

Care and Use Committee (IACUC) of the University of Toledo. C57BL/6 EL4 lymphoma cells were purchased from the ATCC (TIB-39). Fluorescence microscopy was carried out on a Nikon TiU microscope. Flow cytometry was carried out on a BD Accuri

C6 flow cytometer.

4.5.2 Binding Study

4.5.2.1 Liposome Formulation: Stock solutions of DPPC, rhamnose-cholesterol and cholesterol were prepared by dissolving each of them in chloroform. Aliquots taken from the stock solutions were mixed in proportions to obtain a solution with a total lipid concentration of 30 mM in a total volume of 2 mL (Rhamnosyl-ceramide-Liposomes:

DPPC 80%, Cholesterol 20%, and Rhamnosyl Ceramide 1 10 nM; Ceramide-Liposome:

DPPC 80%, cholesterol 20%, Control Ceramide 21 10 nM; Blank Liposome: DPPC 80%,

cholesterol 20% ). Chloroform was evaporated from the lipid solutions by a constant flow of nitrogen to obtain a lipid film. The resulting lipid films were dried under vacuum for

12 hours. After proper drying, the lipid films were crushed and hydrated with 2 mL of

HEPES buffer (pH = 7.4). The suspensions of the lipids in the buffer were incubated at

43 °C for 40 min with continuous agitation. The suspensions were then taken through 10 freeze-thaw cycles using dry ice/acetone and water at 40 °C as freezing and thawing baths, respectively. The liposomes were then subjected to an extrusion process (21 times) using a LipoFast Basic fitted with a 100 nm polycarbonate membrane to obtain liposomes of the desired size.

4.5.2.2 Anti-Rhamnose Antibody Binding to Surface Exposed Rhamnosyl

Ceramide 1 on Liposomes: 50 µL liposomes from each batch was taken in 50 µL phosphate buffered saline (PBS) into a 1.5 mL Eppendorf tube. After the addition of 50

µL of primary antibody (isolated antibody from Rha-Ova immunized mice that is enriched with anti-rhamnose antibodies or isolated antibody from non-immune mice) solution in deionized water (50 µg/mL of antibody concentration), the tubes were incubated on ice for 30 mins. 100 µL PBS containing 0.1% Tween was then added to each tube and vortexed. The tubes were then centrifuged at 14000 rpm in an Eppendorf centrifuge at 4 °C for 5 min to pellet the liposomes. The supernatants were carefully discarded and the liposomes were re-suspended in 100 µL PBS containing 0.1% Tween.

The process was repeated twice and finally was taken in 50 µL of PBS-0.1% Tween. The tubes were then incubated on ice in the dark for 30 min with 50 µL of diluted FITC goat anti-mouse IgG/IgM secondary antibody (30 µg/mL). The unbound second antibodies 96

were removed by washing the liposomes thrice with PBS-0.1% Tween and after the final wash, the liposome pellets were re-suspended in 1 mL PBS-0.1% Tween. 10 µL of the liposome suspensions were taken on glass slides and imaged under a Nikon TiU fluorescence microscope.

4.5.2.3 Anti-Rhamnose Antibody Binding to Surface Exposed Rhamnosyl

Ceramide 1 on EL4 cells; Fluorescence-activated cell sorting (FACS):

EL4 tumor cells were cultured in DMEM medium with 10% fetal calf serum. Cells were then washed with DMEM medium re-suspended in DMEM without serum. The cells were then incubated with 50 µg/mL RhaCer 1 or Cer 2 (prepared by taking 20 µL of

2mg/mL DMSO solution of RhaCer 1 or Cer 2 and diluting it to 2mL with the medium) for 90 min at 37 °C. The cells were then washed five times and taken in Hank’s balanced salt solution (HBSS) and then incubated with anti-Rha antibodies or non-Rha antibodies

(50 µL of 50 µg/mL)for one hour at 0 °C. The cells were washed five times and further incubated with FITC-conjugated goat anti-mouse IgG/IgM secondary antibody for one hour at 0 °C. The cells were again washed five times and taken in HBSS and flow cytometry was performed on an Accuri c6 flow cytometer.

References

1. Galili, U.; Albertini, M.; Sondel, P.; Wigglesworth, K.; Sullivan, M.; Whalen, G.

Cancers. 2010, 2, 773.

2. (a) Oyelaran, O.; McShane, M. L.; Dodd, L.; Gildersleeve, J. C. J. Proteome Res.

2009, 8, 4301. (b) Huflejt, M. E., Vuskovic, M., Vasiliu, D., Xu, H., Obukhova,

P., Shilova, N., Tuzikov, A., Galanina, O., Arun, B., Lu, K., Bovin, N. Mol.

Immunol. 2009, 46, 3037. (c) Schwarz, M.; Spector, L.; Gargir, A.; Shtevi, A.;

Gortler, M.; Alstock, R. T.; Dukler, A. A.; Dotan, N. Glycobiology, 2003, 13,

749.

3. (a) Chen, W.; Gu, L.; Zhang, W.; Motari, E.; Cai, L.; Styslinger, T. J.; Wang, P.

G. ACS Chem. Biol. 2011, 6, 185. (b) Sarkar, S.; Lombardo, S. A.; Herner, D. N.;

Talan, R. S.; Wall, K. A.; Sucheck, S. J. J. Am.Chem. Soc. 2010, 132, 17236.

2013, 24, 363.

4. Long, D. E.; Karmakar, P.; Wall, K. A.;Sucheck, S. J., . Bioorg. Med. Chem.

2014, 22, 527

Appendix A

Supplimentary data from chapter 2

Page 100: HR-MALDI-TOF For Glycopeptide 1

Page 101: RP-HPLC Trace For Glycopeptide 1

Page 102: HR-MALDI-TOF For Glycopeptide 2

Page 103: HR-MALDI-TOF For Pam3Cys MUC1-Tn 4

Page 104: HR-MALDI-TOF For Pam3Cys MUC1-Tn 4 (Peak Expansion)

Page 105: HR-MALDI-TOF For CD8+ T Cell Epitope Glycopeptide 5

HR-MALDI-TOF For Glycopeptide 1:

x104 2355.217

Intens. [a.u.] Intens. 2.0

1.5 2452.273

1.0

2256.114 0.5

1537.748

2062.014 1796.860 2187.076

0.0 500 1000 1500 2000 2500 3000 3500 m/z

100

RP-HPLC Trace For Glycopeptide 1:

101

HR-MALDI-TOF For Glycopeptide 2:

x104

2229.336 Intens. [a.u.] Intens.

568.072

2326.410

1742.715 760.913 1115.161 1411.833 0 500 1000 1500 2000 2500 3000 3500 m/z

102

HR-MALDI-TOF For Pam3Cys MUC1-Tn 4:

3177.797 Intens. [a.u.] Intens.

3000

2000

1000

2000 2250 2500 2750 3000 3250 3500 3750 4000 m/z

103

HR-MALDI-TOF For Pam3Cys MUC1-Tn 4 (Peak Expansion):

3177.797

[a.u.] Intens. 3176.772 3000

3178.768

2000

3175.809

3179.793

1000

3173 3174 3175 3176 3177 3178 3179 3180 3181 m/z

104

HR-MALDI-TOF For CD8+ T Cell Epitope Glycopeptide 5:

x104 1017.940

1.0 Intens. [a.u.] Intens.

0.8

0.6

0.4

0.2

0.0

995 1000 1005 1010 1015 1020 1025 1030 1035 m/z

105

Appendics B

Supplimentary data from chapter 3

Page 108: 1H NMR of dNBS-Gly-Val-Thr-Ser-Ala-OH (3)

Page 109: 1H-1H gCOSY NMR of dNBS-Gly-Val-Thr-Ser-Ala-OH (3)

1 Page 110: H NMR of dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (4)

1 1 Page 111: H- H gCOSY NMR of dNBS-Gly-Val-(Ac3-α-Tn-Thr)-Ser-Ala-OH (4)

1 Page 112: H NMR of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5)

1 1 Page 113: H- H gCOSY NMR of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5)

Page 114: ESI-MS of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5)

Page 115: 1H NMR of N-α-Fmoc-N-im-Trityl-Protected L-Histidine Trityl Thioester

Page 116: 1H-1H gCOSY NMR of N-α-Fmoc-N-im-Trityl-Protected L-Histidine Trityl

Thioester

Page 117: ESI-MS of N-Fmoc-L-prolyl-L-alanine Trityl Thioester (9).

Page 118: ESI-MS of Fmoc-Pro-Ala-SH (10)

1 Page 119: H NMR of Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (7)

1 1 Page 120: H- H gCOSY NMR of Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (7)

Page 121: ESI-MS of Fmoc-Pro-Ala-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (11)

106

Page 122: Analytical RP-HPLC of purified dNBS-Val-Thr-Ser-Ala-OH (2)

Page 123: Analytical RP-HPLC of purified dNBS-Gly-Val-Thr-Ser-Ala-OH (3)

Page 124: Analytical RP-HPLC of purified and dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala- OH (4)

Page 125: Analytical RP-HPLC of purified dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser- Ala-OH (5)

Page 126: Analytical RP-HPLC of purified Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala- OH (7)

Page 127: Analytical RP-HPLC of purified Fmoc-Pro-Ala-His-Gly-Val-(Ac3-Tn-α-Thr)- Ser-Ala-OH (11)

107

1H NMR of dNBS-Gly-Val-Thr-Ser-Ala-OH (3)

OH O N O O 2 O O H H S N N OH N N N H H H O O O O2N OH

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

108

1H-1H gCOSY NMR of dNBS-Gly-Val-Thr-Ser-Ala-OH (3)

OH O N O O 2 O O H H S N N OH N N N H H H O O O O2N OH

109

1 H NMR of dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (4)

AcO OAc O AcO AcHN O O N O O 2 O O H H S N N OH N N N H H H O O O O2N OH

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

110

1 1 H- H gCOSY NMR of dNBS-Gly-Val-(Ac3-α-Tn-Thr)-Ser-Ala-OH (4)

AcO OAc O AcO AcHN O O N O O 2 O O H H S N N OH N N N H H H O O O O2N OH

111

1 H NMR of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5).

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

112

1 1 H- H gCOSY NMR of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5).

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0 9 8 7 6 5 4 3 2 1

Note: The peak at δ = 3.43 for residual water was deemphasized by apodization.

113

ESI-MS of dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (5).

Intens. All, 0.0-0.7min (#1-#149)

x107 1130.6 1.2

1.0

0.8

0.6

1059.5 0.4

0.2

1066.6

1152.6

1105.5 1190.4

0.0 1060 1080 1100 1120 1140 1160 1180 m/z

114

1H NMR of N-α-Fmoc-N-im-Trityl-Protected L-Histidine Trityl Thioester:

N N Trt

STrt FmocHN O

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

115

1H-1H gCOSY NMR of N-α-Fmoc-N-im-Trityl-Protected L-Histidine Trityl Thioester:

N N Trt

STrt FmocHN O

116

ESI-MS of N-Fmoc-L-prolyl-L-alanine Trityl Thioester (9).

Intens. All, 0.0-1.1min (#2-#114) x107

2.0 689.1

1.5

1.0

0.5

705.1 721.0

0.0 300 400 500 600 700 800 900 m/z

117

ESI-MS of Fmoc-Pro-Ala-SH (10).

Intens. All, 0.0-0.4min (#1-#88) x107 1.50

447.4 1.25

1.00

0.75 431.4

0.50

385.3

0.25

339.4

463.3

413.5

425.3

477.3 493.6

467.0

371.6

480.4

304.5

459.5 368.9 0.00 300 320 340 360 380 400 420 440 460 480 m/z

1 H NMR of Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (7):

118

AcO OAc O AcO AcHN O O O O H H FmocHN N N OH N N N H H H O O O N OH

N H

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

119

1 1 H- H gCOSY NMR of Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (7):

AcO OAc O AcO AcHN O O O O H H FmocHN N N OH N N N H H H O O O N OH

N H

120

ESI-MS of Fmoc-Pro-Ala-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-OH (11):

Intens. All, 0.0-0.2min (#1-#47)

x106 1291.1

2.0

1.5

1.0

0.5

1338.0

1266.1

1285.5

1306.5 1346.4

1297.5

1340.0

1277.6

1327.7

1284.0

1273.7

1303.5 1324.1

1314.9

1348.0

1261.5 1301.1 1341.4

1259.5 1299.0

1288.9

1250.4 1255.6 0.0 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 m/z

121

mV Detector A:254nm

2500

O N NO2 OH 2 O O H H N N OH S N N 2000 O H H O O O OH

1500

1000

500

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

Retention time (minute) Figure 1. Analytical RP-HPLC of purified dNBS-Val-Thr-Ser-Ala-OH (2) eluting with

35-90% gradient of

H2O (0.1%TFA) and MeOH ( 0.1% TFA) over a period of 35 minutes, UV detection at

254 nm.

122

mV Detector A:254nm 1250

OH O N O O 2 O O H H S N N OH 1000 N N N H H H O O O O2N OH

750

500

250

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

Retention time (minute)

Figure 2. Analytical RP-HPLC of purified dNBS-Gly-Val-Thr-Ser-Ala-OH (3) eluting with 30-90% gradient of

H2O (0.1%TFA) and MeOH ( 0.1% TFA) over a period of 35 minutes, UV detection at

254 nm.

123

mV Detector A:254nm

900 AcO OAc O 800 AcO AcHN 700 O O N O O 2 O O H H S N N OH 600 N N N H H H O O O O2N OH 500

400

300

200

100

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

Retention time (minute)

Figure 3. Analytical RP-HPLC of purified and dNBS-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-

OH (4) eluting with 30-90% gradient of H2O (0.1%TFA) and MeOH (0.1% TFA) over a period of 35 minutes, UV detection at 254 nm.

124

mV Detector A:254nm 110

100

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min

Retention time (minute)

Figure 4. Analytical RP-HPLC of purified dNBS-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-

OH (5), eluting with 3-55% gradient of H2O (0.1%TFA) and ACN over a period of 50 minutes, UV detection at 254 nm.

125

mV Detector A:254nm 2750 AcO OAc O 2500 AcO AcHN 2250 O O O O H H 2000 FmocHN N N OH N N N H H H O O O 1750 N OH

N 1500 H

1250

1000

750

500

250

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min Retention time (minute)

Figure 5. Analytical RP-HPLC of purified Fmoc-His-Gly-Val-(Ac3-Tn-α-Thr)-Ser-Ala-

OH (7), eluting with 30-90% gradient of H2O (0.1%TFA) and MeOH ( 0.1% TFA) over a period of 35 minutes, UV detection at 254 nm.

126

mV Detector A:254nm 45

0 10 20 30 40 50 min

Retention time (minute)

Figure 6. Analytical RP-HPLC of purified Fmoc-Pro-Ala-His-Gly-Val-(Ac3-Tn-α-Thr)-

Ser-Ala-OH (11), eluting with 3-55% gradient of H2O (0.1%TFA) and ACN over a period of 50 minutes, UV detection at 254 nm.

127